Kallow, S.; Garcia Zuluaga, M.; Fanega Sleziak, N.; Nugraha, B.;
Mertens, A.; Janssens, S.B.;
Gueco, L.; Valle-Descalsota, M.L.; Dang Vu, T.; Toan Vu, D.; Thi Li, L.; Vandelook, F.; Dickie, J.B.; Verboven, P.;
Swennen, R.;
Panis, B.;
Conservation Physiology, 2022 |
Peer Reviewed
The ability of seeds to withstand drying is fundamental to ex situ seed conservation but drying responses are not well known for most wild species including crop wild relatives. We look at drying responses of seeds of Musa acuminata and Musa balbisiana, the two primary wild relatives of bananas and… Show full abstract plantains, using the following four experimental approaches: (i) We equilibrated seeds to a range of relative humidity (RH) levels using non-saturated lithium chloride solutions and subsequently measured moisture content (MC) and viability. At each humidity level we tested viability using embryo rescue (ER), tetrazolium chloride staining and germination in an incubator. We found that seed viability was not reduced when seeds were dried to 4% equilibrium relative humidity (eRH; equating to 2.5% MC). (ii) We assessed viability of mature and less mature seeds using ER and germination in the soil and tested responses to drying. Findings showed that seeds must be fully mature to germinate and immature seeds had negligible viability. (iii) We dried seeds extracted from ripe/unripe fruit to 35-40% eRH at different rates and tested viability with germination tests in the soil. Seeds from unripe fruit lost viability when dried and especially when dried faster; seeds from ripe fruit only lost viability when fast dried. (iv) Finally, we dried and re-imbibed mature and less mature seeds and measured embryo shrinkage and volume change using X-ray computer tomography. Embryos of less mature seeds shrank significantly when dried to 15% eRH from 0.468 to 0.262 mm3, but embryos of mature seeds did not. Based on our results, mature seeds from ripe fruit are desiccation tolerant to moisture levels required for seed genebanking but embryos from immature seeds are mechanistically less able to withstand desiccation, especially when water potential gradients are high. Hide full abstract 
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Thi, L.L.;
Mertens, A.; Vu, D.T.; Vu, T.D.; Minh, P.L.A.; Duc, H.N.; de Backer, S.;
Swennen, R.; Vandelook, F.;
Panis, B.; Amalfi, M.; Decock, C.; Gomes, S.I.F.; Merckx, V.S.F.T.; Janssens, S.B.;
MycoKeys, 2022 |
Peer Reviewed
Fusarium is one of the most important fungal genera of plant pathogens that affect the cultivation of a wide range of crops. Agricultural losses caused by Fusarium oxysporum f. sp. cubense (Foc) directly affect the income, subsistence, and nourishment of thousands of farmers worldwide. For Viet Nam,… Show full abstract predictions on the impact of Foc for the future are dramatic, with an estimated loss in the banana production area of 8% within the next five years and up to 71% within the next 25 years. In the current study, we applied a combined morphological-molecular approach to assess the taxonomic identity and phylogenetic position of the different Foc isolates collected in northern Viet Nam. In addition, we aimed to estimate the proportion of the different Fusarium races infecting bananas in northern Viet Nam. The morphology of the isolates was investigated by growing the collected Fusarium isolates on four distinct nutritious media (PDA, SNA, CLA, and OMA). Molecular phylogenetic relationships were inferred by sequencing partial rpb1, rpb2, and tef1a genes and adding the obtained sequences into a phylogenetic framework. Molecular characterization shows that c. 74% of the Fusarium isolates obtained from infected banana pseudostem tissue belong to F. tardichlamydosporum. Compared to F. tardichlamydosporum, F. odoratissimum accounts for c.10% of the Fusarium wilt in northern Viet Nam, demonstrating that Foc TR4 is not yet a dominant strain in the region. Fusarium cugenangense - considered to cause Race 2 infections among bananas - is only found in c. 10% of the tissue material that was obtained from infected Vietnamese bananas. Additionally, one of the isolates cultured from diseased bananas was phylogenetically not positioned within the F. oxysporum species complex (FOSC), but in contrast, fell within the Fusarium fujikuroi species complex (FFSC). As a result, a possible new pathogen for bananas may have been found. Besides being present on several ABB 'Tay banana', F. tardichlamydosporum was also derived from infected tissue of a wild Musa lutea, showing the importance of wild bananas as a possible sink for Foc. Hide full abstract View article on publisher's site
Kallow, S.;
Mertens, A.; Janssens, S.B.; Vandelook, F.; Dickie, J.;
Swennen, R.;
Panis, B.;
Food and Energy Security, 2022 |
Peer Reviewed
Storing seed collections of crop wild relatives, wild plant taxa genetically related to crops, is an essential component in global food security. Seed banking protects genetic resources from degradation and extinction and provides material for use by breeders. Despite being among the most important… Show full abstract crops in the world, banana and plantain crop wild relatives are largely under-represented in genebanks. Nevertheless, banana crop wild relative seed collections are in fact held in different countries, but these have not previously been part of reporting or analysis. To fill this gap, we firstly collated banana seed accession data from 13 institutions in 10 countries. These included 537 accessions containing an estimated 430,000 seeds of 56 species. We reviewed their taxonomic coverage and seed storage conditions including viability estimates. We found that seed accessions have low viability (25% mean) representing problems in seed storage and processing. Secondly, we surveyed 22 institutions involved in banana genetic resource conservation regarding the key constraints and knowledge gaps that institutions face related to banana seed conservation. Major constraints were identified including finding suitable material and populations to collect seeds from, lack of knowledge regarding optimal storage conditions and germination conditions. Thirdly, we carried out a conservation prioritization and gap analysis of Musaceae taxa, using established methods, to index representativeness. Overall, our conservation assessment showed that despite this extended data set banana crop wild relatives are inadequately conserved, with 51% of taxa not represented in seed collections at all; the average conservation assessment showing high priority for conservation according to the index. Finally, we provide recommendations for future collecting, research, and management, to conserve banana and plantain crop wild relatives in seed banks for future generations. Hide full abstract View article on publisher's site
Mertens, A.; Bawin, Y.; Vanden Abeele, S.; Kallow, S.;
Swennen, R.; Vu, D.T.; Vu, T.D.; Minh, H.T.;
Panis, B.; Vandelook, F.; Janssens, S.B.;
Genetic Resources and Crop Evolution, 2022 |
Peer Reviewed
Collection and storage of crop wild relative (CWR) germplasm is crucial for preserving species genetic diversity and crop improvement. Nevertheless, much of the genetic variation of CWRs is absent in ex situ collections and detailed passport data are often lacking. Here, we focussed on Musa… Show full abstract balbisiana, one of the two main progenitor species of many banana cultivars. We investigated the genetic structure of M. balbisiana across its distribution range using microsatellite markers. Accessions stored at the International Musa Germplasm Transit Centre (ITC) ex situ collection were compared with plant material collected from multiple countries and home gardens from Vietnam. Genetic structure analyses revealed that accessions could be divided into three main clusters. Vietnamese and Chinese populations were assigned to a first and second cluster respectively. A third cluster consisted of ITC and home garden accessions. Samples from Papua New Guinea were allocated to the cluster with Chinese populations but were assigned to a separate fourth cluster if the number of allowed clusters was set higher. Only one ITC accession grouped with native M. balbisiana populations and one group of ITC accessions was nearly genetically identical to home garden samples. This questioned their wild status, including accessions used as reference for wild M. balbisiana. Moreover, most ITC accessions and home garden samples were genetically distinct from wild populations. Our results highlight that additional germplasm should be collected from the native distribution range, especially from Northeast India, Myanmar, China, and the Philippines and stored for ex situ conservation at the ITC. The lack of passport data for many M. balbisiana accessions also complicates the interpretation of genetic information in relation to cultivation and historical dispersal routes. Hide full abstract View article on publisher's site
Kallow, S.;
Panis, B.; Vu, D.T.; Tuong, D.V.;
Paofa, J.;
Mertens, A.;
Swennen, R.; Janssens, S.B.;
BMC Plant Biology, 2021 |
Peer Reviewed
Conservation of plant genetic resources, including the wild relatives of crops, plays an important and well recognised role in addressing some of the key challenges faced by humanity and the planet including ending hunger and biodiversity loss. However, the genetic diversity and representativeness… Show full abstract of ex situ collections, especially that contained in seed collections, is often unknown. This limits meaningful assessments against conservation targets, impairs targeting of future collecting and limits their use. Hide full abstract View article on publisher's site
Kallow, S.; Quaghebeur, K.;
Panis, B.; Janssens, S.B.; Dickie, J.;
Gueco, L.;
Swennen, R.; Vandelook, F.;
Ecology and Evolution, 2021 |
Peer Reviewed
Ecologically meaningful seed germination experiments are constrained by access to seeds and relevant environments for testing at the same time. This is particularly the case when research is carried out far from the native area of the studied species. Here, we demonstrate an alternative--the use of… Show full abstract glasshouses in botanic gardens as simulated-natural habitats to extend the ecological interpretation of germination studies. Our focal taxa were banana crop wild relatives (Musa acuminata subsp. burmannica, Musa acuminata subsp. siamea, and Musa balbisiana), native to tropical and subtropical South-East Asia. Tests were carried out in Belgium, where we performed germination tests in relation to foliage-shading/exposure to solar radiation and seed burial depth, as well as seed survival and dormancy release in the soil. We calibrated the interpretation of these studies by also conducting an experiment in a seminatural habitat in a species native range (M. balbisiana--Los Baños, the Philippines), where we tested germination responses to exposure to sun/shade. Using temperature data loggers, we determined temperature dynamics suitable for germination in both these settings. In these seminatural and simulated-natural habitats, seeds germinated in response to exposure to direct solar radiation. Seed burial depth had a significant but marginal effect by comparison, even when seeds were buried to 7 cm in the soil. Temperatures at sun-exposed compared with shaded environments differed by only a few degrees Celsius. Maximum temperature of the period prior to germination was the most significant contributor to germination responses and germination increased linearly above a threshold of 23℃ to the maximum temperature in the soil (in simulated-natural habitats) of 35℃. Glasshouses can provide useful environments to aid interpretation of seed germination responses to environmental niches. Hide full abstract View article on publisher's site
Eyland, D.;
Breton, C.;
Sardos, J.; Kallow, S.;
Panis, B.;
Swennen, R.;
Paofa, J.; Tardieu, F.; Welcker, C.; Janssens, S.B.;
Carpentier, S.;
Crop Science, 2021 |
Peer Reviewed
Since natural habitats are disappearing fast, there is an urgent need to collect, characterize and phenotype banana crop wild relatives to identify unique genotypes with specific traits that fill the gaps in our gene banks. We report a collection mission in Papua New Guinea carried out in 2019.… Show full abstract Seed containing bunches were collected from Musa peekelii ssp. angustigemma (3), M. schizocarpa (4), M. balbisiana (3), M. acuminata ssp. banksii (14), M. boman (3), M. ingens (2), M. maclayi ssp. maclayi (1) and M. lolodensis (1). This material together with the seeds collected during a previous mission in 2017 form the basis for the development of a wild banana seed bank. For characterization and phenotyping, we focused on the most ubiquitous indigenous species of Papua New Guinea: M. acuminata ssp. banksii, the ancestor of most edible bananas. We calculated that the median genomic dissimilarity of the M. acuminata ssp. banksii accessions was 4% and that they differed at least 5 % from accessions present in the International Transit Centre, the world's largest banana gene bank. High-throughput phenotyping revealed drought avoidance strategies with significant differences in root:shoot ratio, soil water content sensitivity and response towards vapour pressure deficit (VPD). We deliver a proof of principle that the wild diversity is not yet fully covered in the gene banks and that wild acuminata ssp. banksii populations contain individuals with unique traits, useful for drought tolerance breeding programs. Hide full abstract View article on publisher's site
Hill, R.; Llewellyn, T.; Downes, E.; Oddy, J.; MacIntosh, C.; Kallow, S.;
Panis, B.; Dickie, J.B.; Gaya, E.;
Frontiers in Microbiology, 2021 |
Peer Reviewed
Seed banks were first established to conserve crop genetic diversity, but seed banking has more recently been extended to wild plants, particularly crop wild relatives, (e.g. by the Millennium Seed Bank (MSB), Royal Botanic Gardens Kew). Crop wild relatives have been recognised as potential… Show full abstract reservoirs of beneficial traits for our domesticated crops, and with mounting evidence of the importance of the microbiome to organismal health, it follows that the microbial communities of wild relatives could also be a valuable resource for crop resilience to environmental and pathogenic threats. Endophytic fungi reside asymptomatically inside all plant tissues and have been found to confer advantages to their plant host. Preserving the natural microbial diversity of plants could therefore represent an important secondary conservation role of seed banks. At the same time, species that are reported as endophytes may also be latent pathogens. We explored the potential of the MSB as an incidental fungal endophyte bank by assessing diversity of fungi inside stored seeds. Using banana crop wild relatives in the genus Musa as a case-study, we sequenced an extended ITS-LSU fragment in order to delimit operational taxonomic units (OTUs) and used a similarity and phylogenetics approach for classification. Fungi were successfully detected inside just under one third of the seeds, with a few genera accounting for most of the OTUs - primarily Lasiodiplodia, Fusarium and Aspergillus - while a large variety of rare OTUs from across the Ascomycota were isolated only once. Fusarium species were notably abundant - of significance in light of Fusarium wilt, a disease threatening global banana crops - and so were targeted for additional sequencing with the marker EF1α in order to delimit species place them in a phylogeny of the genus. Endophyte community composition, diversity and abundance was significantly different across habitats, and we explored the relationship between community differences and seed germination/viability. Our results show that there is a previously neglected invisible fungal dimension to seed banking that could well have implications for the seed collection and storage procedures, and that collections such as the MSB are indeed a novel source of potentially useful fungal strains. Hide full abstract View article on publisher's site
Mertens, A.;
Swennen, R.; Rønsted, N.; Vandelook, F.;
Panis, B.;
Sachter-Smith, G.; Vu, D.T.; Janssens, S.B.;
Diversity and Distributions, 2021 |
Peer Reviewed
Crop wild relatives (CWR) are an essential source of genetic material for the improvement of certain traits in related crop species. Despite their importance, increasing public, scientific and political support, large gaps exist in the amount of genetic material collected and conserved of many CWR.… Show full abstract Here, we construct a dataset on the distribution of wild banana species (Musa spp.) and assess their risk and conservation status. We deal with the following questions: (a) What areas are potentially suitable for wild banana species? (b) How much of the wild banana diversity is currently at risk or insufficiently conserved ex and in situ? Native distribution area of wild banana species, ranging from the north-eastern states of India to north-eastern Australia. We assessed the potential environmental range of wild species using a species distribution modelling approach with MaxEnt. Extinction risk was evaluated following IUCN criterion B, and the ex and in situ conservation status was assessed using an indicator for biodiversity and sustainable development targets. We found that 11 out of 59 assessed species can be considered as vulnerable and nine as endangered. Highest species richness was found along the border of south China and northern Vietnam, in the north-eastern states of India and on the Malayan peninsula. Our distribution modelling approach indicates that the northern Indo-Burmese region has the highest environmental suitability for most wild banana species and that lowland rain forests in general are highly suitable for bananas. Assessment of in and ex situ conservation status indicates that 56 out of 59 assessed species are currently insufficiently conserved ex situ and that 49 are of high priority for further conservation. Additional in situ conservation is of high priority for six species and of medium priority for 40 species. To date, little of the banana CWR are sufficiently conserved both in and ex situ. Hide full abstract View article on publisher's site
Mertens, A.; Bawin, Y.; Abeele, S.V.; Kallow, S.; Vu, D.T.; Le, L.T.; Vu, T.D.;
Swennen, R.; Vandelook, F.;
Panis, B.; Janssens, S.B.;
PLOS ONE, 2021 |
Peer Reviewed
Crop wild relatives (CWR) are an indispensable source of alleles to improve desired traits in related crops. While knowledge on the genetic diversity of CWR can facilitate breeding and conservation strategies, it has poorly been assessed. Cultivated bananas are a major part of the diet and income… Show full abstract of hundreds of millions of people and can be considered as one of the most important fruits worldwide. Here, we assessed the genetic diversity and structure of Musa balbisiana, an important CWR of plantains, dessert and cooking bananas. Musa balbisiana has its origin in subtropical and tropical broadleaf forests of northern Indo-Burma. This includes a large part of northern Vietnam where until now, no populations have been sampled. We screened the genetic variation and structure present within and between 17 Vietnamese populations and six from China using 18 polymorphic SSR markers. Relatively high variation was found in populations from China and central Vietnam. Populations from northern Vietnam showed varying levels of genetic variation, with low variation in populations near the Red River. Low genetic variation was found in populations of southern Vietnam. Analyses of population structure revealed that populations of northern Vietnam formed a distinct genetic cluster from populations sampled in China. Together with populations of central Vietnam, populations from northern Vietnam could be subdivided into five clusters, likely caused by mountain ranges and connected river systems. We propose that populations sampled in central Vietnam and on the western side of the Hoang Lien Son mountain range in northern Vietnam belong to the native distribution area and should be prioritised for conservation. Southern range edge populations in central Vietnam had especially high genetic diversity, with a high number of unique alleles and might be connected with core populations in northern Laos and southwest China. Southern Vietnamese populations are considered imported and not native. Hide full abstract View article on publisher's site
Panis, B.; Kallow, S.; Janssens, S.B.
In: Kema, G.H.J. (ed.),
Drenth, A. (ed.). Achieving sustainable cultivation of bananas. Volume 2: Germplasm and genetic improvement.
Burleigh Dodds Science Publishing, 2020
Bananas and plantains (Musa spp.) are amongst the most important tropical and subtropical food crops in the world. In order to reduce the impact of biotic and abiotic factors on banana cultivation, it is important to allow for a genetic enrichment of the currently cultivated genepool. Crop Wild… Show full abstract Relatives play an important role in modern plant breeding as they contain important traits for agriculture. Their conservation is therefore essential for future sustainable agriculture and food security. This chapter focusses on the different aspect and techniques that are required to establish seed collections of wild bananas. This covers physiology and morphology of the banana seeds, difficulties encountered during their germination and putative solutions, possibilities for medium and long term storage and population genetics. Hide full abstract 
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Chase, R.;
Blomme, G.;
Carpentier, S.;
Dita, M.;
Ekesa, B.;
Karamura, D.;
Karamura, E.;
Ocimati, W.;
Omondi, B.A.;
Panis, B.;
Rouard, M.;
Ruas, M.;
Sardos, J.;
Staver, C.;
Van Den Houwe, I.;
Zheng, S.J.;
Roux, N.In: Bananas and Plantains - Leading Edge Research and Development - Volume 1: Diversity, Improvement and Protection
. National Research Centre for Banana (ICAR), 2020
Bioversity International is a global research-for-development organization, envisioning agricultural biodiversity nourishing people and sustaining the planet. It delivers scientificevidence, management practices and policy options to use and safeguard agricultural and tree biodiversity to attain… Show full abstract sustainable global food and nutritional security. It works with partners in low-income countries in different regions, where agricultural and tree biodiversity can contribute to improved nutrition, resilience, productivity and climate change adaptation. Although Bioversity International has a broad remit, working in many countries on a wide range of topics, banana remains a key crop and the focus of the banana research program as well as an important element in other areas of research, such as integrated conservation strategies, agricultural landscapes and food systems. Hide full abstract
Van Den Houwe, I.;
Chase, R.;
Sardos, J.;
Ruas, M.; Kempenaers, E.;
Guignon, V.;
Massart, S.;
Carpentier, S.;
Panis, B.;
Rouard, M.;
Roux, N.;
CABI Agriculture and Bioscience, 2020
The CGIAR genebank International Musa Germplasm Transit Centre (ITC) currently holds 1617 banana accessions from 38 countries as an in vitro collection, backed-up by a cryopreserved collection to safeguard global Musa diversity in perpetuity. The ITC also serves as a vital safety backup and transit… Show full abstract centre for national banana genebanks and ensures that germplasm is clean of pests and diseases and freely available under the International Treaty on Plant Genetic Resources for Food and Agriculture. In more than 35 years of activity, the ITC has distributed over 18,000 banana accession samples to researchers and farmers in 113 countries. Ex situ conservation of vegetatively-propagated crops such as banana poses very particular challenges. Maintaining the ITC genebank is labor intense and costly. Efficiencies are sought through research and development of techniques on detecting viruses, the genetic integrity of accessions, and on innovative means of safeguarding banana diversity, such as conserving populations of wild species by seed banking. Although the conservation of global banana diversity is the main objective of the ITC, significant value comes from its holistic approach to better understand and promote its germplasm through numerous research activities and resources. Techniques for morphological and molecular characterization serve to identify and describe the collection, while also determining what gaps should be filled by collecting missions with national partners. The evaluation of desirable agronomic traits inherent in Musa spp. are investigated by a high-throughput phenotyping platform, which helps breeding programs to select cultivars resistant or tolerant to biotic and abiotic stresses. Genomic and bioinformatic studies of several banana wild relatives greatly enhance our understanding of Musa genetic diversity, links to important phenotypic traits and bring new methods for management of the collection. Collectively, these research activities produce enormous amounts of data that require curation and dissemination to the public. The two information systems at the ITC, Musa Genebank Management System and the Musa Germplasm Information System, serve to manage the genebank activities and to make public germplasm-related data for over 30 banana collections worldwide, respectively. By implementing the 10-year workplan set out in the Global Strategy for the Conservation and Use of Musa Genetic Resources, the network MusaNet supports Musa researchers and stakeholders, including the ITC, and most importantly, links to the world's banana-producing countries via three regional banana networks. Hide full abstract View article on publisher's site
Kallow, S.; Davies, R.;
Panis, B.; Janssens, S.B.; Vandelook, F.;
Mertens, A.;
Swennen, R.; Tahir, M.B.; Dickie, J.;
Seed Science Research, 2020 |
Peer Reviewed
Seed conservation of banana crop wild relatives (Musa L. spp.) is limited because of lack of knowledge about their germination ecology. Musa acuminata Colla, the most important banana crop wild relative, is distributed in tropical and subtropical Asian and Pacific rainforests and colonizes… Show full abstract disturbed sites. The role of temperature in stimulating/inhibiting germination to detect disturbance when canopy gaps are formed is not well known. We assessed seed germination thermal requirements of three subspecies of M. acuminata using nine seed accessions which had been stored in the Millennium Seed Bank. Diurnally alternating temperature cycles were almost completely essential for germination compared with constant temperatures. Germination was optimal when the upper temperature of a diurnal cycle was at 35°C; the lower temperature of the cycle was less important. Subspecies occurrence coordinates were used to extract climate temperature data which were then compared against the temperature requirements for germination from our experiment results. Maximum temperatures of the warmest month across subspecies ranges were close to but below optimal germination temperatures, as were diurnal ranges, suggesting soil-warming at the micro-climate level following gap creation is important for M. acuminata seed germination. Additionally, pre-treatment for 3 months at 60% relative humidity at constant 25°C improved germination from 14 ± 10 (mean, standard deviation) to 41 ± 29% suggesting a period in the soil seed bank under the canopy may increase sensitivity to alternating temperature cycles. Overall viability was low (49 ± 28%), and considerable variance was caused by the different accessions. Germination remained somewhat inconsistent. Hide full abstract View article on publisher's site
Zorrilla-Fontanesi, Y.; Pauwels, L.;
Panis, B.; Signorelli, S.;
Vanderschuren, H.;
Swennen, R.;
Nature Food, 2020
The recent emergence of the fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the deadly strain that causes Fusarium wilt of banana, has put the banana production chain for export under threat. Here, we propose research priorities and complementary strategies and challenges for… Show full abstract effective and efficient mitigation management of Fusarium wilt. Our strategies include diversifying the agrosystems to increase crop resilience, as well as using precision breeding approaches to rapidly assess and introduce disease-resistance genes to develop stable and complete Foc resistance in commercial banana cultivars. Hide full abstract View article on publisher's site
Kallow, S.; Longin, K.; Sleziak, N.F.; Janssens, S.B.; Vandelook, F.; Dickie, J.;
Swennen, R.;
Paofa, J.;
Carpentier, S.;
Panis, B.;
Plants, 2020 |
Peer Reviewed
Ex situ seed conservation of banana crop wild relatives (Musa spp. L.), is constrained by critical knowledge gaps in their storage and germination behaviour. Additionally, challenges in collecting seeds from wild populations impact the quality of seed collections. It is, therefore, crucial to… Show full abstract evaluate the viability of seeds from such collecting missions in order to improve the value of future seed collections. We evaluate the seed viability of 37 accessions of seven Musa species, collected from wild populations in Papua New Guinea, during two collecting missions. Seeds from one mission had already been stored in conventional storage (dried for four months at 15% relative humidity, 20 °C and stored for two months at 15% relative humdity, −20 °C), so a post-storage test was carried out. Seeds from the second mission were assessed freshly extracted and following desiccation. We used embryo rescue techniques to overcome the barrier of germinating in vivo Musa seeds. Seeds from the first mission had low viability (19 ± 27% mean and standard deviation) after storage for two months at 15% relative humidity and −20 °C. Musa balbisiana Colla seeds had significantly higher post-storage germination than other species (p < 0.01). Desiccation reduced germination of the seeds from the second collecting mission, from 84 ± 22% (at 16.7 ± 2.4% moisture content) to 36 ± 30% (at 2.4 ± 0.8% moisture content). There was considerable variation between and (to a lesser extent) within accessions, a proportion of individual seeds of all but one species (Musa ingens N.W.Simmonds) survived desiccation and sub-zero temperature storage. We identified that seeds from the basal end of the infructescence were less likely to be viable after storage (p < 0.001); and made morphological observations that identify seeds and infructescences with higher viability in relation to their developmental maturity. We highlight the need for research into seed eco-physiology of crop wild relatives in order to improve future collecting missions. Hide full abstract View article on publisher's site
Bawin, Y.;
Panis, B.; Abeele, S.V.; Li, Z.;
Sardos, J.;
Paofa, J.;
Ge, X.J.;
Mertens, A.; Honnay, O.; Janssens, S.B.;
Plant Genetic Resources, 2019 |
Peer Reviewed
Crop wild relatives (CWRs) play a key role in crop breeding by providing beneficial trait characteristics for improvement of related crops. CWRs are more efficiently used in breeding if the plant material is genetically characterized, but the diversity in CWR genetic resources has often poorly been… Show full abstract assessed. Seven seed collections of Musa balbisiana, an important CWR of dessert and cooking bananas, originating from three natural populations, two feral populations and two ex situ field collections were retrieved and their genetic diversity was quantified using 18 microsatellite markers to select core subsets that conserve the maximum genetic diversity. The highest genetic diversity was observed in the seed collections from natural populations of Yunnan, a region that is part of M. balbisiana's centre of origin. The seeds from the ex situ field collections were less genetically diverse, but contained unique variation with regards to the diversity in all seed collections. Seeds from feral populations displayed low genetic diversity. Core subsets that maximized genetic distance incorporated almost no seeds from the ex situ field collections. In contrast, core subsets that maximized allelic richness contained seeds from the ex situ field collections. We recommend the conservation and additional collection of seeds from natural populations, preferentially originating from the species' region of origin, and from multiple individuals in one population. We also suggest that the number of seeds used for ex situ seed bank regeneration must be much higher for the seed collections from natural populations. Hide full abstract View article on publisher's site
Sardos, J.;
Paofa, J.; Janssens, S.B.; Vanden Abeele, S.;
Roux, N.;
Panis, B.;
2017
The "Exploration of wild banana populations in Papua New Guinea" took place from 7 to 17 June 2017 and focused on the regions around Madang (Madang Province) and Lae (Morobe Province). In the framework of the PhenSeeData project, the aim of the collecting mission was the collection of wild banana… Show full abstract samples that would allow performing population genetics studies and further help to design more efficient conservation strategies specific to wild bananas. In Madang province, we collected samples of M. acuminata banksii, M. schizocarpa and M. peekelii ssp. angustigemma while M. acuminata ssp. banksii, M. schizocarpa, M. maclayi spp. maclayi var maclayi and M. balbisiana were collected in Morobe Province. In addition of leaf samples, seeds were also collected. We report here on the 10 days spent encountering wild bananas of Papua New Guinea. Hide full abstract View article on publisher's site
Kissel, E., Vanhove, A.C., Garcia, S.A.L.,
Panis, B.,
Rouard, M.,
Cenci, A.,
Roux, N.,
Zorrilla, J.,
Swennen, R.,
Carpentier, S. In: Acta Horticulturae 1114
IX International Symposium on Banana: ISHS-ProMusa Symposium on Unravelling the Banana's Genomic Potential, Brisbane, Australia 17-22 August 2014
ISHS, 2016
Evaluating crop biodiversity is a challenging task and needs to integrate knowledge from different levels. This overview paper offers ways to tackle this challenge, illustrated by the case for drought tolerance in banana. KU Leuven hosts the International Musa Germplasm Collection managed by… Show full abstract Bioversity International for safe storage and distribution in order to secure the cropRSQUOs genepool and encourage its use. The latter, however, requires an in-depth knowledge of the variability among the accessions for coping with varying environmental limitations. Our research focuses on variations in drought tolerance by integrating information from different biological levels (genome, transcriptome, metabolome and phenome). Banana originated in the humid tropics, and yields decrease dramatically when the crop is grown in dryer areas. Circumstantial evidence suggests that elements of the Musa balbisiana (B) genome confer greater drought tolerance on banana than those of the Musa acuminata (A) genome. Hence the genomic constitution may affect the stress response. To phenotype different Musa accessions belonging to different genomic groups, we monitored multiple non-destructive and destructive phenotypic plant variables in response to changing water availability. We used multivariate analysis to classify the different variables according to their contribution in explaining the observed variance between accessions. Ultimately, plant phenotype is driven by genes operating to regulate growth combined with environmental limitations. As such, gene and cell function must always be considered in the whole-plant context. Our brief overview of recent applications of -omics approaches in banana research also covers the challenges of applying such -omics approaches to a non-model crop, with a special focus on abiotic stress. As a general workflow, we propose to combine RNA-Seq, proteomics and metabolite analysis to characterize the cellular phenotype and link to the differential genotype. An interdisciplinary network in plant phenotyping should be established to characterize genetic diversity in genebanks and breeding programs. Hide full abstract View PostPrint View article on publisher's site
Panis, B. In: Acta Horticulturae
XXIX International Horticultural Congress on Horticulture::III International Genetically Modified Organisms in Horticulture Symposium - Past, Present and Future
ISHS, 2016
Banana can be considered an ideal crop for improvement through genetic engineering. Firstly, it is an essential staple food for hundreds of millions in the tropics and the number-one fresh fruit crop in the world. Secondly, due to its monoclonal cultivation, the crop is very vulnerable to pests and… Show full abstract diseases, and large amounts of pesticides are thus being used. Next to creating banana cultivars that are resistant to these diseases, other targets for improvement are related to higher production, fruit storage (shelf life), fruit quality (i.e. "fortification" of the fruit, such as increasing vitamin and iron content) and abiotic stress resistance (against wind, cold, salt and drought). Thirdly, edible bananas are highly sterile, which makes classical breeding extremely difficult, but, at the same time, prevents transgene drift via pollen into the environment in the case of genetically engineered plants. All this, together with the fact that efficient transformation protocols have already been available for about 20 years, raises the question of why there are no transgenic bananas yet on the market. Some researchers indicate that, despite enormous investments in genetically modified bananas, the results are still quite poor, and claim that, if the same financial resources had been invested into classical breeding or better cultural practices, many problems could have been solved. This presentation will try to provide an unbiased overview of the past, present and future of genetically modified bananas. Hide full abstract View article on publisher's site
Garcia, S.A.L.;
Panis, B.;
Swennen, R.;
Carpentier, S.;
Ciência e Agrotecnologia, 2014 |
Peer Reviewed
Plasma membrane proteins constitute a very important class of proteins. They are involved in the transmission of external signals to the interior of the cell and selective transport of water, nutrients and ions across the plasma membrane. However, the study of plasma membrane proteins is… Show full abstract challenging because of their poor solubility in aqueous media and low relative abundance. In this work, we evaluated four different strategies for the characterization of plasma membrane proteins from banana roots: (i) the aqueous-polymer two-phase system technique (ATPS) coupled to gelelectrophoresis (gel-based), and (ii) ATPS coupled to LC-MS/MS (gel free), (iii) a microsomal fraction and (iv) a full proteome, both coupled to LC-MS/MS. Our results show that the gel-based strategy is useful for protein visualization but has major limitations in terms of time reproducibility and efficiency. From the gel-free strategies, the microsomal-based strategy allowed the highest number of plasma membrane proteins to be identified, followed by the full proteome strategy and by the ATPS based strategy. The high yield of plasma membrane proteins provided by the microsomal fraction can be explained by the enrichment of membrane proteins in this fraction and the high throughput of the gel-free approach combined with the usage of a fast high-resolution mass spectrometer for the identification of proteins.
[Proteínas da membrana plasmática constituem uma importante classe de proteínas. Elas estão envolvidas na transmissão de
sinais externos para o interior da célula e no transporte seletivo de nutrientes/ions, através da membrana plasmática. Porém, o estudo
dessas proteinas é difícil, porque elas são poucoabundantes e apresentam baixa solubilidade em tampões aquosos. Neste trabalho,
nós
avaliamos quatro estratégias diferentes para extrair proteínas da membrana plasmática de raízes de banana: (i) a técnica de sistema
aquoso de duas fases, constituída porpolímeros(ATPS) combinada com eletroforese em gel e (ii) ATPS sem gel usandoLC-MS/MS,
(iii) uma fração microssomal e (iv) uma fração, contendo o proteoma total celular, ambas as frações avaliadas via LC-MS/MS. Nossos
resultados mostram que a estratégia baseada em eletroforese em gel é útil para a visualização de proteínas, mas apresenta limitações
em termos de reproducibilidade e eficiência. Dentre as estratégias sem o uso de gel, a fração microssomal permitiu a identificação
do maior número de proteínas de membrana plasmática, seguida pela fração de proteoma total e pela técnica de sistema aquoso de
duasfases. O alto rendimento de proteínas de membrana plasmática proporcionado pela fração microssomal pode ser explicado pelo
enriquecimento de proteínas de membrana nessa fração e pela eficiência do espectrômetro de massa na identificação de proteínas.] Hide full abstract View article on publisher's site
Lassois, L.;
Lepoivre, P.;
Swennen, R.;
Van Den Houwe, I.;
Panis, B.In: Lambardi, M. (ed.), Ozudogru, E.A. (ed.),
Jain, S.M. (ed.). Protocols for Micropropagation of Selected Economically-Important Horticultural Plants.
Humana Press, 2013
Bananas that provide a staple food to the millions of people are adversely affected by several viruses such as Banana bunchy Top Virus (BBTV), Banana Streak Virus (BSV), and Cucumber Mosaic Virus (CMV). These viruses are known to have a devastating effect on crop production and constraint to the… Show full abstract international exchange and conservation of banana germplasm—a cornerstone for breeding new cultivars. The viruses are particularly problematic in vegetative propagated crops, like bananas, because of their transmission in the planting material. Different virus eradication techniques have been developed, such as thermotherapy, chemotherapy, and meristem culture for providing virus-free planting material. Meristem culture proved to be the most effective procedure to eradicate phloem-associated viruses. This method requires isolation of meristematic dome of plant under the aseptic conditions and culture in an appropriate nutrient medium to develop new virus-free plants. Thermotherapy is another widely used virus eradication technique, which is initially carried out on in vivo or in vitro plants and eventually combined with meristem culture technique. The plantlets are initially grown at 28°C day temperature and increase it by 2°C per day until reaches 40°C and the night temperature at 28°C; maintain plants at 40°C for 4 weeks; excise meristem and culture onto the regeneration medium. In chemotherapy technique, antiviral chemical compound Virazole® is applied on meristem culture. Combination of these techniques is also applied to improve the eradication rate. Hide full abstract View article on publisher's site
Remy, S., Kovács, G.,
Swennen, R.,
Panis, B. In: Acta Horticulturae 974
II Genetically Modified Organisms in Horticulture Symposium, White River, South Africa, 2012/09/11-15
ISHS, 2013
Edible bananas comprise several characteristics that make them an ideal target for improvement through genetic engineering: (i) they constitute the N° 1 fresh fruit crop in the world, (ii) they are highly sterile which makes classical breeding extremely difficult but at the same time prevents… Show full abstract transgene drift via pollen into the environment, (iii) their cultivation is monoclonal, and (iv) they are susceptible to a wide range of pests and diseases. The latest outbreak of Fusarium wilt disease caused by Fusarium oxysporum f. sp. cubense "Tropical Race 4 or TR4", which is seriously threatening the international banana trade, strengthens the need for developing resistant bananas.
Despite these facts, the number of publications and research groups dealing with genetic engineering of banana remains low compared to other crops. Possible reasons are among others the difficulties in obtaining transformation competent tissues (in case of banana embryogenic cell suspensions or ECS), the duration to obtain fully transformed plants, the cost and effort of maintaining transgenic lines, the lack of a proper and functional legal biosafety system for testing and culturing GMOs in many banana producing countries and the size of individual banana plants rendering large scale testing of transgenes extremely costly.
In this review, we discuss the 19 years history of banana genetic modification. An overview of the different transformation methodologies will be given, from the first reports on protoplast electroporation to Agrobacterium-mediated transformation of ECS. Also the applications of this technology for banana improvement (agronomic treats, quality treats, etc.) will be discussed along with examples of confined field trials of genetically modified bananas.
Finally, a brief overview of the research topics on banana genetic engineering at the Laboratory of Tropical Crop Improvement (LTCI, KU Leuven, Belgium) is discussed. Topics include selectable marker genes, promoter and gene characterization as well as molecular breeding for fungus and drought resistance. Hide full abstract View article on publisher's site
Vos, C.; Claerhout, S.; Mkandawire, R.;
Panis, B.; De Waele, D.; Elsen, A.;
Plant and Soil, 2012 |
Peer Reviewed
Aims
Arbuscular mycorrhizal fungi (AMF) can control root-knot nematode infection, but the mode of action is still unknown. We investigated the effects of AMF and mycorrhizal root exudates on the initial steps of Meloidogyne incognita infection, namely movement towards and penetration of tomato… Show full abstract roots.
Methods
M. incognita soil migration and root penetration were evaluated in a twin-chamber set-up consisting of a control and mycorrhizal (Glomus mosseae) plant compartment (Solanum lycopersicum cv. Marmande) connected by a bridge. Penetration into control and mycorrhizal roots was also assessed when non-mycorrhizal or mycorrhizal root exudates were applied and nematode motility in the presence of the root exudates was tested in vitro.
Results
M. incognita penetration was significantly reduced in mycorrhizal roots compared to control roots. In the twin-chamber set-up, equal numbers of nematodes moved to both compartments, but the majority accumulated in the soil of the mycorrhizal plant compartment, while for the control plants the majority penetrated the roots. Application of mycorrhizal root exudates further reduced nematode penetration in mycorrhizal plants and temporarily paralyzed nematodes, compared with application of water or non-mycorrhizal root exudates.
Conclusions
Nematode penetration was reduced in mycorrhizal tomato roots and mycorrhizal root exudates probably contributed at least partially by affecting nematode motility. Hide full abstract View article on publisher's site
Vanhove, A.C.; Vermaelen, W.;
Panis, B.;
Swennen, R.;
Carpentier, S.;
Frontiers in Plant Science, 2012 |
Peer Reviewed
There is a great need for research aimed at understanding drought tolerance, screening for drought tolerant varieties and breeding crops with an improved water use efficiency. Bananas and plantains are a major staple food and export product with a worldwide production of over 135 million tonnes per… Show full abstract year. Water however is the most limiting abiotic factor in banana production. A screening of the Musa biodiversity has not yet been performed. We at KU Leuven host the Musa International Germplasm collection with over 1200 accessions. To screen the Musa biodiversity for drought tolerant varieties, we developed a screening test for in vitro plants. Five varieties representing different genomic constitutions in banana (AAAh, AAA, AAB, AABp, and ABB) were selected and subjected to a mild osmotic stress. The ABB variety showed the smallest stress induced growth reduction. To get an insight into the acclimation and the accomplishment of homeostasis, the leaf proteome of this variety was characterized via 2D DIGE. After extraction of the leaf proteome of six control and six stressed plants, 2600 spots could be distinguished. A PCA analysis indicates that control and stressed plants can blindly be classified based on their proteome. One hundred and twelve proteins were significantly more abundant in the stressed plants and 18 proteins were significantly more abundant in control plants (FDR α 0.05). Twenty four differential proteins could be identified. The proteome analysis clearly shows that there is a new balance in the stressed plants and that the respiration, metabolism of ROS and several dehydrogenases involved in NAD/NADH homeostasis play an important role. Hide full abstract View article on publisher's site
Henry, I.;
Carpentier, S.; Pampurova, S.; Van Hoylandt, A.;
Panis, B.;
Swennen, R.; Remy, S.;
Planta, 2011 |
Peer Reviewed
Abscisic acid, stress, ripening proteins (ASR) are a family of plant-specific small hydrophilic proteins. Studies in various plant species have highlighted their role in increased resistance to abiotic stress, including drought, but their specific function remains unknown. As a first step toward… Show full abstract their potential use in crop improvement, we investigated the structure and regulation of the Asr gene family in Musa species (bananas and plantains). We determined that the Musa Asr gene family contained at least four members, all of which exhibited the typical two exons, one intron structure of Asr genes and the "ABA/WDS" (abscisic acid/water deficit stress) domain characteristic of Asr genes. Phylogenetic analyses determined that the Musa Asr genes were closely related to each other, probably as the product of recent duplication events. For two of the four members, two versions corresponding to the two sub-genomes of Musa, acuminata and balbisiana were identified. Gene expression and protein analyses were performed and Asr expression could be detected in meristem cultures, root, pseudostem, leaf and cormus. In meristem cultures, mAsr1 and mAsr3 were induced by osmotic stress and wounding, while mAsr3 and mAsr4 were induced by exposure to ABA. mASR3 exhibited the most variation both in terms of amino acid sequence and expression pattern, making it the most promising candidate for further functional study and use in crop improvement. Hide full abstract View article on publisher's site
Vertommen, A.; Moller, A.L.; Cordewener, J.H.;
Swennen, R.;
Panis, B.; Finnie, C.; America, A.H.;
Carpentier, S.;
Journal of Proteomics, 2011 |
Peer Reviewed
Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for… Show full abstract membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified. Hide full abstract View article on publisher's site
Vertommen, A.;
Panis, B.;
Swennen, R.;
Carpentier, S.;
Journal of Proteomics, 2011 |
Peer Reviewed
The workhorse for proteomics in non-model plants is classical two-dimensional electrophoresis, a combination of iso-electric focusing and SDS-PAGE. However, membrane proteins with multiple membrane spanning domains are hardly detected on classical 2-DE gels because of their low abundance and poor… Show full abstract solubility in aqueous media. In the current review, solutions that have been proposed to handle these two problems in non-model plants are discussed. An overview of alternative techniques developed for membrane proteomics is provided together with a comparison of their strong and weak points. Subsequently, strengths and weaknesses of the different techniques and methods to evaluate the identification of membrane proteins are discussed. Finally, an overview of recent plant membrane proteome studies is provided with the used separation technique and the number of identified membrane proteins listed. Hide full abstract View article on publisher's site
Swennen, R.,
Carpentier, S.,
Van Den Houwe, I., Vertommen, A., Kovács, G.,
Sági, L., Remy, S.,
Panis, B. In: Van den Bergh, I. (ed.),
Smith, M. (ed.),
Swennen, R. (ed.),
Hermanto, C. (ed.). Acta Horticulturae 897
International ISHS-ProMusa Symposium on Global Perspectives on Asian Challenges, Guangzhou, China, 14-18/09/2009
ISHS, 2011
The original publication is available at www.actahort.org.
The Musa International Transit Centre (ITC) maintains nearly 1200 banana (Musa spp.) accessions in vitro. Through the Global Conservation Strategy, ITC is linked to field collections, which are used for taxonomy training and data acquisition… Show full abstract on-for example-disease and pest resistance. Bananas exhibit somaclonal variation, and for some accessions, the frequency of somaclonal variation increases dramatically under in vitro conditions. In order to safely store this valuable Musa biodiversity, the in vitro collection is being cryopreserved. Over the years, fundamental research proved instrumental in the successful development of the different cryopreservation protocols. Among others, a proteomics study was performed on meristem cultures to investigate the response of different banana varieties to osmotic stress associated with cryopreservation. Valuable proteomics experience in Musa and other crops was gained based on two-dimensional gel electrophoresis (2DE). Using high-resolution 2DE gels that routinely display about 900-1500 protein spots, individual protein isoforms were separated and an extensive amount of mass spectrometry data accumulated. Ultimately, a 2DE map of the Musa meristem proteome (637 identified proteins) was constructed. Following identification, some of the differentially expressed proteins under osmotic stress were further functionally characterised in engineered plants. This banana transformation platform is also used as a tool for gene and promoter discovery via T-DNA tagging. These biotechnological approaches constitute a significant step towards the development of better performing crops, including more stress-tolerant varieties of banana. Hide full abstract View PostPrint View article on publisher's site
Carpentier, S.;
Panis, B.; Renaut, J.; Samyn, B.; Vertommen, A.; Vanhove, A.C.;
Swennen, R.; Sergeant, K.;
Phytochemistry, 2011 |
Peer Reviewed
Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the… Show full abstract consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties... Hide full abstract View article on publisher's site
Panis, B.; Piette, B.; Andre, E.;
Van Den Houwe, I.;
Swennen, R.In: Panis, B. (ed.), Lynch, P. (ed.). Acta Horticulturae 908.
I International Symposium on Cryopreservation in Horticultural Species, Leuven (BEL), 2009/04/5-8
2011
Cryopreservation procedures are now available for about 150-200 different plant species, but until now for each species and tissue type, cryopreservation protocols needed to be empirically adapted in function of the natural frost/dehydration resistance of the species under investigation, explant… Show full abstract size, explant type and water content. Most of the work on cryopreservation of plants has been performed in the framework of academic studies and involves only one or a few genotypes. Only few plant germplasm collections stored in liquid nitrogen currently exist but the number is increasing. The unavailability of an efficient and robust cryopreservation protocol applicable to many plant species and diverse germplasm types is one of the major bottlenecks for the wide application of cryopreservation to plant germplasm... Hide full abstract View article on publisher's site
Benson, E.E.; Harding, K.; Debouck, D.; Dumet, D.; Escobar, R.; Mafla, G.;
Panis, B.; Panta, A.; Tay, D.;
Van Den Houwe, I.;
Roux, N.;
SGRP, 2011
Among the collective actions of the World Bank-funded Global Public Goods Phase II Project (GPG2), the following collaborative activity: "Refinement and standardization of storage procedures for clonal crops" was given to the CGIAR's In Vitro Genebanks, represented by the Clonal Crop Task Force (… Show full abstractCCTF) composed of genetic resources research staff from the four centres: Bioversity International, CIAT, CIP and IITA. These hold the in trust collections of Musa, cassava, potato, sweetpotato, yam and Andean root and tuber crops (ARTCs). The overarching aims of this activity were to: (1) review the status of vitro conservation in the context of the GPG2 project with an emphasis on the mandated clonal crops; (2) survey the facilities, storage protocols and practices of CGIAR's clonal crop genebanks; (3) collate and review this information with a view to developing quality and risk management systems to support the production and validation of multi-crop best practice guidelines... Hide full abstract View PDF
Carpentier, S.; Vertommen, A.;
Swennen, R.; Witters, E.;
Ferreira, C.F.;
Souza Júnior, M.T.;
Panis, B.;
Journal of Proteome Research, 2010 |
Peer Reviewed
We have designed an in vitro experimental setup to study the role of sucrose in sugar-mediated acclimation of banana meristems using established highly proliferating meristem cultures. It is a first step toward the systems biology of a meristem and the understanding of how it can survive severe… Show full abstract abiotic stress. Using the 2D-DIGE proteomic approach and a meristem-specific EST library, we describe the long-term acclimation response of banana meristems (after 2, 4, 8, and 14 days) and analyze the role of sucrose in this acclimation by setting up a control, a sorbitol, and a sucrose acclimation treatment over time. Sucrose synthase is the dominant enzyme for sucrose breakdown in meristem tissue, which is most likely related to its lower energy consumption. Metabolizing sucrose is of paramount importance to survive, but the uptake of sugar and its metabolism also drive respiration, which may result in limited oxygen levels... Hide full abstract View article on publisher's site
Abdul Rahman, S.A.S.; Zulqarnain, M.;
Othman, R.Y.;
Swennen, R.;
Panis, B.; De Waele, D.; Remy, S.;
Carpentier, S.;
European Journal of Plant Pathology, 2010 |
Peer Reviewed
The polyphagous obligate parasites Meloidogyne spp. devastate a wide range of crop plants including bananas and plantains. Their infestations impact agriculture worldwide. Therefore, an effective combating regime against this nematode species and an in-depth understanding of plant-nematode… Show full abstract interaction are essential. Early detection of infection by visual inspection is not possible. This hampers early control strategy efforts and makes in-depth research of the early infection and plant defence unfeasible. A simple and robust in planta PCR-based nematode detection method is described here as the first crucial step. This PCR-based detection assay exploits the existence of the Internal Transcribed Spacer 1 (ITS 1) region of the ribosomal DNA (rDNA) gene family in the nematodes for early detection of nematode penetration into the roots. The results demonstrate that this detection assay is suitable to serve as a molecular screening tool for plant root diagnostic purposes. Hide full abstract View article on publisher's site
Panis, B.;
Bioversity International, 2009
Cet ouvrage décrit les différentes méthodes actuellement utilisées à KULeuven pour la cryoconservation des Musa. Les avantages et les inconvénients de chaque méthode sont exposés et les domaines requérant davantage de recherche en vue d'optimiser les protocoles sont identifiés. L'objectif de cette… Show full abstract publication est de donner des informations et des conseils sur les méthodologies de cryoconservation appropriées au matériel génétique de Musa. La disponibilité et le type de matériel de départ, les génotypes à cryoconserver et la disponibilité des ressources devront être pris en compte pour déterminer les méthodes les mieux adaptées pour leur utilisation dans d'autres laboratoires.]
[En esta publicación se describen los diversos métodos, desarrollados en la KULeuven para la crioconservación de los cultivos de Musa. Se describen las ventajas y desventajas y se identifican las áreas donde aún se requieren investigaciones para optimizar los protocolos. [The aim of this publication is to provide information and guidance on cryopreservation methodologies suitable for use on Musa germplasm. The different methods currently used at Katholieke Universiteit Leuven (KULeuven) for cryopreserving Musa cultures are described as well as the advantages and disadvantages of each of them. The areas where research is still required to further optimize the protocols are also identified. It is hoped that the detailed descriptions of the methodologies will facilitate their adoption and standard use in different laboratories.] Hide full abstract View PDF
Wang, Q.C.;
Panis, B.; Engelmann, F.; Lambardi, M.; Valkonen, J.P.T.;
Annals of Applied Biology, 2009 |
Peer Reviewed
Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra-low temperature, usually that of liquid nitrogen (-196°C), and is considered as an ideal means for long-term storage of… Show full abstract plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing 'citrus greening'. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy. (Author's abstract). Hide full abstract View article on publisher's site
Panis, B.;
Bioversity International, 2009
The aim of this publication is to provide information and guidance on cryopreservation methodologies suitable for use on Musa germplasm. The different methods currently used at Katholieke Universiteit Leuven (KULeuven) for cryopreserving Musa cultures are described as well as the advantages and… Show full abstract disadvantages of each of them. The areas where research is still required to further optimize the protocols are also identified. It is hoped that the detailed descriptions of the methodologies will facilitate their adoption and standard use in different laboratories.
[Cet ouvrage décrit les différentes méthodes actuellement utilisées à KULeuven pour la cryoconservation des Musa. Les avantages et les inconvénients de chaque méthode sont exposés et les domaines requérant davantage de recherche en vue d'optimiser les protocoles sont identifiés. L'objectif de cette publication est de donner des informations et des conseils sur les méthodologies de cryoconservation appropriées au matériel génétique de Musa. La disponibilité et le type de matériel de départ, les génotypes à cryoconserver et la disponibilité des ressources devront être pris en compte pour déterminer les méthodes les mieux adaptées pour leur utilisation dans d'autres laboratoires.]
[En esta publicación se describen los diversos métodos, desarrollados en la KULeuven para la crioconservación de los cultivos de Musa. Se describen las ventajas y desventajas y se identifican las áreas donde aún se requieren investigaciones para optimizar los protocolos.] Hide full abstract View PDF
Panis, B.;
Bioversity International, 2009
En esta publicación se describen los diversos métodos, desarrollados en la KULeuven para la crioconservación de los cultivos de Musa. Se describen las ventajas y desventajas y se identifican las áreas donde aún se requieren investigaciones para optimizar los protocolos.
[The aim of this… Show full abstract publication is to provide information and guidance on cryopreservation methodologies suitable for use on Musa germplasm. The different methods currently used at Katholieke Universiteit Leuven (KULeuven) for cryopreserving Musa cultures are described as well as the advantages and disadvantages of each of them. The areas where research is still required to further optimize the protocols are also identified. It is hoped that the detailed descriptions of the methodologies will facilitate their adoption and standard use in different laboratories.] Hide full abstract View PDF
Lambert, E.; Goossens, A.;
Panis, B.; Van Labeke, M.C.; Geelen, D.;
Plant Cell, Tissue and Organ Culture (PCTOC), 2009 |
Peer Reviewed
To study the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated… Show full abstract lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1 M sucrose. The encapsulated hairy roots were precultured for 3 days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35-40 percent of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40°C. The survival rates were 90 percent for Maesa and 53 percent for Medicago, which are sufficient to allow implementation in large storage experimental set-ups. (Author's abstract). Hide full abstract View article on publisher's site
Strosse, H.; Andre, E.;
Sági, L.;
Swennen, R.;
Panis, B.;
Plant Cell, Tissue and Organ Culture (PCTOC), 2008 |
Peer Reviewed
Despite their similar morphology, banana and maize shoot tips responded strikingly different with respect to the in vitro formation of homogeneous multiple shoot clusters. While up to 50 small shoots per maize explant could be induced within 1 month, zero to one additional shoot formed starting… Show full abstract from a banana shoot tip. Subsequently, banana shoot tips were subjected to different combinations of five cytokinins (0-100 ?M) and five auxins (0-5 ?M). The cytokinins thidiazuron and benzylaminopurine stimulated multiplication to a higher extent compared to zeatin, kinetin and isopentenyl adenine. The addition of indoleacetic acid, naphthalene acetic acid or indolebutyric acid to cytokinin containing medium did not affect the in vitro response. In contrast, 2,4-dichlorophenoxyacetic acid (1 and 5 ?M) and a higher concentration of picloram (5 ?M) had a detrimental effect on shoot formation and resulted in explant death and globule development. When small (0.1 cm) shoot tips were grown on cytokinin medium without an auxin source, the average number of shoots was generally two to three times lower compared to bigger (0.5 cm) shoot tips. Based on our experience in maize and this large-scale study with banana shoot tips, we conclude that banana is extremely recalcitrant towards adventitious shoot formation. This recalcitrance could not be overcome by any of the 173 different plant growth regulator combinations tested. In vitro multiplication of banana thus appears solely restricted to axillary shoot formation. Hide full abstract View article on publisher's site
Vertommen, A.;
Carpentier, S.; Remmerie, N.; Witters, E.;
Swennen, R.;
Panis, B.In: Laamanen, J. (ed.), Uosukainen, M. (ed.), Hägmann, H. (ed.), Nukari, A. (ed.), Ranrala, S. (ed.). Cryopreservation of Crop Species in Europe.
2008
Abstract of presentation made at
CRYOPLANET - COST Action 871, in Oulu Finland, 20-23 February 2008. View PDF
Carpentier, S.; Coemans, B.; Podevin, N.; Laukens, K.; Witters, E.; Matsumura, H.; Terauchi, R.;
Swennen, R.;
Panis, B.;
Physiologia Plantarum, 2008 |
Peer Reviewed
There is no question that protein- and RNA-based measurements are complementary, but which approach has the highest return in the case of a non-model crop and what is the correlation between mRNA and proteins? We describe and evaluate in detail the advantages and pitfalls of both a proteomics and a… Show full abstract transcriptomics approach. The information on the abundance of transcripts was obtained by serial analysis of gene expression (SAGE), while information on the abundance of proteins was obtained via two-dimensional gel electrophoresis. Hide full abstract View article on publisher's site
Carpentier, S.;
Panis, B.; Vertommen, A.;
Swennen, R.; Sergeant, K.; Renaut, J.; Laukens, K.; Witters, E.; Samyn, B.; Devreese, B.;
Mass Spectrometry Reviews, 2008
Biological research has focused in the past on model organisms and most of the unctional genomics studies in the field of plant sciences are still performed on model species or species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or… Show full abstract energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, that is, high-throughput identification of candidate gene products, tends to be lost in non-model species due to the lack of genomic information or due to the sequence divergence to a related model organism. Nevertheless, a proteomics approach has a great potential to study non-model species. This work reviews non-model plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. The review tackles problems associated with (i) sample preparation, (ii) the analysis and interpretation of a complex data set, (iii) the protein identification via MS, and (iv) data management and integration. We will illustrate the power of 2DE for non-model plants in combination with multivariate data analysis and MS/MS identification and will evaluate possible alternatives. Hide full abstract View article on publisher's site
Strosse, H.; Andre, E.; Sagi, S.;
Swennen, R.;
Panis, B.;
Plant Cell, Tissue and Organ Culture (PCTOC), 2008 |
Peer Reviewed
Despite their similar morphology, banana and maize shoot tips responded strikingly different with respect to the in vitro formation of homogeneous multiple shoot clusters. While up to 50 small shoots per maize explant could be induced within 1 month, zero to one additional shoot formed starting… Show full abstract from a banana shoot tip. Subsequently, banana shoot tips were subjected to different combinations of five cytokinins (0-100 microM) and five auxins (0-5 microM). The cytokinins thidiazuron and benzylaminopurine stimulated multiplication to a higher extent compared to zeatin, kinetin and isopentenyl adenine. The addition of indoleacetic acid, naphthalene acetic acid or indolebutyric acid to cytokinin containing medium did not affect the in vitro response. In contrast, 2,4-dichlorophenoxyacetic acid (1 and 5 microM) and a higher concentration of picloram (5 microM) had a detrimental effect on shoot formation and resulted in explant death and globule development. When small (0.1 cm) shoot tips were grown on cytokinin medium without an auxin source, the average number of shoots was generally two to three times lower compared to bigger (0.5 cm) shoot tips. Based on our experience in maize and this large-scale study with banana shoot tips, we conclude that banana is extremely recalcitrant towards adventitious shoot formation. This recalcitrance could not be overcome by any of the 173 different plant growth regulator combinations tested. In vitro multiplication of banana thus appears solely restricted to axillary shoot formation. (Author's abstract). Hide full abstract View article on publisher's site
Pedreschi, R.; Hertog, M.L.A.T.M.;
Carpentier, S.; Lammertyn, J.; Robben, J.; Noben, J.P.;
Panis, B.;
Swennen, R.; Nicolai, B.M.;
Proteomics, 2008 |
Peer Reviewed
The presence of missing values in gel-based proteomics data represents a real challenge if an objective statistical analysis is pursued. Different methods to handle missing values were evaluated and their influence is discussed on the selection of important proteins through multivariate techniques.… Show full abstract The evaluated methods consisted of directly dealing with them during the multivariate analysis with the nonlinear estimation by iterative partial least squares (NIPALS) algorithm or imputing them by using either k-nearest neighbor or Bayesian principal component analysis (BPCA) before carrying out the multivariate analysis. These techniques were applied to data obtained from gels stained with classical postrunning dyes and from DIGE gels. Before applying the multivariate techniques, the normality and homoscedasticity assumptions on which parametric tests are based on were tested in order to perform a sound statistical analysis. From the three tested methods to handle missing values in our datasets, BPCA imputation of missing values showed to be the most consistent method. (Author's abstract). Hide full abstract View article on publisher's site
Panis, B.;
Van Den Houwe, I.; Piette, B.;
Swennen, R.;
Advances in Horticultural Science, 2007 |
Peer Reviewed
Cryopreservation is the ultimate method to preserve large collections of vegetatively propagated
plant species, like banana, for the long term. Two cryopreservation protocols, i.e. vitrification of proliferating
meristem clumps (called “scalps”) and vitrification of apical meristems were… Show full abstract investigated. The protocols were
successfully applied to 540 accessions belonging to 22 different banana groups. The regeneration rate after
thawing strongly depends on the genomic group to which the accession belongs and the cryopreservation method
that is used. For safe long-term storage, we opted for three independent successful experiments. An experiment
is judged successful if at least one shoot from the stored material regenerates with more than 95% probability. Hide full abstract
Helliot, B.;
Panis, B.; Busogoro, J.P.; Sobry, S.; Poumay, Y.; Raes, M.;
Swennen, R.;
Lepoivre, P.;
European Journal of Histochemistry, 2007 |
Peer Reviewed
The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem… Show full abstract cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2 percent paraformaldehyde/0.25 percent glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R.White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem- tip culture alone. Hide full abstract View article on publisher's site
Carpentier, S.; Witters, E.; Laukens, K.; Van Onckelen, H.;
Swennen, R.;
Panis, B.;
Proteomics, 2007 |
Peer Reviewed
Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-… Show full abstractmediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using nonparametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energyconserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop. Hide full abstract View article on publisher's site
Vertommen, A.;
Carpentier, S.; Remmerie, N.; Witters, E.;
Swennen, R.;
Panis, B.;
Communications in Agricultural and Applied Biological Sciences, 2007 |
Peer Reviewed
The preferred method to safely preserve the banana biodiversity is cryopreservation (or storage at -196°C). Successful application of this technique requires sufficient removal of tissue water (dehydration phase), generally preceded by an acclimation phase (Panis et al., 1996). To get more insight… Show full abstract into the physiological processes during that acclimation period, it is important to understand which proteins are involved and how they interact with each other. To analyze proteins of poorly sequenced species, like banana (Musa spp.), two dimensional gel electrophoresis (2DE), protein quantification and identification through tandem mass spectrometry (MS/MS) is the method of preference (Carpentier et al., 2005; Samyn et al., 2007; Carpentier et al., 2007). However, classical 2DE is highly denaturating, which results in loss of information about the organization of protein complexes and/or protein-protein interactions. Moreover, hydrophobic proteins are difficult to analyze since they tend to precipitate during first dimension iso-electric focusing. To solve these problems, Schägger and von Jagow (1991) developed Blue native PAGE (BN-PAGE) which allows separation of protein complexes as well as hydrophobic proteins in the mass range of 10 kDa to 1 MDa. This technique comprises (i) the use of mild, neutral detergents for solubilisation and (ii) the application of Coomassie Brilliant Blue G250 to give a negative charge to proteins and protein complexes. This permits separation according to their mass. In plants, protein complexes of organelles have been most intensively investigated. Till now, there are no publications on protein complexes from whole cellular lysates of plants which makes optimizing BNPAGE to study complexes in banana meristems a real challenge. Even more since, we are dealing with limited amounts of tissue of a recalcitrant nonmodel crop. Hide full abstract View article on publisher's site
Panis, B.; Lambardi, M.
In: Ruane, J. (ed.), Sonnino, A. (ed.). The role of biotechnology in exploring and protecting agricultural genetic resources.
FAO, 2006
Over the past decades, plant cryopreservation technologies have been evolving rapidly, opening the door to the possibility of bong-term storage of valuable genetic resources of many crop and forest species. From the original slow-cooling approach, research has moved to easier and more reproducible… Show full abstract techniques, which allow the complete vitrification of extra- and intra-cellular liquids. This chapter describes concisely the procedures that have been proposed in time for the cryopreservation of a wide range of tissues and organs, such as cell suspensions, embryogenic callus, pollen, meristematic tissues, seeds and embryo axes. In addition, the most important achievements in the cryopreservation of herbaceous, hardwood and softwood species are discussed. (Author's abstract). Hide full abstract View article on publisher's site
Zhu, G.Y.; Geuns, J.M.C.; Dussert, S.;
Swennen, R.;
Panis, B.;
Physiologia Plantarum, 2006 |
Peer Reviewed
To understand the mechanisms of sucrose-induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem… Show full abstract cultures of different banana varieties. The four banana varieties that were selected show different post-thaw shoot regeneration rates (0-53.4 percent). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2-week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose-pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the postthaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrosepretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post-thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process. (Author's abstract). Hide full abstract View article on publisher's site
Van Den Houwe, I.;
Panis, B.; Arnaud, E.; Markham, R.;
Swennen, R.In: Segers, H. (ed.), Desmet, P. (ed.), Baus, E. (ed.). Tropical biodiversity: Science, data, conservation .
3rd GBIF Science Symposium, Brussels (BEL), 2005/04/18-19
2006
The international banana germplasm collection is managed by the IPGRI/INIBAP Transit Centre in Belgium since 1985. This unique collection, placed under the auspices of FAO in 1994, consists of approximately 1200 accessions of wild, cultivated and improved bananas, introduced from 44 countries in… Show full abstract the world. Samples of these accessions are permanently maintained in vitro as proliferating shoot cultures under slow growth conditions at low temperature (16°C) and reduced light intensity (25µmol/m²/s). Incoming germplasm undergoes a standardized indexing process for five virus diseases in collaboration with 3 indexing centres (Australia, France and South Africa) and pathogen-free accessions are made freely available for international distribution. Throughout the last five years, over 25,000 samples of germplasm have been delivered worldwide to hundreds of institutes and individuals involved in development projects with farmers, for various research activities or to underpin specific gene bank activities such as cytological studies and virus eradication research. Currently, also a long-term base collection is being established, using a cryopreservation protocol developed at the Laboratory of Tropical Crop Improvement, KULeuven, Belgium. At present, more then one quarter of the entire collection is safely stored in liquid nitrogen (-196°C). Evidently, the effective management and use of this unique collection strongly depends on its documentation status. To facilitate data maintenance and processing relevant for the day-to-day activities of the gene bank, a tailored-made information system using bar-codes has been put in place recently. All passport data, and available characterization and evaluation data are documented in the INIBAP's Musa Germplasm Information System (MGIS). This decentralized system contains standardized information on banana varieties managed in 18 banana gene banks around the world and is readily accessible for users ( mgis.grinfo.net or mgis.inibap.org). The data of the INIBAP Transit Centre, which are part of the MGIS database, are also available through SINGER (System-Wide Information Network on Genetic Resources), an on-line database containing information on genetic resources held by the CGIAR Centres ( singer.cgiar.org). Hide full abstract View PDF
Samyn, B.; Sergeant, K.;
Carpentier, S.; Debyser, G.;
Panis, B.;
Swennen, R.; Van Beeumen, J.;
Journal of Proteome Research, 2006 |
Peer Reviewed
We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a non-model plant with unsequenced genome. Furthermore, this approach… Show full abstract allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm. (Author's abstract).
[Nous rapportons l'utilisation de la dérivation chimique avec l'analyse de MALDI-MS/MS pour l'analyse des séquences de novo. En utilisant trois algorithmes basés sur l'homologie fréquemment utilisés dans la recherche, nous pouvons identifier plus de 40 protéines de bananier, une plante non-modèle avec un génome non séquencé le génome. En outre, cette approche a permis l'identification de différents isoformes. Nous avons également observé que les taux d'identification obtenus varient en fonction de la position des séquences peptidiques en utilisant l'algorithme MS.] Hide full abstract View article on publisher's site
Geuns, J.M.C.; Orriach, M.L.;
Swennen, R.; Zhu, G.;
Panis, B.; Compernolle, F.; Van der Auweraer, D.;
Analytical Biochemistry, 2006 |
Peer Reviewed
The diamines putrescine (PUT) and diaminopropane (DAP), the polyamines spermidine (SPD) and spermine (SPM), and the arylalkyl amines phenethylamine (PEA), tyramine (TYR), dopamine (DA), and salsolinol (SAL) were dansylated and baseline separated by LC using a Waters ODS-2 column. The dansyl… Show full abstract derivatives were detected by fluorescence (Labda ex: 337 nm; Labda em: 520 nm). Besides the amine function, the phenolic OH groups of TYR, DA, and SAL were also dansylated (LC-MS, formation of N,O-didansyl
[TYR] and N,O,O Labda-tridansyl derivatives
[DA and SAL]). Calibration curves revealed response factors being appreciably lower for (N,O-didansyl) aminophenol TYR and (N,O,O Labda-tridansyl) DA and SAL than for N-dansylamines. However, the method is suitable as a cheap alternative to LC-MS for the simultaneous determination of polyamines and arylalkyl amines of large quantities of samples. (Author's abstract). Hide full abstract View article on publisher's site
Strosse, H.; Schoofs, H.;
Panis, B.; Andre, E.; Reyniers, K.;
Swennen, R.;
Plant Science, 2006 |
Peer Reviewed
Multiple meristem cultures of 18 varieties belonging to 5 genome types in Musa (AA, AAA, AAA-h, AAB and ABB) were established by culturing elongated shoots on MS medium supplemented with 100 mM BAP. The top layers comprising the most meristematic tissue, i.e. scalps, were excised and induced for… Show full abstract embryogenesis on media containing 1-50 mM 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic responses were obtained for each of the tested concentrations, with an optimum at 5 mM 2,4-D. From the 24,375 scalps tested, only 3.3 percent resulted in an embryogenic response. The average embryogenic frequency was 6.0 percent for cooking bananas (ABB), 3.8 percent for Cavendish-type bananas (AAA) and 1.8 percent for plantains (AAB). Once embryogenic complexes were transferred to liquid maintenance medium, embryogenic cell suspensions with high regeneration capacity were obtained. (Author's abstract). Hide full abstract View article on publisher's site
Xu, C.X.;
Panis, B.; Strosse, H.; Li, H.P.;
Xiao, H.; Fan, H.Z.;
Swennen, R.;
Journal of Horticultural Science & Biotechnology, 2005 |
Peer Reviewed
'Williams' is one of the most important dessert banana cultivars in the World. Improvement through genetic engineering depends on the development of embryogenic cell suspension (ECS) cultures. Yellow, compact meristematic globules and yellow-whiteish, friable embryogenic calli were induced from… Show full abstract scalps (i.e., the top layer of highly proliferating meristem cultures). About 10 percent of induced explants gave rise to embryogenic calli. Embryogenic calli were used subsequently to establish ECS cultures. After 3 months of selection and sub-culturing, homogeneous ECS cultures with a high regeneration capacity were obtained. More than 80 percent of the cell culture consisted of embryogenic cell aggregates. Regenerable somatic embryos emerged about 3 weeks after the ECS was plated onto regeneration medium. Their weight increased 4.8- to 13-fold after 8 weeks of culture. The number of somatic embryos regenerated per ml settled cell volume (SCV) of embryogenic cells (i.e., somatic embryo recovery frequency) ranged between 0.92-2.17 X 105, depending on incubation and regeneration conditions. The conversion frequency from somatic embryo to plant varied between 42.5-55.5 percent, and was independent of regeneration conditions. (Author's abstract). Hide full abstract View article on publisher's site
Panis, B., Lambardi, M.
In: The role of biotechnology for the characterization and conservation of crop, forestry, animal and fishery genetic resources
The Role of Biotechnology, International Workshop, Villa Gualino, Turin (ITA), 2005/03/05-07
2005
Over the past decades, plant cryopreservation technologies have been evolving rapidly, opening the door to the possibility of long-term storage of valuable genetic resources of many crop and forest species. From the original slow-cooling approach, research has moved to easier and more reproducible… Show full abstract techniques which allow a complete vitrification of extra- and intra-cellular liquids through the direct immersion of explants in liquid nitrogen. This report describes concisely most of the procedures which have been proposed in time for the cryopreservation of a wide range of tissues and organs, such as cell suspensions, embryogenic callus, pollen, meristematic tissues, seeds and embryo axes. Also the most important achievements in the cryopreservation of herbaceous, hardwood and softwood species are discussed. Hide full abstract View article on publisher's site
Carpentier, S.; Witters, E.; Laukens, K.; Deckers, P.;
Swennen, R.;
Panis, B.;
Proteomics, 2005 |
Peer Reviewed
This study focuses on the specific problems of protein extraction from recalcitrant plant tissues and evaluates several methods to bypass them. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is absolutely essential for good results. We evaluated… Show full abstract four methods: the classical trichloroacetic acid (TCA)/acetone precipitation, TCA/acetone precipitation and fractionation, an alternative based on fractionation and without precipitation, and phenol extraction methanol/ammonium acetate precipitation. We optimized the phenol extraction protocol for small amounts of tissue, which is essential when the study material is limited. The protocol was optimized for banana (Musa spp.) and was subsequently applied to two other plant species: apple (Malus domestica L.) and potato (Solanum tuberosum L.). Banana (Musa spp.) is a good representative of a "difficult" plant species since it contains many interfering metabolites. Only classical TCA/acetone precipitation and phenol extraction methods proved useful as standard methods. Both methods are associated with a minor but reproducible loss of proteins. Every extraction method and the subsequent analytical procedure have their physicochemical limitations; both methods should be investigated before selecting an appropriate protocol. The study, which is presented in this paper, is useful for guiding the experimental setup of many other non-model species, containing various interfering elements. (Author's abstract). Hide full abstract View article on publisher's site
Roux, N.; Strosse, H.; Toloza, A.;
Panis, B.;
Dolezel, J.In: Hvoslef-Eide, A.K. (ed.), Preil, W. (ed.). Liquid culture systems for
in vitro plant propagation.
Springer, 2005
Cell suspensions are the material of choice for rapid multiplication and for genetic engineering strategies such as in vitro mutagenesis and genetic transformation. Effective use of cell suspension cultures relies on the knowledge of several key parameters, which include genetic stability, kinetics… Show full abstract of the cell cycle and a mode of plant regeneration. Here we report on the use of DNA flow cytometry for quality monitoring of banana cell suspension cultures. The method facilitates detection of ploidy changes and the occurrence of aneuploidy, which result in somaclonal variation of cell-suspension-derived plants. Flow cytometry could also be used to analyse the cell cycle kinetics by calculating the ratio of cells in the G2 and G1 phase of the cell cycle. This is important to determine the most appropriate moment for mutagenic treatment or for genetic transformation but also as an indicator on the proportion of cycling cells. In addition, the unicellular origin of somatic embryos was verified by treating embryogenic cell suspensions with colchicine and by determining the ploidy of regenerated plants by flow cytometric analysis. None of the plants regenerated from colchicine-treated embryogenic cell suspensions were mixoploid (chimeric). The application of flow cytometry will be discussed in relation to (a) the monitoring of genetic instability in DNA content of cell suspensions (b) the analysis of cell cycle and (c) the origin of somatic embryos of bananas and plantains. (Author's abstract).
[Les cellules en suspension sont un matériel de choix pour la multiplication rapide et les stratégies d'ingénierie génétique comme la mutagenèse in vitro et la transformation génétique. L'utilisation effective des cultures de cellules en suspension dépend de la connaissance de plusieurs paramètres-clés, qui incluent la stabilité génétique, la cinétique du cycle cellulaire et le mode de régénération des plantes. Nous rapportons ici l'utilisation de la cytométrie en flux de l'ADN pour contrôler la qualité des cultures en suspension des cellules du bananier. La méthode facilite la détection des changements de ploïdie et l'occurrence des aneuploïdies, qui occasionnent des variations somaclonales des plantes issues des suspensions cellulaires. La cytométrie en flux pourrait aussi être utilisée pour analyser la cinétique du cycle cellulaire par le calcul du ratio cellulaire pendant les phases G2 et G1 du cycle. Cela est important pour déterminer le moment le plus approprié pour un traitement mutagénique ou la transformation génétique, mais aussi comme un indicateur de la proportion de cellules dans le cycle. En plus, l'origine unicellulaire des embryons somatiques a été vérifiée en traitant les suspensions de cellules embryogéniques avec de la colchichine et en déterminant la ploïdie des plantes régénérées par l'analyse cytométrique. Aucune des plantes régénérées à partir des suspensions de cellules traitées à la colchicine n'était mixoploïde (chimérique). L'application de la cytométrie en flux sera discutée en relation avec (a) la surveillance de la stabilité génétique du contenu d'ADN des cellules en suspension, (b) l'analyse du cycle cellulaire et (c) l'origine des embryons somatiques des bananiers et bananiers plantain.] Hide full abstract View article on publisher's site
Panis, B.; Helliot, B.; Strosse, H.; Remy, S.;
Lepoivre, P.;
Swennen, R.In: Chang, W.C. (ed.), Drew, R. (ed.). Acta Horticulturae 692.
Proceedings of the Second International Symposium on Biotechnology of Tropical and Subtropical Species, Taipei (TWN), 2001/11/05-09
ISHS, 2005
Currently cryopreservation is applied mainly for safe long-term germplasm conservation of seedless and thus vegetatively propagated crops like banana (Musa spp.). Three cryopreservation methods for shoot-tip cultures of banana are currently available. The first method relies on rapid freezing of… Show full abstract highly proliferating meristem cultures precultured for 2 weeks on 0.4 M sucrose. The second method is based on vitrification of tiny meristems excised from rooted in vitro plants. The third, and until now most successful protocol, is a combination of the previous ones; vitrification of highly proliferating, sucrose-precultured meristem cultures. Post thaw regeneration rates are up to 75 percent, depending on the cryopreservation protocol and the cultivar. Besides its traditional application for germplasm storage, cryopreservation of meristem cultures can also result in virus eradication of BSV and CMV from infected plants. Only the most meristematic, and thus the least virus infected, part of these cultures regenerates after freezing. Finally, cryopreservation can also be applied to plant material with specific characteristics, such as medicinal and alcohol producing cell limes, genetically transformed tissues and transformation competent tissues. The safe storage of transformation competent embryogenic cell suspensions of banana la of utmost importance since their initiation is difficult and time-consuming and their morphogenic capacity decreases with time. Cell cultures were recovered after 4 years of storage in liquid nitrogen. We showed that viability and regeneration capacity remained intact, as well as competence to Agrobacterium mediated transformation. (Author's abstract). Hide full abstract View article on publisher's site
Criel, B.; Panta, A.;
Carpentier, S.; Renaut, J.;
Swennen, R.;
Panis, B.; Hausman, J.F.;
Communications in Agricultural and Applied Biological Sciences, 2005 |
Peer Reviewed
Cryopreservation involves the storage of biological material in liquid nitrogen (-196°c). At this temperature all the chemical and physical processes are arrested, allowing a safe storage over an unlimited period of time. In this was, cryopreservation provides a good alternative for in vitro… Show full abstract culture and field collections, since it is less labor intensive and less susceptible to pests, somaclonal variation and human error. Standardized cryopreservation protocols are not yet available for potato. At CIP more than 400 accessions of potato landraces have been tested with a cryopreservation method derived from the vitrification protocol (straw treatment, see below) developed by Steponkus et al. (1992). ClP reported a high variability in the recovery rates, which was linked to genotype characteristics. In order to avoid protocol adjustments for each genotype separately (which is costly, time consuming, and unpredictable due to a lack of insight into the cellular reactions in potato towards cryopreservation), studies on the role of biochemical cell components on cryopreservation are necessary and have been initiated. Hide full abstract View article on publisher's site
Carpentier, S.; Witters, E.; Laukens, K.;
Panis, B.;
Swennen, R.;
Communications in Agricultural and Applied Biological Sciences, 2005 |
Peer Reviewed
Proteomics is currently flourishing. Two-dimensional electrophoresis (2DE) was established by O'Farrel in 1975 and is still one of the most adequate techniques for functional analysis. The introduction and commercialization of a broad range of IPG-strips, the improvement of electrophoresis… Show full abstract techniques and equipment, the progression in bioinformatics and high throughput identification of proteins by mass spectrometry were major breakthroughs. Gene expression profiling based on mRNA is a powerful approach, yet post-translational modifications can not be analysed. Moreover, several stress studies have shown that changes in mRNA transcript levels do not automatically imply corresponding changes in protein amount or activity and those posttranslational modifications can occur in response to stress. A proteomic approach complements a genomic approach by looking at the actual protein population. Mass spectrometry identification of proteins separated by 2DE is now the most commonly used method in proteome analysis. Although it's intrinsic limitations (poor recovery of hydrophilic and now abundant proteins, restricted MW and pi range) 2DE is quantitatively the strongest technique, is cost-effective and is the most suitable proteomic approach for species of which the genome is not (fully) sequenced. Along with the general limitations of the currently available techniques, plant proteome approaches face specific problems. Sample extraction and preparation is the most critical step and is absolutely essential for good results. Most plant tissues do not provide a ready source of proteins and thus need special precautions. Hide full abstract View article on publisher's site
Davey, M.W.; Stals, E.;
Panis, B.; Keulemans, J.;
Swennen, R.;
Analytical Biochemistry, 2005 |
Peer Reviewed
Malondialdehyde (MDA) is a widely used marker of oxidative lipid injury whose concentration varies in response to biotic and abiotic stress. Commonly, MDA is quantified as a strong light-absorbing and fluorescing adduct following reaction with thiobarbituric acid (TBA). However, plant tissues in… Show full abstract particular contain many compounds that potentially interfere with this reaction and whose concentrations also vary according to the tissue type and stress conditions. As part of our studies into the stress responses of plant tissues, we were interested in developing a rapid, accurate, and robust protocol for MDA analysis using reverse-phased HPLC to avoid these problems with reaction specificity. We demonstrate that a partitioning step into n-butanol during sample preparation is essential and that gradient HPLC analysis is necessary to prevent sample carryover between injections. Furthermore, the starting composition of the mobile phase must be sufficiently hydrophobic to allow direct injection of the n-butanol extracts without peak splitting, tailing, and other artifacts. To minimize analysis times, we used a short, so-called "Rocket" HPLC column and high flow rates. The optimized HPLC separation has a turnaround time of 2.5 min per sample. Butanolic extracts of MDA(TBA)2 were stable for at least 48 h, and recoveries were linear between 0.38 and 7.5 pmol MDA added. Importantly, this procedure proved to be compatible with existing extraction procedures for L-ascorbate and glutathione analysis in different plant species, allowing multiple "stress metabolite" analyses to be carried out on a single tissue extract. (Author's abstract). Hide full abstract View article on publisher's site
Carpentier, S.; Witters, E.; Laukens, K.;
Swennen, R.;
Panis, B.;
Molecular and Cellular Proteomics, 2005 |
Peer Reviewed
Bananas and plantains (Musa spp.), with an annual production of about 100 million tons, are important throughout the developing countries of the (sub)tropics both as a subsistence and export crop. Through breeding and farmer selection, varieties are available with different degrees of tolerance… Show full abstract towards (a)biotic stresses. Proteomic research in non-model plants is often hampered by the lack of routine sample preparation procedures and the dependence of protein identification on orthologous proteins. Banana is a difficult plant species since it contains extremely high amounts of interfering metabolites and its genome is poorly characterized. Using a 2DE protocol developed for small amounts of tissue (Carpentier et al., Proteomics 2005, in press) and cross species MS/MS identification (Witters et al., RCMS 2003), we investigate the influence of sucrose preculture on the proteome of meristematic cells in a drought and cryopreservation tolerant and a susceptible cultivar. We have shown that a two-week preculture on 0.4M sucrose is essential for the acquisition of tolerance towards the severe dehydration prior to cryopreservation (Panis et al., Plant Science 1996). Up till now, we were able to classify 40 proteins that were significantly up-regulated and 19 proteins that were down-regulated due to the sucrose treatment. We classified the abiotic stress response proteins within different categories and put forward some intra- and inter-categorical protein interactions. Sucrose up-regulated categories are: (i) energy (35 percent), (ii) protein destination (20 percent), (iii) disease and defense (13 percent), (iv) signal transduction (10 percent), (v) transporters (5 percent"), (vi) transcription (5 percent), (vii) secondary metabolism (5 percent), (viii) metabolism (3 percent) and (ix) cell structure (3 percent). Sucrose down-regulated categories are: (i) metabolism (39 percent), (ii) storage (39 percent) and (iii) protein synthesis (22 percent). This work proves that quantitative differential proteome analysis is also successful for recalcitrant non-model crops and sheds new light on sugar metabolism and the adaptation towards dehydration tolerance. (Author's abstract). Hide full abstract
Strosse, H.;
Van Den Houwe, I.;
Panis, B.In: Jain, S.M. (ed.),
Swennen, R. (ed.). Banana improvement: cellular, molecular biology, and induced mutations.
Science Publishers, 2004
The International Musa germplasm collection is sited at the INIBAP (International Network for the Improvement of Banana and Plantain) Transit Centre at K.U.Leuven. By now, more than 1000 different accessions of shoot-tip cultures have been initiated in vitro, multiplied and maintained at reduced… Show full abstract temperature conditions (16+1°C). Shoot cultures are grown on MS (Murashige and Skoog) medium, supplemented with 30 g/l sucrose, 2.25 mg/l BA (6-benzyladenine) and 0.175 mg/l IAA (indole-3-acetic acid). In comparison with the culture medium on which shoot-tips are maintained, a tenfold decrease in cytokinin content (0.225 mg/l HA) induces regeneration of rooted plants. In contrast, adding 22.5 mg/l HA to the culture medium results in suppression of the apical dominance hi shoot-tip cultures and a reduction of corm and leaf tissue between meristematic tissue. Highly proliferating meristem cultures are obtained and used as starting material in the scalp methodology, the technique mass commonly applied for the development of embryogenic cell suspensions at the Laboratory of Tropical Crop Improvement, K.U.Leuvcn. Initiation and maintenance of cell cultures is rather labour intensive and lime consuming. However, since 1 ml of settled cells of a highly regenerable cell suspension can yield more than 100,000 plants, cell cultures are most suitable for mass clonal propagation. Moreover, embryogenic cell suspensions are highly preferred as target material for protoplast culture and genetic engineering since the risk of chimerism is circumvented because of the unicellular origin of regenerated plants. (Author's abstract). Hide full abstract View article on publisher's site
Panis, B.; Strosse, H.; Remy, S.;
Sági, L.;
Swennen, R.In: Jain, S.M. (ed.),
Swennen, R. (ed.). Banana improvement: cellular, molecular biology, and induced mutations.
Science Publishers, 2004
The world's largest banana collection (1141 accessions) is stored at the international Musa germplasm collection at the INIBAP (International Network for the Improvement of Banana and Plantain) Transit Centre at K.U.Leuven. In vitro proliferating shoot tips are currently maintained under slow-… Show full abstractgrowth conditions at reduced temperatures and light intensity. However, for the long-tam conservation of germplasm of banana, cryopreservation of meristem cultures is considered to be the only practicable solution. Three cryopreservation protocols for meristem cultures have been developed. This paper gives an overview of pros and cons for each cryopreservation method. The initiation of embryogenic cell suspension cultures of banana is still difficult and time-consuming, irrespective of the starting material used. Moreover, once established, these cell suspensions are subject to somaclonal variation and microbial contamination, and a prolonged culture period may result in total loss of morphogenic capacity. Up to now, most successful banana transformation procedures rely on embryogenic cell suspensions. The safe preservation of these valuable suspensions through cryopreservation is thus of utmost importance. A cryopreservation technique bas been developed which involves cryoprotection followed by slow freezing and plunging into liquid nitrogen. Currently, 51 independent cell lines and 15 different cultivars are in safe long-term liquid nitrogen storage. Recently, banana cell suspensions were recovered after 5 years storage in liquid nitrogen. Their competence for Agrobacterium-mediated transformation was tested. Both transient [3-glucuronidase frequency and stable transformation frequency were unaffected. (Author's abstract). Hide full abstract View article on publisher's site
Roux, N.; Toloza, A.;
Dolezel, J.;
Panis, B.In: Jain, S.M. (ed.),
Swennen, R. (ed.). Banana improvement: cellular, molecular biology, and induced mutations.
Science Publishers, 2004
In bananas shoot-tip cultures are traditionally used for mutagenesis. The main problem with this type of culture is the presence of chimerism. In contrast, embryogenic cell suspensions allow the handling of large populations under controlled conditions and simultaneously avoid chimerism of embryos… Show full abstract have a single-cell origin. In this study, we verified the unicellular origin of somatic embryos by treating embryogenic cell suspensions (ECS) with colchicine and determining the ploidy of regenerated plants by flow cytometry. We have found that none of the plants regenerated from colchicine-treated ECS was mixoploid (chimeric). To optimise a protocol for the irradiation of ECS, several experiments were performed. Flow cytometric analysis of cell-cycle kinetics proved to be more informative than growth curves for the determination of the optimal timing of irradiation. The optimal irradiation dose was determined using three different parameters. Among them, the fresh weight gain and the regeneration capacity indicated that the optimal irradiation dose lies between 50 and 75 Gy for embryogenic cell suspensions of both 'Williams' (AAA) and 'Three Hand Planty' (AAB plantain). (Author's abstract). Hide full abstract View article on publisher's site
Lopez, J.; Strosse, H.; Ventura, J.C.; Sanchez, R.; Rodríguez, S.;
Swennen, R.;
Panis, B.; Afza, R.
In: Jain, S.M. (ed.),
Swennen, R. (ed.). Banana improvement: cellular, molecular biology, and induced mutations.
Science Publishers, 2004
Bananas and plantains provide an important carbohydrate source in the Cuban diet. Low yields aid susceptibility to diseases (mainly black Sigatoka, caused by the fungus Mycosphaerella fijiensis) of current cultivars render the development of new varieties a high priority. Biotechnological and… Show full abstract nuclear techniques have been successfully applied on shod tips, resulting in the generation of improved cultivars. Potential induced mutants derived after gamma irradiation were tested in the field. A dwarf-like mutant of the cultivar 'Parecido al Rey' (clone V6-44) was obtained. Some mutants ('Parecido al Rey' variant 6-32 and 6-44 and 'Gran Enano' variant 3-2) were selected for further field evaluation due to their tolerance to black Sigatoka disease, as well as their higher yield compared to non-irradiated control plants. These characters appeared to lie unstable during following field cycles. To reduce the frequency of chimeras obtained after irradiation and to obtain a higher multiplication rate of irradiated samples, cell suspensions were established and used as starting material for mutation induction. The regeneration capacity of the established 'Navolean' cell culture was quantified. Homogenized samples of 500 microl cell suspension gave rise to between 1300 and 2650 embryos with a germination frequency of 20.7 percent. Percentages of survival ex vitro amounted to 95 percent. Field performance and genetic stability of plants regenerated from irradiated cell suspensions are currently under investigation. (Author's abstract). Hide full abstract View article on publisher's site
Helliot, B.;
Panis, B.; Hernandez, R.;
Swennen, R.;
Lepoivre, P.; Frison, E.A.
In: Jain, S.M. (ed.),
Swennen, R. (ed.). Banana improvement: cellular, molecular biology, and induced mutations.
Science Publishers, 2004
Bananas and plantains (Musa spp.) are threatened by various pests and diseases, including a number of important viral diseases which represent a constraint on banana production and on germplasm movement from country to country. The latter is especially important for farmers who are waiting to… Show full abstract benefit from pest- and disease-resistant varieties, either naturally occurring germplasm or improved varieties produced by breeding programmes or by mutation techniques, as produced in the framework of the IAEA Co-ordinated Research Programme. INIBAP has therefore been very active in establishing a system for the safe international movement of Musa germplasm using different virus-indexing centres (South Africa, Australia and France). At present, about 25 percent of the International Musa Germplasm Collection maintained at K.U.Leuven (Belgium) by INIBAP, and in particular a significant number of potentially important and improved varieties from breeding programmes, are infected with viruses. Most of this germplasm is infected with BSV but also with CMV, BBTV, BanMMV and BBrMV. To make these accessions available, a programme of virus elimination is currently carried on in the Plant Pathology Unit (FUSAGx, Belgium) in collaboration with the Laboratory of Tropical Crop Improvement (K.U.Leuven, Belgium). Different in vitro techniques, such thermotherapy, chemotherapy, electrotherapy or meristem culture, as well as more innovative ones, such as cryotherapy, were tested for their virus elimination capacity. For this, Williams BSJ banana plants (AAA) mechanically infected with CMV were used. Initially, the health status of regenerated material was checked on in vitro plants through ELISA. The putative virus-free material was then tested a second time after greenhouse acclimatisation. (Author's abstract). Hide full abstract View article on publisher's site
Roux, N.; Strosse, H.; Toloza, A.;
Panis, B.;
Dolezel, J.In: Jain, S.M. (ed.),
Swennen, R. (ed.). Banana improvement: cellular, molecular biology, and induced mutations.
Science Publishers, 2004
During micropropagation of bananas and plantains, somaclonal variation can occur in regenerated plantlets. This variation may interfere with the use of these cultures for physical or chemical mutagenesis and/or genetic transformation. Although the causes of genetic instability are poorly understood,… Show full abstract chromosome instability is believed toe be one of the most common causes of tissue culture-induced variation. Using flow cytometry, variation in chromosome number could be detected in embryogenic cell suspensions and in plants regenerated from them. Results obtained by flow cytometry were verified by chromosome counting in meristem root-tip cells. Abnormalities in DNA content such as polyploidy and aneuploidy were detected at the cell suspension level. For the first time, a hypotriploid banana embryogenic cell lime with 2n = 28 (i.e. with loss of five chromosomes) was reported. Factors affecting the genetic stability of embryogenic cell suspensions are discussed. (Author's abstract). Hide full abstract View article on publisher's site
Xu, C.X.;
Panis, B.; Strosse, H.;
Swennen, R.; Li, H.P.;
Xiao, H.; Fan, H.Z.;
Plant Physiology Communications, 2004
Embryogenic cell suspensions (ECSs) of 'Agbagaba' and 'Orishele' (Musa spp., AAB Group) were used to regenerate embryos after 1-week and 2-week pre-culture in liquid medium respectively. The ECSs were inoculated on regeneration medium RD1 or M3 and incubated under light or in dark. The amount of… Show full abstract regenerable somatic embryos that could be regenerated from per milliliter settled cell volume (SCV) of ECS depended on pre-culture time in liquid media, the types of regeneration media and incubation conditions. Plant regeneration frequency from the somatic embryos and plantlets could be regenerated from 1 mL SCV of ECS were indirectly affected with those conditions for the regeneration of somatic embryos mentioned above. (Author's abstract).
[Des suspensions cellulaires embryogéniques (ECS) de 'Agbagaba' et 'Orishele' (Musa spp., Groupe AAB) ont été employées pour régénérer des embryons après une pré-culture de, respectivement, 1 et de 2 semaines en milieu liquide. Les ECS ont été placées sur milieu de régénération RD1 ou M3 et incubées en lumière ou dans l'obscurité. La quantité d'embryons somatiques régénérables par millilitre de suspension cellulaire établie (SCV) d'ECS a dépendu du temps de pré-culture en milieu liquide, du type de milieu de régénération et des conditions d'incubation. La fréquence de régénération de plantes issues d'embryons somatiques et le nombre de plantules régénérées à partir de 1 mL SCV d'ECS ont été indirectement affectés par ces conditions de régénération des embryons somatiques mentionnées ci-dessus. (Résumé d'auteur).] Hide full abstract
Xu, C.X.;
Panis, B.; Strosse, H.;
Swennen, R.; Li, H.P.;
Xiao, H.; Fan, H.Z.;
Journal of South China Agricultural University, 2004 |
Peer Reviewed
Embryogenic cell suspensions (ECS) of 'Grande Naine' (Musa AAA group) were plated on RD1 or M3 medium for the regeneration of somatic embryos, 1 to 2 weeks after last subculture. The first regenerable somatic embryos were observed about 3 weeks after inoculation. After 8 weeks of culture, the mass… Show full abstract of the embryogenic mass increased about 5 10 18 times and the number of somatic embryos that could be regenerated from 1 mL settled cell volume (SCV) of ECS ranged between 0.71 x 105 and 3.07 x 105, depending on pre-culture time in liquid medium before regeneration, regeneration media and incubation conditions (light/dark). The frequency of plant recovery and the amount of plantlets regenerated from 1 mL SCV of ECS were indirectly affected by somatic embryos regeneration conditions that were studied. (Author's abstract).
[Des suspensions cellulaires embryogéniques (ECS) de 'Grande Naine' (Musa groupe AAA) ont été cultivées sur milieu RD1 ou M3 pour la régénération d'embryons somatiques, 1 à 2 semaines après la dernière sous-culture. Les premiers embryons somatiques régénérés ont été observés environ 3 semaines après l'inoculation. Après 8 semaines de culture, la masse embryogène a augmenté environ de 5 10 18 fois et le nombre des embryons somatiques régénérés à partir de 1 mL de volume de suspension cellulaire établie (SCV) d'ECS a été entre 0,71 x 105 et 3,07 x 105, selon le temps de pré-culture en milieu liquide avant la régénération, les milieux de régénération et les conditions d'incubation (lumière/obscurité). La fréquence de régénération de plantes et la quantité de plantules régénérées à partir de 1 mL SCV d'ECS ont été indirectement affectées par les conditions de régénération des embryons somatiques étudiées. (Résumé d'Auteur).] Hide full abstract View article on publisher's site
Xu, C.X.;
Panis, B.; Strosse, H.;
Swennen, R.; Li, H.P.;
Xiao, H.; Fan, H.Z.;
Journal of South China Agricultural University, 2004 |
Peer Reviewed
Embryogenic callus was successfully induced starting from immature male flowers in 2 out of 5 banana cultivars and starting from scalps in 2 out of 3 cultivars. All these 4 cultivars from which embryogenic callus could be induced belonged to Musa AAA group. The frequency of embryogenic callus… Show full abstract induction was depended on genotypes, cultivars and incubation conditions etc. Embryogenic cell suspensions (ECSs) were initiated successfully from embryogenic callus of all these 4 cultivars. The possibility of getting ECSs from embryogenic callus was also cultivar-dependent. (Author's abstract).
[Des cals embryogéniques ont été induits avec succès à partir de fleurs mâles immatures de deux cultivars de bananier sur cinq et à partir de scalps de deux cultivars sur trois. Ces quatre cultivars ont pu permettre l'induction de cals embryogéniques appartenant au groupe AAA de Musa. La fréquence d'induction des cals embryogéniques a dépendu des génotypes, des variétés cultivées et des conditions d'incubation, etc. Des suspensions de cellules embryogéniques (ECS) ont été induites avec succès à partir des cals embryogéniques de ces quatre cultivars. La probabilité d'obtenir des ECS à partir de cals embryogéniques a aussi été dépendante du cultivar. (Résumé d'Auteur).] Hide full abstract View article on publisher's site
Helliot, B.;
Swennen, R.; Poumay, Y.; Frison, E.A.;
Lepoivre, P.;
Panis, B.;
Plant Cell Reports, 2003 |
Peer Reviewed
Cryopreservation has been shown to improve the frequency of virus elimination - specifically cucumber mosaic virus and banana streak virus - from banana (Musa spp.) plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the ultrastructure of… Show full abstract meristem tips at each step of the cryopreservation process. Excised meristematic clumps produced from infected banana plants belonging to cv. 'Williams' (AAA, 'Cavendish' subgroup) were cryopreserved through vitrification using the PVS-2 solution. We demonstrated that the cryopreservation method used only allowed survival of small areas of cells in the meristematic dome and at the base of the primordia. Cellular and subcellular changes occurring during the cryopreservation process are discussed. Hide full abstract View article on publisher's site
Helliot, B.;
Panis, B.; Frison, E.A.; De Clercq, E.;
Swennen, R.;
Lepoivre, P.; Neyts, J.;
Antiviral Research, 2003 |
Peer Reviewed
We report that the anti-retroviral and anti-hepadnavirus molecules, adefovir, tenofovir and 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP), efficiently eradicate the episomal form of Banana streak virus (BSV) from banana plants. Up to 90 percent of plants regenerated from BSV-infected… Show full abstract highly proliferating meristems were virus free following a 6-month treatment period with 10 µg/ml (a non-phytotoxic concentration) of either compounds (Author's abstract).
[Il est rapporté que les molécules anti-rétrovirales et anti-hepadnavirus, adefovir, tenofovir et 9-(2-phosphonométhoxyéthyl)-2,6-diaminopurine (PMEDAP), éradiquent efficacement la forme épisomale du virus de la mosaïque en tirets du bananier (BSV) dans les bananiers. Jusqu'à 90 pourcent des plants régénérés à partir de méristèmes en prolifération active infectés se sont révélés exempts de virus après un traitement de 6 mois en présence d'une concentration non-phytotoxique de 10 µg/ml de l'un de ces composés.]
[Reportamos que las moléculas antiretrovirales y antihepadnavirales, adefovir, tenofovir y 9-(2-fosfonometoxietil)-2,6-diaminopurina (PMEDAP), erradican eficazmente la forma episomal del Virus del rayado del banano (BSV) de las plantas de banano. Hasta un 90 por ciento de las plantas regeneradas a partir de los meristemas altamente proliferantes infectados con el BSV estaba libre de virus después de un tratamiento de 6 meses con 10 µg/ml (una concentración no fitotóxica) de cualesquiera de los compuestos.] Hide full abstract View article on publisher's site
Lepoivre, P.; Busogoro, J.P.; Etame, J.J.; El Hadrami, A.;
Carlier, J.; Harelimana, G.;
Mourichon, X.;
Panis, B.; Stella Riveros, A.; Salle, G.; Strosse, H.;
Swennen, R.In: Jacome, L. (ed.),
Lepoivre, P. (ed.),
Marin, D.H. (ed.),
Ortiz, R. (ed.),
Romero, C.R.A (ed.), Escalant, J.V. (ed.). #Mycosphaerella# leaf spot diseases of bananas: present status and outlook.
INIBAP, 2003
Using standard testing procedures, banana genotypes were classified as 1) highly resistant (HR) cultivars characterized by an early blockage of leaf infection (incompatible interactions), 2) partially resistant cultivars exhibiting a slow rate of symptom development (compatible reactions) and 3)… Show full abstract susceptible cultivars, characterized by rapid development of necrotic lesions (compatible reaction). Most information on incompatible reactions comes from observations of early necrosis of stomatal guard cells and the deposit of electron-dense compounds around the penetration sites of M. fijiensis on the cultivar 'Yangambi km5'. Such rapid death of a few host cells, associated with the blockage of the progression of the infecting agent is usually defined as a hypersensitive reaction. Such a reaction often operates within a gene-for-gene relationship and as a consequence the resulting resistance may be unstable. As regards compatible interactions, cytological studies showed that M. fijiensis behaves first as a biotrophic parasite which colonizes exclusively the intercellular spaces without the formation of haustoria. Two main mechanisms have been investigated to explain the slow development of a lesion in partially resistant genotypes: preformed antifungal compounds and tolerance to putative toxin(s) produced by M. fijiensis. The mechanisms will be presented in relation to their possible use as early screening markers for selecting banana genotypes for durable resistance to M. fijiensis.
[En utilisant des procédures de test standard, des génotypes de bananier ont été classés en : 1) cultivars hautement résistants (HR) caractérisés par un blocage rapide de l'infection foliaire (interactions incompatibles), 2) cultivars partiellement résistants (PR) montrant un développement lent des symptômes (réactions compatibles) et 3) cultivars susceptibles (S) caractérisés par un développement rapide de lésions nécrotiques (réaction compatible). L'essentiel des informations sur les réactions incompatibles provient d'observations de nécrose précoce des cellules de garde des stomates et du dépôt de composés denses aux électrons autour des sites de pénétration de M. fijiensis chez le cultivar 'Yangambi km5'. La mort aussi rapide d'un petit nombre de cellules hôtes, associée avec le blocage de la progression de l'agent infectieux, est habituellement définie comme une réaction hypersensible. Une telle réaction se produit souvent dans le cadre d'une relation gène pour gène et, en conséquence, la résistance qui en résulte peut être instable. Pour ce qui concerne les interactions compatibles, les études cytologiques ont montré que M. fijiensis se comporte d'abord comme un parasite biotrophique qui colonise exclusivement les espaces intercellulaires sans formation d'haustoria. Deux mécanismes principaux ont été étudiés pour expliquer le développement lent des lésions chez les génotypes partiellement résistants : des composés antifongiques préformés et la tolérance à une(des) toxine(s) putative(s) produite(s) par M. fijiensis. Les mécanismes sont présentés en relation avec leur utilisation possible comme marqueurs lors de criblage précoce pour sélectionner des génotypes de bananiers possédant une résistance durable à M. fijiensis.]
[Utilizando los procedimientos de evaluación estándar, los genotipos de banano fueron clasificados en tres categorías: 1) cultivares altamente resistentes (HR) caracterizados por un bloqueo temprano de la infección foliar (interacciones incompatibles), 2) cultivares parcialmente resistentes (PR) que exhiben una evolución lenta de los síntomas (reacciones compatibles), y 3) cultivares susceptibles (S), caracterizados por un desarrollo rápido de las lesiones necróticas (reacción compatible). La mayor parte de la información sobre las reacciones incompatibles proviene de los estudios del cultivar 'Yangambi km5'. Se observaron la necrosis de las células de guarda estomatales y los depósitos de los compuestos con alta densidad de electrones alrededor de los sitios de penetración de M. fijiensis. La muerte tan rápida de unas cuantas células hospedantes asociada con el bloqueo de la progresión del agente infectante se define usualmente como una reacción hipersensible. Esta reacción a menudo opera dentro de una relación de gen por gen y podría convertir la resistencia en inestable. Con las reacciones compatibles, los estudios citológicos revelaron que M. fijiensis se comporta primero como un parásito biotrófico que coloniza exclusivamente los espacios intercelulares sin formar los haustorios. Dos mecanismos principales podrían estar involucrados en el desarrollo lento de las lesiones observado en los genotipos resistentes parcialmente: compuestos antifungosos sintetizados de manera constitutiva o tolerancia a la(s) toxina(s) putativa(s) producidas por M. fijiensis. Estos mecanismos se presentarán en relación con su posible utilidad como marcadores de cribado temprano en la selección de los genotipos de banano con respecto a la resistencia duradera a M. fijiensis.] Hide full abstract View PDF
Swennen, R.;
Arinaitwe, G.; Cammue, B.P.A.; François, I.;
Panis, B.; Remy, S.;
Sági, L.;
Santos-Ordóñez, E.; Strosse, H.;
Van Den Houwe, I.In: Jacome, L. (ed.),
Lepoivre, P. (ed.),
Marin, D.H. (ed.),
Ortiz, R. (ed.),
Romero, C.R.A (ed.), Escalant, J.V. (ed.). #Mycosphaerella# leaf spot diseases of bananas: present status and outlook.
INIBAP, 2003
In smallholdings, average banana and plantain yields per unit have not increased significantly in the last 30 years. Increases in production are due almost exclusively to an increase in the area under cultivation. Increasing pest and disease pressure, especially from leaf spot diseases, and the… Show full abstract deteriorating natural resource base have collectively been responsible for these low yields. Resistant high yielding bananas have been bred and supplied to smallholders in the 1990s after nearly 70 years of conventional breeding. This very slow progress was due to the high sterility, poor seed germination rate, need for interploidy crosses, the long generation cycle, which are inherent to bananas and plantains. A breeding program can only supply a few promising hybrids per year for further evaluation. The few selected hybrids are high yielding and resistant to some diseases but have usually lost other desired characteristics such as shelf life or pulp texture. Genetic transformation tools offer an opportunity for plant breeders to overcome the constraints imposed by the high level of sterility of the most popular cultivars. Good progress has been made in the development of a molecular toolbox for bananas and plantains in the areas of 1) cell suspension, 2) genetic transformation (particle bombardment and Agrobacterium-mediated transformation), 3) high expression of foreign genes, 4) insertion of multiple genes and 5) identification of genes for resistance to fungal disease.
[Dans les exploitations de petite taille, les rendements en bananes et bananes plantain n'ont pas significativement augmenté au cours des 30 dernières années. L'augmentation de la production est due presque exclusivement à une augmentation de la surface cultivée. L'accroissement de la pression des maladies et ravageurs, et particulièrement des maladies foliaires, et la détérioration de la base de la ressource naturelle ont été collectivement responsables de ces faibles rendements. Des bananiers résistants et à rendement élevé ont été produits et distribués aux petits producteurs dans les années 90, après près de 70 ans d'amélioration conventionnelle. Ces progrès très lents sont dus à la stérilité élevée, au faible taux de germination des semences, au besoin de réaliser des croisements interploïdes et au long cycle de génération, qui sont propres aux bananiers et aux bananiers plantain. Un programme d'amélioration ne peut produire que quelques hybrides prometteurs par an pour leur évaluation ultérieure. Les quelques hybrides sélectionnés ont une production élevée et sont résistants à certaines maladies mais ont généralement perdu d'autres caractéristiques désirées, telles que la durée de conservation ou la texture de la pulpe. Les outils de transformation génétique offrent une opportunité aux sélectionneurs de surmonter les contraintes imposées par le niveau élevé de stérilité des cultivars les plus populaires. Des progrès importants ont été faits dans le développement d'une boîte à outils moléculaires pour les bananiers et les bananiers plantain dans les domaines : 1) des suspensions cellulaires ; 2) de la transformation génétique (bombardement de particules et transformation avec Agrobacterium ; 3) du niveau d'expression élevé de gènes étrangers ; 4) de l'insertion de gènes multiples et 5) de l'identification de gènes de résistance aux maladies fongiques.]
[Durante los últimos 30 años, los rendimientos promedio de los bananos y plátanos no han aumentado significativamente en las pequeñas fincas y los aumentos de producción se deben casi exclusivamente a un aumento del área bajo cultivo. El aumento de la presión de plagas y enfermedades, especialmente de las enfermedades de las manchas foliares, y el deterioro de la base de recursos naturales han sido responsables de manera colectiva de estos rendimientos tan bajos. En la década de los 90, se seleccionaron bananos resistentes de alto rendimiento los cuales fueron suministrados a los pequeños productores, después de casi 70 años de mejoramiento convencional. Este progreso tan lento se debió a una alta esterilidad, una tasa pobre de germinación de las semillas, una necesidad de cruzamientos interploídicos, un largo ciclo de regeneración, etc., inherentes a los bananos y plátanos. Básicamente, un programa de mejoramiento puede proporcionar solo unos pocos híbridos prometedores por año para realizar las evaluaciones consiguientes. Los pocos híbridos seleccionados son de alto rendimiento y resistentes a algunas enfermedades, pero usualmente pierden otras características deseadas como vida verde, textura de la pulpa, etc. Las herramientas de la transformación genética ofrecen una oportunidad a los fitomejoradores para vencer las limitaciones impuestas por el alto nivel de esterilidad en las variedades más populares. También se alcanzó un buen progreso en el desarrollo de una serie de herramientas moleculares para los bananos y plátanos en las áreas de (1) desarrollo de las suspensiones celulares; (2) tecnologías de transformación genética (bombardeo con partículas o transformación con Agrobacterium); (3) alta expresión de genes foráneos; (4) inserción de genes múltiples; (5) identificación de genes para la resistencia a enfermedades fungosas.] Hide full abstract View PDF
Roux, N.; Toloza, A.; Busogoro, J.P.;
Panis, B.; Strosse, H.;
Lepoivre, P.;
Swennen, R.; Zapata-Arias, F.J.
In: Jacome, L. (ed.),
Lepoivre, P. (ed.),
Marin, D.H. (ed.),
Ortiz, R. (ed.),
Romero, C.R.A (ed.), Escalant, J.V. (ed.). #Mycosphaerella# leaf spot diseases of bananas: present status and outlook.
INIBAP, 2003
Mycosphaerella leaf spot diseases can reduce fruit yield by up to 50 percent. Chemical strategies exist to combat these diseases, but they are environmentally unsound, hazardous and very expensive for many farmers. The only sustainable means to reduce the use of pesticides is breeding for tolerant… Show full abstract cultivars. Whereas breeders are looking for genetic variability to develop new varieties, edible Musa cultivars are multiplied vegetatively, polyploid and sterile. Spontaneous somatic mutation has already contributed largely to obtaining new cultivars of Musa. Nevertheless, the rate of occurrence is too low to satisfy practical breeding needs. Mutations can be induced by physical or chemical mutagens. With the development of tissue culture techniques, in vitro mutagenesis and somaclonal variation raised hopes in the 1980-1990s. In spite of this, very few useful and stable mutants/somaclones were obtained. The multicellular structure of meristems which leads to chimerism is certainly an impeding factor. Additionally, the random process in mutation induction calls for the screening of several thousand plants after treatment. Recently, following a five-year FAO/IAEA/DGIC coordinated research project, it has been possible to overcome these two barriers by mutagenic treatment of embryogenic cell suspensions and by establishing an early mass screening method resting on infiltration of juglone, a toxic metabolite of M. fijiensis. After screening approximately 4000 plants, 15 putative mutants showed tolerance to this metabolite. These plants must be evaluated for their resistance to M. fijiensis infection under controlled conditions and field experiment.
[Les maladies foliaires causées par Mycosphaerella spp. peuvent réduire le rendement de jusqu'à 50 pourcent. Des moyens chimiques existent pour lutter contre ces maladies, mais ils sont nocifs pour l'environnement, dangereux et très coûteux pour nombre de fermiers. La seule manière durable de réduire le recours aux insecticides est de créer des cultivars tolérants. Sauf que pour y arriver, les sélectionneurs ont besoin de variabilité génétique et que les cultivars de bananiers sont polyploïdes, stériles et multipliés végétativement. Les mutations somatiques spontanées ont déjà passablement contribuées à l'obtention de nouveaux cultivars, mais leur fréquence est trop faible pour satisfaire les besoins des sélectionneurs. Des agents mutagènes chimiques et physiques peuvent provoquer des mutations. Dans les années 1980-1990, le développement des techniques de culture de tissus, mutagenèse in vitro et variation somaclonale ont soulevé bien des espoirs. Malgré cela, peu de mutants/somaclones ont été obtenus. La structure multicellulaire des méristèmes, qui produit des chimères, est sans doute un facteur limitant. De plus, l'induction de mutations est un processus aléatoire qui nécessite le criblage de plusieurs milliers de plants. Récemment, suite à un projet de recherche de cinq ans coordonné par FAO/IAEA/DGIC, il a été possible de contourner ces deux obstacles en faisant subir un traitement mutagène à des suspensions de cellules embryogéniques et en mettant au point une méthode de criblage précoce basée sur l'infiltration de juglone, un métabolite toxique de M. fijiensis. Après avoir criblé 4000 plants, 15 mutants présomptifs ont montré une tolérance à ce métabolite. Ces plants doivent être évalués pour leur résistance à M. fijiensis sous des conditions contrôlées et en champ.]
[Las enfermedades de las manchas foliares causadas por Mycosphaerella spp. afectan significativamente el cultivo bananero y puede reducir el rendimiento de la fruta en hasta un 50 porciento. Existen estrategias de control químico para combatir estas enfermedades, pero estas causan daños al ambiente, son peligrosas y muy costosas para muchos agricultores. El único medio sostenible para reducir el uso de plaguicidas es el mejoramiento de los cultivares tolerantes. Los mejoradores que están investigando la variabilidad genética para desarrollar nuevas variedades, toman en cuenta que las variedades comestibles de Musa se multiplican vegetativamente, son poliploides y estériles. La mutación somática espontánea es una fuente de variación que ya ha contribuido en gran medida en la obtención de las nuevas variedades en Musa spp. Sin embargo, la tasa de ocurrencia es muy baja para satisfacer las necesidades prácticas de mejoramiento. Las mutaciones pueden ser inducidas por mutágenos físicos o químicos. Con el desarrollo de las técnicas del cultivo de tejidos, la mutagénesis y variación somaclonal in vitro elevaron las esperanzas en las décadas de los 80 y 90. A pesar de esto, se obtuvieron pocos mutantes o somaclones útiles y estables. La estructura multicelular de los meristemas que conduce al quimerismo es ciertamente un factor de impedimento. Adicionalmente, el proceso aleatorio para inducir la mutación requiere el cribado de varios miles de plantas después del tratamiento. Recientemente, después de realizar un proyecto de cinco años investigación de coordinado por FAO/IAEA/DGCI, fue posible vencer estas dos barreras mediante un tratamiento mutagénico de las suspensiones de células embriogénicas y el establecimiento de un método de cribado masivo temprano que se basa en la infiltración de juglone, un metabolito tóxico de M. fijiensis. Después de realizar el cribado de aproximadamente 4000 plantas, 15 mutantes putativos mostraron tolerancia a este metabolito.] Hide full abstract View PDF
Strosse, H.; Domergue, R.;
Panis, B.; Escalant, J.V.;
Côte, F.;
INIBAP, 2003
These guidelines present two protocols to produce embryogenic cell suspensions by using scalps or immature male flowers. The protocols have been developed by the Laboratory of Tropical Crop Improvement of Katholieke Universiteit Leuven (KULeuven) and the cellular biology laboratory of the Centre de… Show full abstract coopération internationale en recherche agronomique pour le développement (Cirad).
[Ce guide technique présente deux protocoles qui expliquent comment produire des suspensions cellulaires embryogènes à partir de scalps ou de fleurs immatures. Les protocoles ont été développés par des chercheurs du Laboratory of Tropical Crop Improvement de la Katholieke Universiteit Leuven (KULeuven) et du laboratoire de biologie cellulaire du Centre de coopération internationale en recherche agronomique pour le développement (Cirad).]
[Estas guías técnicas presentan dos protocolos para producir suspensiones de células a partir de escalpos o de flores inmaduras. Los protocolos fueron desarrollados por investigadores del Laboratory of Tropical Crop Improvement de la Katholieke Universiteit Leuven (KULeuven) y del Laboratorio de biología celular del Centre de coopération internationale en recherche agronomique pour le développement (Cirad).] Hide full abstract View PDF
Strosse, H.; Domergue, R.;
Panis, B.; Escalant, J.V.;
Côte, F.;
INIBAP, 2003
These guidelines present two protocols to produce embryogenic cell suspensions by using scalps or immature male flowers. The protocols have been developed by the Laboratory of Tropical Crop Improvement of Katholieke Universiteit Leuven (KULeuven) and the cellular biology laboratory of the Centre de… Show full abstract coopération internationale en recherche agronomique pour le développement (Cirad).
[Ce guide technique présente deux protocoles qui expliquent comment produire des suspensions cellulaires embryogènes à partir de scalps ou de fleurs immatures. Les protocoles ont été développés par des chercheurs du Laboratory of Tropical Crop Improvement de la Katholieke Universiteit Leuven (KULeuven) et du laboratoire de biologie cellulaire du Centre de coopération internationale en recherche agronomique pour le développement (Cirad).]
[Estas guías técnicas presentan dos protocolos para producir suspensiones de células a partir de escalpos o de flores inmaduras. Los protocolos fueron desarrollados por investigadores del Laboratory of Tropical Crop Improvement de la Katholieke Universiteit Leuven (KULeuven) y del Laboratorio de biología celular del Centre de coopération internationale en recherche agronomique pour le développement (Cirad).] Hide full abstract View PDF
Strosse, H.; Domergue, R.;
Panis, B.; Escalant, J.V.;
Côte, F.;
Vezina, A. (ed.);
Picq, C. (ed.)
INIBAP, 2003
These guidelines present two protocols to produce embryogenic cell suspensions by using scalps or immature male flowers. The protocols have been developed by the Laboratory of Tropical Crop Improvement of Katholieke Universiteit Leuven (KULeuven) and the cellular biology laboratory of the Centre de… Show full abstract coopération internationale en recherche agronomique pour le développement (Cirad).
[Ce guide technique présente deux protocoles qui expliquent comment produire des suspensions cellulaires embryogènes à partir de scalps ou de fleurs immatures. Les protocoles ont été développés par des chercheurs du Laboratory of Tropical Crop Improvement de la Katholieke Universiteit Leuven (KULeuven) et du laboratoire de biologie cellulaire du Centre de coopération internationale en recherche agronomique pour le développement (Cirad).]
[Estas guías técnicas presentan dos protocolos para producir suspensiones de células a partir de escalpos o de flores inmaduras. Los protocolos fueron desarrollados por investigadores del Laboratory of Tropical Crop Improvement de la Katholieke Universiteit Leuven (KULeuven) y del Laboratorio de biología celular del Centre de coopération internationale en recherche agronomique pour le développement (Cirad).] Hide full abstract View PDF
Agrawal, A.; Sharma, H.;
Swennen, R.;
Panis, B.In: Singh, H.P. (ed.), Dadlani, N.K. (ed.). Global conference on banana and plantain: abstracts [Conférence globale sur les bananiers et bananiers plantain: résumés] [Conferencia global sobre banano y plátano : resúmenes].
Global Conference on Banana and Plantain, Bangalore (IND), 2002/10/28-31
AIPUB, 2002
The present work was carried out to determine the optimal PVS2 treatment time required for optimal post-thaw survival. Following a pre-treatment with 0.4 M sucrose for 2 weeks, proliferating meristem clumps of 7 cultivars were subjected to PVS2 for 30, 60, 90, 120 and 150 minutes at 0°C. The… Show full abstract following cultivars, belonging to different genomics groups were tested: 'Cachaco' (AAB), 'Burro Cemsa' (AAB), 'Prata' (AAB), 'Obino l'Ewai' (AAB-plantain), 'Cemsa 3/4' (AAB-plantain), 'Guyod' (AA) and 'Kamarasange' (AB). For all genotypes tested, PVS2 did not prove to be toxic to the meristems (controls). In case of ABB bananas, controls remained almost 100 percent viable even up to 150 minutes. However in all others, viability decreased with increase in treatment time of PVS2. Hide full abstract View PDF
Panis, B.; Strosse, H.; Van Den Hende, S.;
Swennen, R.;
CryoLetters, 2002 |
Peer Reviewed
A simple cryopreservation method is described for proliferating meristem cultures of banana (Musa spp.). It relies on a 2-week preculture on media containing 0.4 M sucrose followed by rapid cooling in liquid nitrogen. Different preculture media were screened for efficient protection of banana… Show full abstract meristems during cryopreservation. Sucrose can be replaced by both fructose and glucose without significantly affecting post-thaw survival. A high BA concentration (100 microM) in the preculture medium results in less material available for cryopreservation, but does not affect cryoprotection. Culture in liquid media significantly improved post-thaw regeneration. The optimized cryopreservation protocol was applied on 36 banana accessions belonging to 8 different genomic groups. Lt is shown that post-thaw regeneration frequencies (ranging between 0 and 66 percent) are highly dependent on the genomic constitution of the banana cultivar. Hide full abstract View article on publisher's site
Ramon, M.; Geuns, J.M.C.;
Swennen, R.;
Panis, B.;
CryoLetters, 2002 |
Peer Reviewed
Polyamines and fatty acids were studied in proliferating meristem cultures of 3 banana cultivars with high ('Cachaco'), medium ('Williams Bronze Free') and low ('Mbwazirume') survival rates after cryopreservation. A 2-week preculture on medium containing 0.4 M sucrose that is essential to obtain… Show full abstract survival after cryopreservation resulted in increased polyamine levels, especially putrescine. This increase in putrescine content was positively correlated with the survival rate after simple freezing or after vitrification. The total fatty acid content also increased after 0.4 M sucrose pre-treatment. However, only the ratio of unsaturated/saturated fatty acids correlated positively with the survival rate after cryopreservation. This is the first report showing a correlation of both putrescine increase and level of unsaturation of membrane lipids after sucrose treatment with survival rate after cryopreservation. Hide full abstract View article on publisher's site
Helliot, B.;
Panis, B.; Poumay, Y.;
Swennen, R.;
Lepoivre, P.; Frison, E.A.;
Plant Cell Reports, 2002 |
Peer Reviewed
The utilisation of cryopreservation for the eradication of cucumber mosaic virus (CMV) or banana streak virus (BSV) from Musa spp. was investigated. Banana plants, cv. Williams (AAA, Cavendish subgroup), were mechanically infected with CMV or naturally infected with BSV and proliferating meristems… Show full abstract were produced from the infected plants. Excised meristematic clumps were cryopreserved through vitrification using PVS-2 solution. The health status of regenerated in vitro plants was first checked by means of ELISA. The putative virus-free material was subsequently tested a second time following greenhouse acclimatisation. The frequency of virus eradication for CMV and BSV was 30 percent and 90 percent, respectively, following cryopreservation. In comparison, the frequency of virus-free plants regenerated directly from highly proliferating meristems, corresponding to a spontaneous eradication rate, reached 0 percent and 52 percent for CMV and BSV respectively. The conventional meristem culture resulted in 0 percent CMV-free plants and 76 percent BSV-free plants, while the cryoprotective treatment resulted in 2 percent CMV-free plants and 87 percent BSV-free plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the structure of the meristern lips by light microscopy. The cryopreservation method used only allowed survival of small areas of cells located in the meristematic dome and of the base of the primordia.
[L'emploi de la cryoconservation pour l'éradication du virus de la mosaïque du concombre (CMV) ou de celui de la mosaïque en tirets du bananier (BSV) issu de Musa spp. a été étudié. Des plants de bananier cv. 'Williams (AAA, sous-groupe 'Cavendish') ont été infectés mécaniquement avec le CMV ou naturellement avec le BSV et les méristèmes en prolifération active ont été produits par ces plants. Les amas méristématiques excisés ont été cryoconservés par vitrification avec une solution de PVS-2. L'état sanitaire des vitroplants régénérés a d'abord été vérifié par ELISA. Le matériel supposé indemne de virus a été ensuite testé une seconde fois après acclimatation en serre. La fréquence d'éradication a été de 30 pourcent pour le CMV et de 90 pourcent pour le BSV après la cryoconservation. En comparaison, la fréquence de plantes saines obtenues après régénération directe de méristèmes en prolifération active, correspondant au taux d'éradication spontanée, atteint 0 pourcent pour le CMV et 52 pourcent pour le BSV. La culture conventionnelle de méristèmes a donné 0 pourcent de plants sains pour le CMV et 76 pourcent pour le BSV alors que le traitement cryoprotecteur a donné 2 pourcent de plants sains pour le CMV et 87 pourcent pour le BSV. Pour comprendre le mode d'action de la cryoconservation pour l'éradication des particules virales, nous avons examiné la structure des lèvres méristématiques en microscopie optique. La méthode de cryoconservation employée a permis seulement la survie de petites surfaces de cellules situées dans le dôme méristématique et à la base des primordia.]
[Se investigó la utilización de la crioconservación para la erradicación de los virus del mosaico del pepino (CMV) o del rayado del banano (BSV) de las Musa spp. Las plantas de banano, cv. Williams (AAA, subgrupo Cavendish), fueron infectadas mecánicamente con el CMV o infectadas naturalmente con el BSV y de las plantas infectadas se produjeron meristemas proliferantes. Los racimos meristemáticos cortados fueron crioconservados a través de la vitrificación utilizando la solución PVS-2. El estado sanitario de las plantas regeneradas in vitro primero fue verificado mediante la prueba ELISA. El material libre de virus putativo fue posteriormente examinado por segunda vez después de una aclimatación en el invernadero. La frecuencia de la erradicación de virus para el CMV y BSV fue de 30 por ciento y 90 por ciento, respectivamente, después de la crioconservación. En comparación, la frecuencia de las plantas libres de virus regeneradas directamente de los meristemas altamente proliferantes que corresponden a una tasa de erradicación espontánea, alcanzó 0 por ciento y 52 por ciento para el CMV y BSV, respectivamente. El cultivo de meristemas convencional dio como resultado 0 por ciento de plantas libres del CMV y 76 por ciento de plantas libres del BSV, mientras que el tratamiento crioprotector dio como resultado 2 por ciento de plantas libres del CMV y 87 por ciento de plantas libres del BSV. Para entender el modo de acción de la crioconservación para la erradicación de las partículas virales, hemos examinado la estructura de los labios del meristema con la ayuda del microscopio luminoso. El método de crioconservación utilizado solo permitió la supervivencia de unas pequeñas áreas de células localizadas en el domo meristemático y en la base del primordio.] Hide full abstract View article on publisher's site
Escalant, J.V.,
Panis, B. In: XV ACORBAT meeting, Cartagena de Indias, Colombia, 2002/10/27-11/02
AUGURA, 2002
Important progress has been made in the genetic improvement of Musa in recent years and new varieties are now becoming available from breeding programmes. This paper provides a summary of the status of Musa improvement and provides information on the results to be expected in the short to medium… Show full abstract term. It is clear that there is a growing interest in Musa improvement and research efforts are focused not only on 'Cavendish', but also on the other major groups of bananas and plantains. However, considering the global importance of the crop, the number of Musa breeding programmes in existence remains small. Despite this, research is progressing both in the areas of conventional breeding and in the use of genetic transformation technologies. Technologies that can help to increase the efficiency of breeding, such as tissue culture and embryo rescue are routinely used by most breeding programmes, while research on the use of molecular markers as a breeding tool and the molecular characterization of germplasm is advancing. Some programmes are also employing the creation of new types using somaclonal variation. A mechanism for collaboration and information exchange between researchers involved in Musa genetic improvement has been put in pace in the form of PROMUSA, the Global Programme for Musa Improvement. This has allowed the global prioritization of research needs and the acceleration of progress through the formation of synergistic partnerships.
[Les biotechnologies pour l'amélioration génétique des #Musa#]
[Ces dernières années, l'amélioration génétique de Musa a vu s'accomplir d'importants progrès et des nouvelles variétés issues des programmes de sélection sont maintenant disponibles. Cet article résume l'état de l'amélioration des Musa et expose les résultats prévus à court et moyen termes. L'amélioration de Musa fait clairement l'objet d'un intérêt croissant et les actions de recherche ne se sont pas concentrées sur le seul 'Cavendish', mais ont porté aussi sur les autres groupes majeurs de bananier et de plantain. Cependant, si l'on tient compte de l'importance mondiale de cette culture, le nombre de programmes d'amélioration existant reste faible. Néanmoins, la recherche progresse tant dans les domaines de la sélection conventionnelle que dans l'emploi des technologies de transformation génétique. Des technologies capables d'optimiser la sélection, telles que la culture de tissu et le sauvetage d'embryon, servent couramment dans de nombreux programmes. Dans le même temps, la recherche sur l'emploi des marqueurs moléculaires comme outil de sélection et sur la caractérisation moléculaire du matériel génétique avancent. Quelques programmes s'intéressent aussi à la création de nouveaux types par variation somaclonale. Un système de collaboration et d'échange de l'information entre les chercheurs impliqués dans l'amélioration génétique de Musa a été mis en place sous la forme de PROMUSA, le Programme mondial pour l'amélioration des Musa. Ainsi, des priorités de recherche ont pu être définies au niveau mondial et les progrès s'en sont trouvés accélérés grâce à la création de partenariats synergiques.] Hide full abstract View PDF
Roux, N.; Strosse, H.; Toloza, A.;
Panis, B.;
Dolezel, J.;
Swennen, R.Picq, C. (prep.).
In: 3rd international symposium on molecular and cellular biology of bananas. Programme and abstracts [Troisième symposium international sur la biologie moléculaire et cellulaire du bananier. Programme et résumés] [Tercero simposio international dedicado a la biología molecular y celular de los bananos. Programa y resúmenes]
Third International Symposium on Molecular and Cellular Biology of Bananas, Leuven (BEL), 2002/09/09-11
INIBAP, 2002
View PDF
Schoofs, H.,
Panis, B., Strosse, H., Mayo Mosqueda, A.,
Lopez Torres, J.,
Roux, N.,
Dolezel, J.,
Swennen, R. In: Third FAO/IAEA Research Co-ordination Meeting, Colombo (LKA), 1999/10/04-08
IAEA, 2001
This paper deals with the major bottlenecks in the generation and maintenance of morphogenic banana cell suspensions and plant regeneration via somatic embryogenesis therefrom. It further highlights shortcomings in our knowledge and interesting future research topics. View PDF
Helliot, B.;
Panis, B.; Locicero, A.; Reyniers, K.; Muylle, H.; Vandewalle, M.; Michel, C.;
Swennen, R.;
Lepoivre, P.In: Sorvari, S. (ed.), Karhu, S. (ed.), Kanervo, E. (ed.), Pihakaski, S. (ed.). Acta Horticulturae 560.
Fourth International Symposium on In Vitro Culture and Horticultural Breeding, Tampere (FIN), 2001/10/30
2001
The efficiency of meristem culture for virus disease eradication from Musa was studied. 'Williams' BSJ banana plants were used for this study. Plants were infected by one of the 3 following viruses: the Cucumber Mosaic Virus (CMV), the Banana Bunchy Top Virus (BBTV) and the Banana Streak Virus (BSV)… Show full abstract. Meristems were excised from in vivo plants, from in vitro plants and from highly proliferating meristems. Eradication rates varied according first to the virus and secondly to the plant material from which the meristem was excised.
[Développement de techniques in vitro pour l'éradication des viroses sur Musa]
[L'efficacité de la culture de méristèmes pour l'éradication des viroses sur Musa a été étudiée en utilisant des plants de bananier BSJ 'Williams'. Le matériel a été infecté par l'un des virus suivants : le virus de la mosaïque du concombre (CMV), le virus du Bunchy Top du bananier (BBTV) et le virus de la mosaïque en tirets du bananier (BSV). Les méristèmes ont été excisés de plants in vivo, de vitroplants et de méristèmes en prolifération active. Les taux d'éradication varient en fonction du virus et du matériel dont le méristème est issu.]
[Desarrollo de las técnicas in vitro para la eliminación de las enfermedades virales de Musa]
[Se estudió la eficacia del cultivo de meristemas para la erradicación de las enfermedades virales de Musa. Para este estudio se utilizaron las plantas de banano 'Williams' BSJ. Las plantas fueron infectadas por uno de los 3 virus siguientes: Virus del Mosaico del Pepino (CMV), Virus Bunchy Top del Banano (BBTV) y Virus del Rayado del Banano (BSV). Los meristemas fueron recortados de las plantas in vivo, plantas in vitro y de los meristemas altamente proliferantes. Las tasas de erradicación variaron primero de acuerdo al virus, y segundo, de acuerdo al material vegetal del cual se extrajo el meristema.] Hide full abstract View article on publisher's site
Panis, B.;
Swennen, R.; Engelmann, F.
In: Sorvari, S. (ed.), Karhu, S. (ed.), Kanervo, E. (ed.), Pihakaski, S. (ed.). Acta Horticulturae 560.
Fourth International Symposium on In Vitro Culture and Horticultural Breeding, Tampere (FIN), 2001/10/30
2001
Plant germplasm stored in liquid nitrogen (-196°C) does not undergo cellular divisions. In addition, metabolic and most physical processes are stopped at this temperature. As such, plants can be stored for very long time periods and both the problem of genetic instability and the risk of loosing… Show full abstract accessions due to contamination or human error during subculturing are overcome. Most cryopreservation endeavours deal with recalcitrant seeds, in vitro tissues from vegetatively propagated crops, species with a particular gene combination (elite genotypes) and dedifferentiated plant cell cultures. Sofar, cryopreservation procedures are developed for in vitro tissues and recalcitrant seeds of about 100 and 40 species, respectively. There is still a limited number of cases where cryopreservation is used routinely for plant germplasm conservation, mainly because the techniques need to be adapted for each species in function of its natural freezing resistance, explant size and type, and its water content. Care must be taken to avoid ice crystallisation during the freezing process, which otherwise would cause physical damage to the tissues. The existing cryogenic strategies rely on air-drying, freeze dehydration, osmotic dehydration, addition of penetrating cryoprotective substances and adaptive metabolism (hardening) or combinations of these processes (Author's abstract).
[Cryoconservation de matériel génétique]
[Le matériel génétique stocké dans l'azote liquide (-196°C) n'entre pas en division cellulaire et la plupart des processus métaboliques et physiques sont stoppés. Ainsi, les plantes peuvent être conservées pendant très longtemps et l'on évite les problèmes liés à l'instabilité génétique et le risque de perte des accessions par contamination ou erreur humaine lors des subcultures. La plupart des tentatives de cryoconservation ont concerné les graines récalcitrantes, les tissus d'espèces à propagation végétative, les espèces possédant une combinaison génique particulière (génotype élite) et les suspensions de cellules végétales dédifférenciées. Jusque là, les procédures de cryoconservation ont été développées pour les tissus in vitro d'une centaine d'espèces et les graines récalcitrantes d'environ 40 espèces. Il est rare que la cryoconservation soit employée en routine pour la conservation du matériel génétique, surtout parce que la technique doit être adaptée à chaque espèce en fonction de sa résistance naturelle à la congélation, de la nature et de la taille de l'explant et de la teneur hydrique. Un soin particulier doit être apporté pour éviter la formation de cristaux de glace durant le processus de congélation qui, autrement, entraînerait des dommages physiques des tissus. Les stratégies cryogéniques existantes reposent sur le séchage à l'air, la déshydratation au froid, la déshydratation osmotique, l'addition de cryoprotectants et le métabolisme adaptatif (endurcissement) ou la combinaison de ces procédés (Résumé d'auteur).]
[Crioconservación de germoplasma de las plantas]
[El germoplasma de las plantas almacenado en nitrógeno líquido (-196°C) no experimenta divisiones celulares. En adición, el proceso metabólico y la mayoría de los procesos físicos se detienen a esta temperatura. Como tal, las plantas pueden ser almacenadas por períodos de tiempo muy largos y los problemas de la inestabilidad genética y del riesgo de perder accesiones debido a la contaminación o error humano durante el subcultivo están resueltos. La mayoría de los esfuerzos se concentran en las semillas recalcitrantes, tejidos in vitro de los cultivos que se propagan vegetativamente, especies con una combinación génica particular (genotipos elite) y cultivos de células diferenciadas de las plantas. Hasta ahora, los procedimientos de crioconservación han sido desarrollados para los tejidos in vitro y semillas recalcitrantes de unas 100 y 40 especies, respectivamente. Aún existe una cantidad limitada de casos donde la crioconservación se utiliza habitualmente para la conservación de germoplasma de las plantas, debido principalmente a que las técnicas necesitan ser adaptadas para cada especie en función de su resistencia natural a la congelación, tamaño y tipo de explantes, y su contenido hídrico. Se debe tener cuidado para evitar la cristalización de hielo durante el proceso de congelación que de otra manera podría causar daños físicos a los tejidos. Las estrategias criogénicas existentes cuentan con el secado al aire, deshidratación durante la congelación, deshidratación osmótica, adición de sustancias crioprotectoras penetrantes y metabolismo adaptativo (aclimatación) o combinaciones de estos procesos (Resumen del autor).] Hide full abstract View article on publisher's site
Panis, B.; Schoofs, H.; Remy, S.;
Sági, L.;
Swennen, R.In: Engelmann, F. (ed.),
Takagi, H. (ed.). Cryopreservation of tropical plant germplasm : Current research progress and application.
IPGRI/JIRCAS, 2000
Banana (Musa spp.) embryogenic cell suspensions can be initiated from either immature male flowers or highly proliferating meristem cultures. In both cases, it is very time consuming and labour intensive. Moreover, in both cases, the efficiency of embryogenic cell suspension production is very low… Show full abstract and cultivar-dependent. Embryogenic cell suspensions are mostly monocots, especially in the case of banana where no immature embryos are available, the material of choice for genetic engineering. Currently, embryogenic cell suspensions are the only source of regenerable protoplasts in banana. These protoplasts can be transformed through electroporation. Numerous transgenic banana plants have been obtained by subjecting embryogenic cells to particle bombardment and Agrobacterium infection. Once initiated, cell suspensions are subject to somaclonal variation and contamination. Moreover, a prolonged culture period results in a decrease and finally even in the loss of their morphogenic capacity. It is therefore of the utmost importance that these valuable cell suspensions are safely stored through cryopreservation. Therefore, we developed a cryopreservation protocol which relies on slow freezing (1°C/min) to -40°C in the presence of a cryoprotectant solution containing 180 g/L sucrose and 7.5 percent dimethylsulphoxide (DMSO). Currently, 19 cell lines belonging to 7 different cultivars are safely stored in liquid nitrogen for the long term. After thawing, cell suspensions can again be initiated for use in genetic engineering.
[Cryoconservation de suspensions cellulaires embryogéniques de bananier : un atout pour la transformation génétique]
[Des suspensions cellulaires embryogéniques de bananier (Musa spp.) peuvent être initiées à partir de fleurs mâles immatures ou de méristèmes en prolifération active. Dans les deux cas, cela demande beaucoup de travail et de temps. De plus, la productivité de ces suspensions reste très faible et dépend du cultivar. Des suspensions cellulaires embryogéniques ont été surtout effectuées sur des monocotylédones, particulièrement pour le bananier où aucun embryon immature, matériel de choix du génie génétique, n'est disponible. Actuellement, les suspensions cellulaires embryogéniques sont la seule source de protoplastes régénérables pour le bananier. Ces protoplastes peuvent être transformés par électroporation. Plusieurs plants de bananiers transgéniques ont été obtenus en soumettant des cellules embryogéniques à un bombardement de particules ou à une infection au moyen d'Agrobacterium. Une fois initiée, les suspensions cellulaires sont sujettes à la variation somaclonale et à la contamination. De plus, une période de culture prolongée entraîne le déclin, puis finalement la perte de leur capacité morphogénique. Il est de la plus haute importance que ces précieuses suspensions cellulaires soient stockées en toute sécurité par cryoconservation. C'est pourquoi, nous avons développé un protocole de cryoconservation qui repose sur une congélation lente (1°C/min) jusqu'à -40°C en présence d'une solution cryoprotectrice contenant 180 g/l de sucrose et 7.5 pourcent de diméthylsulfoxyde (DMSO). Actuellement, 19 lignées cellulaires appartenant à 7 cultivars différents sont stockées, à long terme et en sécurité, dans l'azote liquide. Après décongélation, les suspensions cellulaires peuvent être réactivées pour leur emploi en génie génétique.]
[Crioconservación de las suspensiones de células embriogénicas de banano: Una ayuda para la transformación genética]
[Las suspensiones de células embriogénicas de banano (Musa spp.) pueden ser iniciadas a partir de flores masculinas inmaduras o cultivos de meristemas altamente proliferantes. En ambos casos, este trabajo es muy intenso y requiere de mucho tiempo. Además, en ambos casos, la eficacia de la producción de suspensiones de células embriogénicas es muy baja y depende del cultivar. Las suspensiones de células embriogénicas en su mayoría son monocotiledóneas, especialmente en el caso del banano donde no se dispone de embriones inmaduros, material de selección para la ingeniería genética. Comúnmente, las suspensiones de células embriogénicas son la única fuente de protoplastos regenerables en banano. Estos protoplastos pueden ser transformados mediante electroporación. Se han obtenido numerosas plantas transgénicas de banano sometiendo las células embriogénicas al bombardeo con partículas e infección con Agrobacterium. Una vez iniciadas, las suspensiones celulares están sujetas a la variación somaclonal y contaminación. Además, un período de cultivo prolongado da como resultado una disminución y finalmente incluso la pérdida de su capacidad morfogénica. Por lo tanto, es de suma importancia que estas valiosas suspensiones celulares sean seguramente almacenadas a través de la crioconservación. Hemos desarrollado un protocolo de crioconservación que cuenta con una congelación lenta (1°C/min) hasta -40°C en presencia de una solución crioprotectora que contiene 180 g/L de sacarosa y 7.5 porciento de dimetilsulfóxido (DMSO). Actualmente, 19 líneas de células pertenecientes a 7 cultivares diferentes están almacenadas sin peligro en nitrógeno líquido para un término largo. Después de la descongelación, las suspensiones celulares pueden ser iniciadas nuevamente para ser utilizadas en la ingeniería genética.] Hide full abstract View PDF View article on publisher's site
Van Den Houwe, I.;
Panis, B.;
Swennen, R.In: Engelmann, F. (ed.),
Takagi, H. (ed.). Cryopreservation of tropical plant germplasm : Current research progress and application.
IPGRI/JIRCAS, 2000
The cultivation of banana (Musa spp.) is threatened by several pests and diseases. As such, edible cultivars as well as their wild ancestors, all native to South East Asia, need to be collected. Once safely stored, they are available for classical breeding programmes, genetic engineering and as… Show full abstract short-term replacement of disease-affected banana. The global and world's largest Musa germplasm collection (1141 accessions) is currently stored at the INIBAP Transit Centre (ITC) (Laboratory of Tropical Crop Improvement, K.U. Leuven, Belgium) under the auspices of FAO. In vitro proliferating shoot-tips are stored under slow-growth conditions at reduced temperatures and light intensity. As such, the shoot-tip cultures need to be subcultured on average once per year, depending on the cultivar. Since 1985, nearly 6000 accessions have been provided to research scientists, plant breeders and other users in developed and developing countries. Although in vitro collections have proven their value, somaclonal variation, loss of morphogenic potential, contamination, etc. remain serious impediments to conservation. Moreover, the maintenance of such a large collection is still a labour-intensive endeavour. Therefore, to complement the active collection, a base collection using cryopreservation is under development. As such, germplasm can be safely stored for unlimited periods. Currently, three cryopreservation methods are under investigation. Post-thaw regeneration frequencies, availability of starting material and user-friendliness of the protocol will finally decide which cryopreservation method will be chosen for routine application at the ITC. Hide full abstract View PDF View article on publisher's site
Panis, B.; Schoofs, H.; Thinh, N.T.;
Swennen, R.In: Engelmann, F. (ed.),
Takagi, H. (ed.). Cryopreservation of tropical plant germplasm : Current research progress and application.
IPGRI/JIRCAS, 2000
Two types of highly meristematic and regenerable tissue can be obtained in banana; (1) tiny meristems excised from in vitro plants and (2) proliferating 'cauliflower like' meristem clumps. Both tissue types are currently subject to cryopreservation experiments. In this report, we present two… Show full abstract cryopreservation techniques applied on proliferating meristem clumps, i.e. simple freezing and vitrification. 'Cauliflower-like' meristems are obtained on media containing a high concentration of benzylaminopurine (BAP). Simple freezing involves a 2-week preculture on a semisolid medium with a high (0.4 M) sucrose concentration, after which the meristem clumps are plunged into liquid nitrogen. Post-thaw viability rates could be improved by transferring thawed meristems to liquid medium instead of semisolid medium. As such, viability rates between 0 and 68 percent were obtained, depending on the cultivar. ABB cooking bananas responded well, unlike AAA highland bananas and AAB plantains, which responded poorly to this cryopreservation protocol. Therefore, we tested the vitrification technique on the same material. Sucrose precultured and non-precultured meristem clumps were subjected to the PVS2 solution at 0°C and subsequently frozen. We observed that sucrose precultured meristems are less sensitive to the PVS solution and most importantly that these precultured meristems became more resistant to freezing. For the 21 accessions under investigation, post-thaw viability rates ranged between 14 and 79 percent. For both techniques we found that a sucrose preculture period is essential to obtain high levels of survival after freezing.
[Cryoconservation de cultures méristématiques de bananier en prolifération active]
[Deux types de tissu fortement méristématiques et régénérables peuvent être obtenus pour le bananier ; (1) des méristèmes minuscules excisés de plants in vitro et (2) des amas méristématiques en prolifération de type 'chou-fleur'. Les deux types de tissu sont soumis actuellement à des expériences de cryoconservation. Dans ce rapport, nous présentons deux techniques de cryoconservation appliquées sur des amas méristématiques en prolifération active : une simple congélation et une vitrification. Des méristèmes de type 'chou-fleur' sont obtenus sur des milieux contenant une forte concentration de benzylaminopurine (BAP). La congélation simple implique une phase de préculture de deux semaines sur un milieu semi-solide enrichi fortement en sucrose (0,4 M), après quoi, les amas méristématiques sont plongés dans l'azote liquide. Les taux de viabilité post-décongélation pourraient être améliorés en transférant les méristèmes décongelés en milieu liquide plutôt qu'en milieu semi-solide. Des taux de viabilité de 0 à 68 pourcent ont été ainsi obtenus, selon le cultivar. Les bananiers à cuire ABB ont bien répondu à ce protocole de cryoconservation, contrairement aux bananiers d'altitude et aux plantains AAB, dont la réponse a été faible. Par conséquent, la technique de vitrification a été appliquée à ce matériel. Des amas méristématiques, précultivés ou non sur sucrose, ont été soumis à une solution PVS2 à 0°C, puis congelés. Les méristèmes précultivés sur sucrose s'avèrent moins sensibles à la solution PVS et surtout, résistent mieux à la congélation. Pour les 21 accessions testées, les taux de survie post-décongélation ont varié de 14 pourcent à 79 pourcent. La préculture sur sucrose s'est révélée essentielle, dans les deux techniques, à l'obtention de bons taux de survie après congélation.]
[Crioconservación de los cultivos de meristemas proliferantes de banano]
[En banano se puede obtener dos tipos de tejidos altamente meristemáticos y regenerables: (1) meristemas minúsculos recortados de las plantas cultivadas in vitro y (2) racimos de meristemas proliferantes 'parecidos a coliflor'. Ambos tipos de tejidos se someten actualmente a experimentos de crioconservación. En este informe, presentamos dos técnicas de crioconservación aplicadas a los racimos de meristemas proliferantes, es decir, una congelación simple y vitrificación. Los meristemas 'parecidos a coliflor' se obtienen en los medios que contienen una alta concentración de benzilaminopurina (BAP). La congelación simple involucra un precultivo de 2 semanas en un medio semisólido con una alta concentración (0.4 M) de sacarosa, después de lo cual los racimos de meristemas se sumergen en nitrógeno líquido. Las tasas de viabilidad después de descongelación podrían ser mejoradas transfiriendo los meristemas a un medio líquido en vez de semisólido. Como tales, se obtuvieron tasas de viabilidad entre 0 y 68 porciento, dependiendo del cultivar. Los bananos de cocción ABB respondieron bien, a diferencia de los bananos de altiplanos AAA y plátanos AAB, que respondieron pobremente a este protocolo de crioconservación. Por lo tanto, hemos examinado la técnica de vitrificación en el mismo material. Los racimos de meristemas precultivados en sacarosa y sin precultivo fueron sujetos a una solución de PVS2 a 0°C y subsecuentemente congelados. Hemos observado que los meristemas precultivados en sacarosa son menos sensibles a la solución PVS y lo que es más importante es que estos meristemas precultivados se hicieron más resistentes a la congelación. Para las 21 accesiones bajo investigación, las tasas de viabilidad después de la descongelación variaron entre 14 y 79 porciento. Para ambas técnicas hemos encontrado que es esencial efectuar un precultivo para obtener altos niveles de supervivencia después de la congelación.] Hide full abstract View PDF View article on publisher's site
Sági, L.; Remy, S.; Cammue, B.P.A.; Maes, K.; Raemaekers, T.;
Panis, B.; Schoofs, H.;
Swennen, R.In: Craenen, K. (ed.),
Ortiz, R. (ed.),
Karamura, E.B. (ed.), Vuylsteke, D. (ed.). Acta Horticulturae 540.
First International Conference on Banana and Plantain for Africa, Kampala (UGA), 1996/10/14-18
ISHS, 2000
Genetic transformation of banana and plantain has been performed in our laboratory by (i) particle bombardment of embryogenic cell suspensions (ECS), (ii) electroporation of protoplasts isolated from ECS, and (iii) cocultivation of meristematic tissues with Agrobacterium tumefaciens. So far, the… Show full abstract following cultivars have been transformed successfully: 'Williams' (AAA), 'Three Hand Planty' (AAB), 'Bluggoe' (ABB), 'Cardaba' (ABB), 'Monthan' (ABB) and 'Tani' (BB). Transient expression of the gusA reporter gene has been observed in all these cultivars following at least one of the three transformation techniques. Transgenic plants have been regenerated after particle bombardment of 'Bluggoe', 'Three Hand Planty' and 'Williams', representing the three economically important genomic groups in Musa. Molecular and biochemical analysis has confirmed integration and expression of the transgenes in the banana and plantain genome. At present, more than 200 independent transgenic lines, generated from more than 1000 shoots, are growing in the greenhouse. Practical applications of molecular breeding of banana and plantain to create resistance to pathogens and pests are in progress.
[Producción de bananos y plátanos transgénicos]
[Production de bananiers et de bananiers plantain transgéniques]
[La transformation génétique des bananiers et des bananiers plantain a été exécutée dans notre laboratoire par : (i) bombardement de particules des suspensions cellulaires embryogéniques (SCE), (ii) électroporation de protoplastes isolés des SCE, et (iii) co-cultivation des tissus méristématiques en présence d'Agrobactériium tumefaciens. Jusqu'ici, les cultivars suivants ont été transformés avec succès : 'Williams' (AAA.), 'Three Hand Planty' (AAB), 'Bluggoe' (ABB), 'Cardaba'(ABB), 'Monthan' (ABB) et 'Tani' (BB). L'expression transitoire du gène gusA a été observée dans tous ces cultivars à la suite de l'une au moins de ces trois techniques de transformation. Des plants transgéniques ont été régénérés après bombardement de particules chez 'Bluggoe', 'Three Hand Planty' et 'Williams', représentant les trois groupes génomiques économiquement importants chez Musa. Une analyse moléculaire et biochimique a confirmé l'intégration et l'expression des transgènes dans les génomes de bananier et de bananier plantain. Actuellement, plus de 200 lignées transgéniques indépendantes, générées par plus de 1000 tiges, se développent en serre. Les applications pratiques de l'amélioration moléculaire du bananier et du bananier plantain afin de créer une résistance aux pathogènes et aux ravageurs sont en cours.]
[La transformación genética del banano y plátano se realizó en nuestro laboratorio mediante (i) bombardeo con partículas de suspensiones de células embriogénicas (ECS), (ii) electroporación de los protoplastos aislados de los ECS, y (iii) cocultivo de los tejidos meristemáticos con el Agrobacterium tumefaciens. A la fecha, los siguientes cultivares fueron transformados exitosamente: 'Williams' (AAA), 'Three Hand Planty' (AAB), 'Bluggoe' (ABB), 'Cardaba' (ABB), 'Monthan' (ABB) y 'Tani' (BB). La expresión transitoria del gen reportero gusA se observó en todos estos cultivares aplicando al menos una de las tres técnicas de transformación. Las plantas transgénicas se regeneraron después del bombardeo con partículas de 'Bluggoe', 'Three Hand Planty' y 'Williams', que representan tres grupos genómicos económicamente importantes en Musa. Los análisis molecular y bioquímico han confirmado la integración y la expresión de los transgenes en el genoma del banano y plátano. Actualmente, más de 200 líneas transgénicas independientes generadas de más de 1000 brotes, están siendo cultivadas en el invernadero. Se están desarrollando aplicaciones prácticas de mejoramiento molecular del banano y plátano para crear la resistencia a los patógenos y plagas.] Hide full abstract View article on publisher's site
Côte, F.; Goue, O.; Domergue, R.;
Panis, B.;
Jenny, C.;
CryoLetters, 2000 |
Peer Reviewed
This study describes the in-field behavior of bananas (Musa AA sp.) obtained after regeneration of cryopreserved embryogenic cell suspensions. Observations were focused on the classical vegetal development descriptors. We observed no significant differences between the cryopreserved-derived plants… Show full abstract and the control plants with respect to the plant height and circumference, the number of leaves, the number of fruits, the fruit length, the fruit diameter and weight, the bunch weight and the date of harvest. During the first culture cycle, 2 out of 11 descriptors analyzed were however found to be différent between the control and the cryopreserved suspensions derived plants. These were the number of nodal clusters of the inflorescence (usually called 'hands') and the date of flowering. These différences were, however, quite minor as the two cases together amounted to only 2 percent of the control value. During the second cycle of culture, no significant difference between the two groups of plants was found whatever the parameter analysed. These results suggest that, with the experimental conditions of the study, there is no différence at the agronomic level between plants produced from cryopreserved embryogenic cell suspensions and control plants (Author's abstract).
[Comportement au champ de bananiers (Musa AA sp.) obtenus après régénération de suspensions cellulaires embryogéniques cryoconservées]
[Cette étude décrit le comportement au champ de bananiers (Musa AA sp.) issus de suspensions cellulaires embryogéniques cryoconservées. Les observations ont porté sur les descripteurs classiques du développement végétal. Aucune différence significative n'est apparue entre les plants cryoconservés et les plants témoins en termes de hauteur de plante et circonférence, du nombre de feuilles, du nombre de fruits, de longueur du fruit, de diamètre et de poids du fruit, de poids du régime et de date de récolte. Durant le premier cycle de culture, 2 des 11 descripteurs analysés se sont distingués entre le témoin et les plants cryoconservés : le nombre de mains et la date de floraison. Ces différences, assez mineures, ont représenté ensemble seulement 2 pourcent de la valeur du témoin. Durant le second cycle de culture, aucune différence significative n'a été notée, quel que soit le paramètre analysé. Ces résultats suggèrent que, dans ces conditions expérimentales, il n'existe aucune différence au plan agronomique entre les plants issus de suspensions cellulaires embryogéniques cryoconservées et les plants témoins (Résumé d'auteur).]
[Comportamiento en campo de las plantas de banano (Musa AA sp.) obtenidas después de la regeneración de las suspensiones de células embriogénicas crioconservadas]
[Este estudio describe el comportamiento en el campo de los bananos (Musa AA sp.) obtenidos después de la regeneración de las suspensiones de células embriogénicas crioconservadas. Las observaciones se enfocaron en los descriptores del desarrollo vegetal clásico. No hemos observado diferencias significativas entre las plantas derivadas de la crioconservación y las plantas testigo, con respecto a la altura y circunferencia de la planta, cantidad de hojas, cantidad de frutas, largo de frutas, diámetro y peso de frutas, peso del racimo y fecha de cosecha. No obstante, durante el primer ciclo de cultivo, 2 de los 11 descriptores analizados resultaron diferentes entre las plantas testigo y las plantas derivadas de las suspensiones crioconservadas. Estos descriptores fueron los siguientes: la cantidad de racimos nodales de la inflorescencia (usualmente llamados 'manos') y la fecha de floración. Sin embargo, estas diferencias fueron realmente pequeñas ya que los dos casos juntos sumaron sólo el 2 porciento del valor de control. Durante el segundo ciclo de cultivo, no hubo diferencias significativas entre los dos grupos de plantas con respecto a los parámetros analizados. Estos resultados sugieren que en el marco de las condiciones experimentales del estudio, no existen diferencias a escala agronómica entre las plantas producidas de las suspensiones de células crioconservadas y las plantas testigo (Resumen del autor).] Hide full abstract View article on publisher's site
Schoofs, H.;
Panis, B.; Strosse, H.; Mayo Mosqueda, A.;
Lopez Torres, J.;
Roux, N.;
Dolezel, J.;
Swennen, R.;
Infomusa, 1999
During the last decade, Musa embryogenic cell suspensions were successfully initiated from scalps and (fe)male flowers of many genotypes and landraces. The initiation of a banana suspension takes 9 to 26 months depending on the type of explant and landrace. Low embryogenic responses hamper the… Show full abstract optimization of the induction and early initiation steps. Only one out of two to one out of five good embryogenic calluses result thus far in a "good" i.e. highly regenerable and transformation-competent suspension. Clearly, their initiation is still far from routine. The main problem related to the use of scalps is the need for a prolonged culture in the presence of very high BA concentrations and its possible effect on the ploidy level. Flow cytometry analysis provides a very powerful tool to quickly determine the ploidy level of starting material and suspensions. This is important, as the initiation of suspensions is so time and labour consuming. Also of prime importance is the cryopreservation of cell suspensions at an early stage as they can get contaminated very quickly, loose their morphogenic potential with time and are prone to somaclonal variation. The first data on somaclonal variation among suspension-derived plants are now available.
[Durant les dix dernières années, des suspensions cellulaires embryogéniques de Musa ont été initiées avec succès à partir de scalps et de fleurs mâle et femelle pour de nombreux génotypes et variétés locales. L'initiation d'une suspension de bananier prend de 9 à 26 mois suivant le type d'explant et la variété. Des réponses embryogéniques faibles ont entravé l'optimisation de l'induction et les premières étapes de l'initiation. Seulement de un sur deux à un sur cinq cals embryogéniques convenables évoluent en une « bonne » suspension hautement régénérable et compétente pour la transformation. En clair, leur initiation est encore loin d'être une routine. Le principal problème relatif à l'utilisation des scalps est la nécessité d'une culture prolongée en présence de concentrations très élevées de BA et de ses effets possibles sur le niveau de ploïdie. L'analyse de cytométrie en flux procure un outil très puissant pour la détermination rapide du niveau de ploïdie du matériel de départ et des suspensions. Ceci est important au vu du temps passé et du travail fourni. Il est aussi de première importance d'obtenir une cryoconservation des suspensions cellulaires à un stade précoce pour se prémunir des contaminations qui peuvent très rapidement survenir, des pertes du potentiel morphogénique qui peuvent s'installer avec le temps et de la variation somaclonale. Les premiers rapports sur la variation somaclonale de plants issus de suspensions cellulaires sont à présent disponibles.]
[Durante la última década, las suspensiones de células embriogénicas de Musa se iniciaban con éxito a partir de los cortes de las flores femeninas y masculinas de muchos genotipos y variedades indígenas. La iniciación de una suspensión toma de 9 a 26 meses dependiendo del tipo de explante y variedad indígena. Las bajas respuestas embriogénicas impiden la optimización de la inducción y los primeros pasos de iniciación. Hasta la fecha, sólo uno de dos o uno de cinco buenos callos embriogénicos dan como resultado una suspensión "buena", es decir altamente regenerable y capaz de transformarse. Está claro que su iniciación está todavía lejos de formar un procedimiento rutinario. El problema principal relacionado con el uso de los cortes consiste en la necesidad de un cultivo prolongado en presencia de muy altas concentraciones de BA y su posible efecto sobre el nivel de ploidia. El análisis mediante la citometría de flujo proporciona una herramienta muy poderosa para determinar con rapidez el nivel de ploidia del material inicial y de las suspensiones. Esto es importante, ya la iniciación de las suspensiones consume mucho tiempo y trabajo. También, de primera importancia es la crioconservación de las suspensiones celulares en una etapa temprana, ya que ellas pueden ser contaminadas rápidamente, pueden perder su potencial morfogénico con el tiempo y estar predispuestas a la variación somaclonal. Actualmente, tenemos los primeros datos sobre la variación somaclonal entre las plantas derivadas de suspensiones.] Hide full abstract View PDF
Schoofs, H.;
Panis, B.; Strosse, H.; Mayo Mosqueda, A.;
Lopez Torres, J.;
Roux, N.;
Dolezel, J.;
Swennen, R.;
Infomusa, 1999
During the last decade, Musa embryogenic cell suspensions were successfully initiated from scalps and (fe)male flowers of many genotypes and landraces. The initiation of a banana suspension takes 9 to 26 months depending on the type of explant and landrace. Low embryogenic responses hamper the… Show full abstract optimization of the induction and early initiation steps. Only one out of two to one out of five good embryogenic calluses result thus far in a "good" i.e. highly regenerable and transformation-competent suspension. Clearly, their initiation is still far from routine. The main problem related to the use of scalps is the need for a prolonged culture in the presence of very high BA concentrations and its possible effect on the ploidy level. Flow cytometry analysis provides a very powerful tool to quickly determine the ploidy level of starting material and suspensions. This is important, as the initiation of suspensions is so time and labour consuming. Also of prime importance is the cryopreservation of cell suspensions at an early stage as they can get contaminated very quickly, loose their morphogenic potential with time and are prone to somaclonal variation. The first data on somaclonal variation among suspension-derived plants are now available.
[Durant les dix dernières années, des suspensions cellulaires embryogéniques de Musa ont été initiées avec succès à partir de scalps et de fleurs mâle et femelle pour de nombreux génotypes et variétés locales. L'initiation d'une suspension de bananier prend de 9 à 26 mois suivant le type d'explant et la variété. Des réponses embryogéniques faibles ont entravé l'optimisation de l'induction et les premières étapes de l'initiation. Seulement de un sur deux à un sur cinq cals embryogéniques convenables évoluent en une « bonne » suspension hautement régénérable et compétente pour la transformation. En clair, leur initiation est encore loin d'être une routine. Le principal problème relatif à l'utilisation des scalps est la nécessité d'une culture prolongée en présence de concentrations très élevées de BA et de ses effets possibles sur le niveau de ploïdie. L'analyse de cytométrie en flux procure un outil très puissant pour la détermination rapide du niveau de ploïdie du matériel de départ et des suspensions. Ceci est important au vu du temps passé et du travail fourni. Il est aussi de première importance d'obtenir une cryoconservation des suspensions cellulaires à un stade précoce pour se prémunir des contaminations qui peuvent très rapidement survenir, des pertes du potentiel morphogénique qui peuvent s'installer avec le temps et de la variation somaclonale. Les premiers rapports sur la variation somaclonale de plants issus de suspensions cellulaires sont à présent disponibles.]
[Durante la última década, las suspensiones de células embriogénicas de Musa se iniciaban con éxito a partir de los cortes de las flores femeninas y masculinas de muchos genotipos y variedades indígenas. La iniciación de una suspensión toma de 9 a 26 meses dependiendo del tipo de explante y variedad indígena. Las bajas respuestas embriogénicas impiden la optimización de la inducción y los primeros pasos de iniciación. Hasta la fecha, sólo uno de dos o uno de cinco buenos callos embriogénicos dan como resultado una suspensión "buena", es decir altamente regenerable y capaz de transformarse. Está claro que su iniciación está todavía lejos de formar un procedimiento rutinario. El problema principal relacionado con el uso de los cortes consiste en la necesidad de un cultivo prolongado en presencia de muy altas concentraciones de BA y su posible efecto sobre el nivel de ploidia. El análisis mediante la citometría de flujo proporciona una herramienta muy poderosa para determinar con rapidez el nivel de ploidia del material inicial y de las suspensiones. Esto es importante, ya la iniciación de las suspensiones consume mucho tiempo y trabajo. También, de primera importancia es la crioconservación de las suspensiones celulares en una etapa temprana, ya que ellas pueden ser contaminadas rápidamente, pueden perder su potencial morfogénico con el tiempo y estar predispuestas a la variación somaclonal. Actualmente, tenemos los primeros datos sobre la variación somaclonal entre las plantas derivadas de suspensiones.] Hide full abstract View PDF
Schoofs, H.;
Panis, B.; Strosse, H.; Mayo Mosqueda, A.;
Lopez Torres, J.;
Roux, N.;
Dolezel, J.;
Swennen, R.;
Infomusa, 1999
During the last decade, Musa embryogenic cell suspensions were successfully initiated from scalps and (fe)male flowers of many genotypes and landraces. The initiation of a banana suspension takes 9 to 26 months depending on the type of explant and landrace. Low embryogenic responses hamper the… Show full abstract optimization of the induction and early initiation steps. Only one out of two to one out of five good embryogenic calluses result thus far in a "good" i.e. highly regenerable and transformation-competent suspension. Clearly, their initiation is still far from routine. The main problem related to the use of scalps is the need for a prolonged culture in the presence of very high BA concentrations and its possible effect on the ploidy level. Flow cytometry analysis provides a very powerful tool to quickly determine the ploidy level of starting material and suspensions. This is important, as the initiation of suspensions is so time and labour consuming. Also of prime importance is the cryopreservation of cell suspensions at an early stage as they can get contaminated very quickly, loose their morphogenic potential with time and are prone to somaclonal variation. The first data on somaclonal variation among suspension-derived plants are now available.
[Durant les dix dernières années, des suspensions cellulaires embryogéniques de Musa ont été initiées avec succès à partir de scalps et de fleurs mâle et femelle pour de nombreux génotypes et variétés locales. L'initiation d'une suspension de bananier prend de 9 à 26 mois suivant le type d'explant et la variété. Des réponses embryogéniques faibles ont entravé l'optimisation de l'induction et les premières étapes de l'initiation. Seulement de un sur deux à un sur cinq cals embryogéniques convenables évoluent en une « bonne » suspension hautement régénérable et compétente pour la transformation. En clair, leur initiation est encore loin d'être une routine. Le principal problème relatif à l'utilisation des scalps est la nécessité d'une culture prolongée en présence de concentrations très élevées de BA et de ses effets possibles sur le niveau de ploïdie. L'analyse de cytométrie en flux procure un outil très puissant pour la détermination rapide du niveau de ploïdie du matériel de départ et des suspensions. Ceci est important au vu du temps passé et du travail fourni. Il est aussi de première importance d'obtenir une cryoconservation des suspensions cellulaires à un stade précoce pour se prémunir des contaminations qui peuvent très rapidement survenir, des pertes du potentiel morphogénique qui peuvent s'installer avec le temps et de la variation somaclonale. Les premiers rapports sur la variation somaclonale de plants issus de suspensions cellulaires sont à présent disponibles.]
[Durante la última década, las suspensiones de células embriogénicas de Musa se iniciaban con éxito a partir de los cortes de las flores femeninas y masculinas de muchos genotipos y variedades indígenas. La iniciación de una suspensión toma de 9 a 26 meses dependiendo del tipo de explante y variedad indígena. Las bajas respuestas embriogénicas impiden la optimización de la inducción y los primeros pasos de iniciación. Hasta la fecha, sólo uno de dos o uno de cinco buenos callos embriogénicos dan como resultado una suspensión "buena", es decir altamente regenerable y capaz de transformarse. Está claro que su iniciación está todavía lejos de formar un procedimiento rutinario. El problema principal relacionado con el uso de los cortes consiste en la necesidad de un cultivo prolongado en presencia de muy altas concentraciones de BA y su posible efecto sobre el nivel de ploidia. El análisis mediante la citometría de flujo proporciona una herramienta muy poderosa para determinar con rapidez el nivel de ploidia del material inicial y de las suspensiones. Esto es importante, ya la iniciación de las suspensiones consume mucho tiempo y trabajo. También, de primera importancia es la crioconservación de las suspensiones celulares en una etapa temprana, ya que ellas pueden ser contaminadas rápidamente, pueden perder su potencial morfogénico con el tiempo y estar predispuestas a la variación somaclonal. Actualmente, tenemos los primeros datos sobre la variación somaclonal entre las plantas derivadas de suspensiones.] Hide full abstract View PDF
Panis, B.; Vandenbranden, K.; Schoofs, H.;
Swennen, R.In: Drew, R. (ed.). Acta Horticulturae 461.
International Symposium on Biotechnology of Tropical and Subtropical Species, Brisbane, Queensland, Australia, 29 September 1997
ISHS, 1998
Since most banana landraces (Musa spp.) do not produce seed and are vegetatively propagated, germplasm must be maintained clonally. Therefore field and in vitro collections were established. Currently, the world's largest banana collection (1080 accessions) is kept as in vitro proliferating… Show full abstract meristems under limited growth conditions at the Laboratory of Tropical Crop Improvement of K.U.Leuven (Katholieke Universiteit Leuven, Belgium). Cryopreservation is a welcome alternative since during storage at ultra-low temperatures (-196 °C), all metabolical, chemical and physical processes are arrested. As such. the material under storage does not need to be subcultured, thus avoiding risks of contamination and human error, and no somaclonal variation will take place. We developed an extremely simple cryopreservation system relying on a two-week-preculture phase of highly proliferating meristems on media with elevated sugar concentrations followed by rapid freezing in liquid nitrogen. This cryopreservation protocol was successfully tested on 20 accessions belonging to different genomic groups. Normal plants were regenerated from each of them. Regeneration frequencies vary from 7.4 to 68.9 percent, depending on the cultivar. The mode of action of the sugar preculture phase with respect to the cryopreservation ability of different cultivars is under investigation. It is demonstrated that lipid, sugar and protein contents of non-precultured and sugar-precultured plants differ (Author's abstract).
[Conservation de matériel génétique de bananier par cryoconservation]
[Du fait de la stérilité de la plupart des races locales de bananier (Musa spp.) et de leur propagation par voie végétative, le matériel génétique doit être maintenu sous forme de clones. Par conséquent, des collections en champ et in vitro ont été établies. Actuellement, la plus grande collection de bananier au monde (1080 accessions) est maintenue sous forme de méristèmes en prolifération active in vitro, en conditions de croissance ralentie au Laboratoire d'amélioration des espèces tropicales de K.U.Leuven (Katholieke Universiteit Leuven, en Belgique). La cryoconservation est une alternative intéressante puisque, pendant le stockage aux températures ultra basses (-196°C), tous les processus métaboliques, chimiques et physiques sont suspendus. Ainsi, les risques inhérents au repiquage de contamination, d'erreur humaine et d'apparition de variation somaclonale sont évités. Nous avons développé un système très simple de cryoconservation fondé sur une phase de préculture de deux semaines des méristèmes en prolifération active sur des milieux fortement enrichis en sucre, suivie d'une congélation rapide dans l'azote liquide. Ce protocole de cryoconservation a été appliqué avec succès à 20 accessions appartenant à différents groupes génomiques. Des plantes normales ont été régénérées pour chacune d'entre elles. Les fréquences de régénération ont varié de 7,4 pourcent à 68,9 pourcent, selon le cultivar. Le mode d'action de la phase de préculture sucrée est à l'étude, en tenant compte des capacités de cryoconservation des différents cultivars. Il s'avère que les teneurs en lipides, en sucres et en protéines des plants non précultivés et précultivés diffèrent (Résumé d'auteur).]
[Conservación del germoplasma de banano a través de técnicas criogénicas]
[Ya que la mayoría de las razas indígenas de banano (Musa spp.) no produce semillas y se propaga vegetativamente, el germoplasma debe mantenerse de manera clonal. Por esta razón, se establecieron colecciones de campo e in vitro. Actualmente, la colección de banano más grande del mundo (1080 accesiones) se mantiene en forma de meristemas proliferantes in vitro bajo condiciones de crecimiento limitado en el Laboratorio de Mejoramiento de Cultivos Tropicales de la Universidad Católica de Lovaina (Katholieke Universiteit Leuven, Bélgica). La crioconservación es una alternativa bien recibida, ya que durante el almacenamiento a temperaturas ultrabajas (-196 °C), todos los procesos metabólicos, químicos y físicos se detienen. Por lo tanto, el material bajo almacenamiento no necesita subcultivarse, evitando de esta manera los riesgos de contaminación y error humano, y no aparecerá variación somaclonal. Hemos desarrollado un sistema de crioconservación extremadamente sencillo, que consiste de una fase de precultivo de meristemas altamente proliferantes de dos semanas en un medio con elevadas concentraciones de azúcar, seguida por una congelación rápida en nitrógeno líquido. Este protocolo de crioconservación fue examinado exitosamente en 20 accesiones pertenecientes a diferentes grupos genómicos. Se regeneraron plantas normales de cada una de ellas. Las frecuencias de regeneración varían de 7.4 a 68.9 porciento, dependiendo del cultivar. Se investiga el modo de actuar de la fase de precultivo en azúcar con respecto a la habilidad de crioconservación de diferentes cultivares. Se demostró que los contenidos de lípidos, azúcares y proteínas de las plantas precultivadas en azúcar y sin la fase de precultivo difieren (Resumen del autor).] Hide full abstract View article on publisher's site
In: Schoofs, H., Reyniers, K.,
Panis, B.,
Swennen, R. . Cellular biology and biotechnology including mutation techniques for creation of new useful banana genotypes: Report of the second research co-ordination meeting of FAO/IAEABADC co-ordinated research project - Kuala Lumpur, Malaysia, 13-17 October 1997
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, 1998
A set of twenty-one different banana cultivars covering all possible diploid and triploid genome groups and including the most important edible Musa types was selected for an in-depth study of the embryogenic pathway from scalps and its stumbling blocks. Embryogenic cell material was found to arise… Show full abstract from the starchy rich cells in the peripheral layers of meristematic globules. It started by the formation of distinct somatic embryos and most often resulted in the establishment of rapidly growing embryogenic cell cultures after 6 to 16 weeks. Out of the set of twenty-one cultivars, eighteen cultivars finally produced embryogenic cell cultures. Embryogenic cell suspensions could be initiated for fifteen among them. Competence of scalps for somatic embryogenesis was not genome dependent given that scalps were prepared from highly proliferating meristem cultures.
[Origine des cellules embryogéniques chez les Musa]
[On a sélectionné 21 cultivars de bananier différents, représentant tous les groupes génomiques diploïdes et triploïdes possibles et incluant les principaux types de Musa comestibles, afin de procéder à une étude approfondie de l'itinéraire de l'embryogénèse à partir des scalps et d'identifier les pierres d'achoppement. Il a été constaté que le matériel cellulaire embryogénique naissait des cellules riches en amidon situées dans les couches périphériques des globules méristématiques. Il y avait tout d'abord formation d'embryons somatiques distincts, qui donnaient le plus souvent lieu à l'établissement de cultures cellulaires embryogéniques à croissance rapide au bout de 6 à 16 semaines. Sur les 21 cultivars étudiés, 18 ont finalement produit des cultures cellulaires embryogéniques. Chez une partie des suspensions cellulaires embryogéniques établies, on a testé en serre la conformité des plants en phase végétative.]
[Origen de células embriogénicas en Musa]
[Una serie de veintidos cultivares de banano que cubren todos los posibles grupos genómicos diploides y triploides e incluyen los tipos comestibles más importantes de Musa, fueron seleccionados para un estudio profundo del método embriogénico a partir de los cortes, así como sus inconvenientes. Se descubrió que el material de las céluas embriogénicas creció a partir de las células ricas en almidón en las capas periféricas de los glóbulos meristemáticos. Este proceso empezó formando distintos embriones somáticos y, en la mayoría de las veces, dio como resultado el establecimiento de cultivos de células embriogénicas que crecían rápidamente entre las semanas 6 a 16. De la serie de 21 cultivares, dieciocho de ellos produjeron finalmente cultivos de células embriogénicas. La conformidad de algunas plantas procedentes de suspensiones de células embriogénicas establecidas se examinó a nivel vegetativo en el invernadero.] Hide full abstract View PDF
Surga Rivas, J.G.;
Swennen, R.;
Panis, B.In: Guzmán Chaves, J.A. (ed.). .
XII ACORBAT meeting, Santo Domingo, Dominican Republic, 27 October-2 November, 1996
ACORBAT, 1998
In order to optimise the regeneration of banana meristems, the cryopreservation technique developed by Panis (1995) was applied to four banana cultivars: 'Bluggoe' (ABB group), 'Dominico Harton' (AAB group), 'Kisubi' (AB group) and 'Grande Naine' (AAA group). Two types of regeneration media (liquid… Show full abstract and semisolid medium) at two sucrose concentrations (0.1 end 0.2M) were compared. The best results were always obtained for the cv. 'Bluggoe', followed by 'Dominico Harton', 'Kisubi' and ''Grande Naine''. Highest viability rates were obtained for 'Bluggoe' (67.8 percent) and ''Grande Naine'' (23.7 percent) using a liquid regeneration medium containing 0.2 M sucrose. For 'Dominico Harton' (35 percent) and 'Kisubi' (29.5 percent), the liquid regeneration medium with 0.1 M sucrose was optimal.
[Dans le but d'optimiser la régénération des explants de Musa spp., la technique de cryoconservation développée par Panis (1995) a été appliquée sur quatre cultivars, 'Bluggoe' (groupe ABB), 'Dominico Harton' (groupe AAB), 'Kisubi' (groupe AB) et 'Grande Naine' (groupe AAA). Deux types de supports de milieux (liquide et semi-solide), chacun d¦eux enrichi en saccharose à deux concentrations (0,1 et 0,2M), ont été essayés. Les réactions des explants traités ont suivi dans tous les cas le même ordre de variation du taux de survie. Les meileurs résultats ont été obtenus avec 'Bluggoe' suivis de 'Dominico Harton', 'Kisubi' et 'Grande Naine'. Les meilleurs taux de survie ont été observés pour 'Bluggoe' (67,8 pourcent) et 'Grande Naine' (23.7 pourcent) en milieu liquide aux concentrations en saccharose de 0,2M et 'Dominico Harton' (35 pourcent) et 'Kisubi' (29,5) à la concentration en saccharose de 0.1 M.]
[Con el objeto de buscar progreso en la regeneración de meristemos de Musa spp, la técnica de la criopreservación desarrollada por Panis, fue aplicada en cuatro cultivaressrzs 'Bluggoe' (grupo ABB), 'Dominico Hartón' (grupo AAB), 'Kisubi' (grupo AB) y 'Gran Enano' (grupo AAA). Dos tipos de soportes (liquido y semi sólido) en el medio de regeneración y dos concentraciones en sacarosa (0.1 y 0.2M) fueron comparados. Los mejores resultados fueron siempre obtenidos en el 'Bluggoe' seguido por 'Dominico Hartón', 'Kisubi' y ''Gran Enano'. Las tasas más altas de viabilidad fueron obtenidas para 'Bluggoe' (67,8 porciento) y 'Gran Enano' (23,7 porciento) cuando se usó medio de regeneración liquido conteniendo 0,2M de sacarosa. Para los clones 'Dominico Hartón' (35 porciento) y 'Kisubi' (29,5 porciento) el medio de regeneración líquido con 0,1 M de sacarosa fue el óptimo.] Hide full abstract View PDF
In: Panis, B. . INIBAP annual report 1997
INIBAP, 1998
Previous research has shown that the starting material for successful cryopreservation must exhibit a high proliferation. This type of material is the same as the required for the initiation of embryogenic cell suspensions. The simple cryopreservation protocol developed at the KUL is based on a… Show full abstract preculture of proliferating meristems on media containing high (0,4 M) sucrose concentrations. After two weeks, surviving tissues are excised and rapidly frozen in liquid nitrogen for storage. It has been show that the post-thaw viability rate is very genotype dependent, ranging from 10 to 67 percent. The pattern of regrowth is also cultivars specific, with some cultivars producing meristems directly without an intermediate callus phase, while others produced only non-regenerate callus. Finally, viability rates between repetitions of the same accession also varied considerably.
[Cryoconservation, une opportunité de conservation à long terme du matériel génétique de #Musa#. Des recherches antérieures ont montré que, pour une cryoconservation réussie, le matériel de départ devait être en prolifération active. Ce type de matériel est identique à celui requis pour l'initiation de suspensions cellulaires embryogéniques. Le protocole simple de cryoconservation développé à la KUL se fonde sur une pré-culture de méristèmes en prolifération active sur un milieu contenant des concentrations de sucrose élevées (0,4M). Après deux semaines, les tissus survivants sont excisés et congelés rapidement dans l'azote liquide pour le stockage. Il s'avère que le taux de viabilité post congélation dépend beaucoup du génotype, allant de 10 pourcent à 67 pourcent. Le processus de reprise de la croissance est aussi spécifique du cultivar ; certains cultivars produisant directement des méristèmes sans phase de cal intermédiaire, d'autres produisant seulement des cals non régénérants. Enfin, les taux de viabilité des répétitions pour une même accession varient considérablement.]
[Crioconservación, una oportunidad para la conservación del germoplasma de #Musa# a largo plazo. Las investigaciones previas han mostrado que el material inicial para una crioconservación exitosa debe mostrar un alto nivel de proliferación. Este tipo de material es el mismo que se requiere para la iniciación de las suspensiones de células embriogénicas. El protocolo de crioconservación, muy sencillo, desarrollado en la KUL, está basado en un precultivo de los meristemas proliferantes en un medio que contiene altas concentraciones de sacarosa (0,4 M). Después de dos semanas, los tejidos sobrevivientes se recortan y se congelan rápidamente en nitrógeno líquido para su almacenamiento. Se demostró que la tasa de viabilidad después de la descongelación depende del genotipo y varía de 10 a 67 por ciento. El patrón de un nuevo crecimiento también es específico del cultivar, donde algunos cultivares producen meristemas directamente sin una fase intermedia de callos, mientras que otros producen solo callos no regenerados. Finalmente, las tasas de viabilidad entre las repeticiones de la misma accesión también varían considerablemente.] Hide full abstract View PDF
Schoofs, H.;
Panis, B.;
Swennen, R.In: Galán Saúco, V. (ed.). Acta Horticulturae 490.
First International Symposium on Banana in the Subtropics, Puerto de la Cruz, Tenerife, Spain, 10-14 November 1997
ISHS, 1998
During the past 5 years, much progress has been made in the establishment of embryogenic cell cultures in Musa. Explants: such as seeds and male flowers have limited value because only wild bananas produce seeds and male flowers cannot be derived from 'Horn' and 'False Horn' plantains. Scalps,… Show full abstract somatic explants derived from in vitro proliferating meristem cultures, can however be generated from any given banana or plantain landrace (Musa spp.). At the Laboratory of Tropical Crop Improvement a widely applicable methodology has been developed for the initiation of embryogenic cell suspensions using such explants. A preculture of meristems on a medium with 100 microM N6-benzylaminopurine was found to enhance significantly the competence for somatic embryogenesis. Out of 21 landraces covering all diploid and triploid genome groups and the most important banana types, 18 cultivars showed an embryogenic response. From 15 accessions an embryogenic cell suspension could be established. Embryogenic frequencies of responding accessions varied between 15 and 80 percent. For some of the cultivars the induction period was reduced by 8-10 weeks as compared with the protocol without preculture. Less than 2 percent off-types were observed at the vegetative level but complete evaluation is proceeding in the field. Many of the cell suspensions were cryopreserved and used in genetic transformation programs. (Author's abstract).
[Capacité des explants pour l'embryogenèse somatique chez Musa]
[Durant les cinq dernières années, d'énormes progrès ont été accomplis dans l'établissement de cultures de cellules embryogènes chez Musa. Les explants tels que les graines et les fleurs mâles ont une valeur limitée puisque seuls les bananiers sauvages produisent des graines et que des fleurs mâles ne peuvent provenir des bananiers plantain 'Horn' et 'False Horn'. Des scalps, explants somatiques provenant de cultures méristématiques in vitro en prolifération active, peuvent cependant être obtenus à partir de n'importe quelle race locale de bananier ou de bananier plantain (Musa spp.). Une méthodologie largement applicable a été établie pour l'initiation de suspensions cellulaires embryogènes à partir de tels explants. Une pré-culture de méristèmes sur un milieu additionné de N6-Benzylaminopurine 100 µM a augmenté significativement la potentialité d'embryogenèse somatique. Sur les 21 variétés rassemblant tous les groupes génomiques diploïdes et triploïdes et les types de bananiers les plus importants, 18 cultivars ont présenté une réponse embryogénique. Une suspension cellulaire embryogène a pu être établie à partir de 15 accessions. Les fréquences embryogènes de ces accessions varient de 15 à 80 pour cent. Pour certains des cultivars, la période d'induction s'est trouvée réduite de 8 à 10 semaines par rapport au protocole sans pré-culture. Moins de 2 pour cent de variants ont été observés au niveau végétatif et une évaluation complète se poursuit au champ. La plupart des suspensions cellulaires ont été cryoconservées et utilisées dans des programmes de transformation génétique (Résumé d'auteur).]
[Competencia de tejidos para la embriogénesis somática en Musa]
[Durante los últimos 5 años, se avanzó bastante en el establecimiento de los cultivos de células embriogénicas en Musa. Los explantes como semillas y flores masculinas tienen un valor limitado ya que sólo los bananos silvestres producen semillas, y las flores masculinas no pueden ser derivadas de los plátanos 'Cuerno' y 'Falso Cuerno'. Sin embargo, los tejidos, explantes somáticos derivados de los cultivos in vitro de meristemas en proliferación, pueden ser generados a partir de cualquier banano o plátano (Musa spp.)dados. En el Laboratorio de Mejoramiento de Cultivos Tropicales se ha desarrollado una metodología de amplia aplicación para la iniciación de las suspensiones de células embriogénicas utilizando estos explantes. Se descubrió que un precultivo de meristemas en un medio de 100 microM de N6-benzilaminopurina mejora significativamente la competencia por la embriogénesis somática. De las 21 especies indígenas que cubren todos los grupos genómicos diploides y triploides y los tipos más importantes de banano, 18 cultivares mostraron una respuesta embriogénica. De 15 accesiones se pudo establecer una suspensión de células embriogénicas. Las frecuencias embriogénicas de las accesiones que respondieron variaron entre 15 y 80 por ciento. Para algunos de los cultivares el período de introducción se redujo en 8-10 semanas en comparación con el protocolo sin precultivo. Se observó menos de 2 por ciento de tipos anormales a nivel vegetativo, pero la evaluación completa se está realizando en el campo. Muchas de las suspenciones celulares fueron criopreservadas y utilizadas en los programas de transformación genética. (Resumen del autor).] Hide full abstract View article on publisher's site
Panis, B.; Peeters, M.C.;
Swennen, R.;
African Crop Science Conference Proceedings, 1997
The cultivation of banana (Musa) spp. and cotton (Gossypium spp.) is threatened by several pests and diseases. It is therefore important to develop more resistant varieties and thus to store safely germplasm needed for breeding. The world's largest Musa germplasm collection under the auspices of… Show full abstract Food and Agriculture Organisation (FAO) is currently stored at the INIBAP transit centre (Laboratory of Tropical Crop Improvement, K.U.Leuven, Belgium) as proliferating in vitro meristems under slow growth conditions. In addition to this active collection, a base collection using cryopreservation is under development. As such, the material can be preserved for an unlimited period. In theory, any regenerable tissue is suitable for storage of germplasm tbrough cryopreservation. We developed cryopreservation protocols for embryogenic cell suspension cultures of tbe monocot, banana and the dicot, cotton and in vitro meristems of banana. A secure storage of true-to-type and morphogenic competent cell suspensions is important since it is the material of choice for genetic engineering. The cryopreservation procedure for cell suspensions involves slow freezing (1 °C/min) in the presence of a cryoprotective solution (dimethylsulphoxide). In view of germplasm preservation, cryopreservation of in vitro meristems holds great promise, since no suspensions need to be initiated for each accession. Banana meristems can be cryopreserved using a simple and fast protocol which does not rely on sophisticated equipment. It involves a preculture on MS media containing high concentrations of sucrose followed by rapid freezing. This protocol has successfully been applied to 18 accessions belonging to distinct Musa groups (AA, BB, AB, AAA, AAB, AAB plantain and ABB) (Author's abstract).
[Cryoconservation du bananier et du cotonnier : un outil pour la sécurité de la conservation du matériel génétique]
[La culture du bananier (Musa spp.) et du coton (Gossypium spp.) est menacée par plusieurs maladies et ravageurs. Il est donc nécessaire de développer des variétés plus résistantes et de conserver sans risque le matériel génétique. A l'heure actuelle, la plus grande collection mondiale de Musa est conservée in vitro sous forme de méristèmes proliférants en conditions de croissance ralentie au centre de transit de L'INIBAP (Laboratoire des Plantes Tropicales, K.U.Leuven). Malgré l'obtention d'une collection active, une collection de base est préférable. On peut ainsi conserver le matériel pendant des périodes illimitées. En théorie, tous les tissus régénérables sont utilisables pour la cryoconservation. Nous avons développé des protocoles de cryoconservation pour des suspensions cellulaires embryogéniques du monocotylédone, banane et du dicotylédone, coton et pour des méristèmes in vitro du bananier. Le stockage sans risque des suspensions est très important parce qu' elles servent comme matériel de départ pour le génie génétique. Les cellules embryogéniques peuvent être conservées dans l'azote liquide après une congélation lente en présence d'une solution cryoprotectrice (diméthylsulphoxide). Pour conserver une banque de germplasm, la cryoconservation de méristèmes in vitro est très prometteuse, parce qu' ainsi on n'est pas obligé d'initier des suspensions cellulaires pour chaque nouvelle acquisition. Les méristèmes du bananier peuvent être cryoconservés en utilisant un protocole simple et rapide qui n'a pas besoin d'équipement sophistiqué. Cette méthode implique une phase de préculture du bouillon MS contenant des concentrations élevées en saccharose, suivie par une congélation rapide dans l'azote liquide. Ce protocole a été appliqué avec succès sur 18 variétés appartenant à des groupes distincts de Musa (AA, BB, AB, AAA, AAB, AAB plantain et ABB) (Résumé d'auteur).]
[Crioconservación del banano y algodón: una herramienta para la conservación segura de germoplasma]
[El cultivo de banano (Musa) spp. y algodón (Gossypium spp.) es amenazado por varias plagas y enfermedades. Por lo tanto, es importante desarrollar variedades más resistentes y almacenar el germoplasma necesario para el mejoramiento de manera segura. La colección de germoplasma de Musa más grande del mundo bajo los auspicios de la Organización para la Alimentación y Agricultura (FAO), se mantiene actualmente en el Centro de Tránsito de INIBAP (Laboratorio de Mejoramiento de Cultivos Tropicales, Universidad Católica de Lovaina, Bélgica) en forma de meristemas proliferantes in vitro bajo condiciones de crecimiento lento. En adición a esta colección activa, se está desarrollando una colección de base utilizando la crioconservación. Por lo tanto, el material puede ser conservado por un período ilimitado. En teoría, cualquier tejido regenerable es adecuado para el almacenamiento de germoplasma a través de crioconservación. Hemos desarrollado protocolos de crioconservación para los cultivos de suspensiones de células embriogénicas de las monocotiledóneas, banano y dicotiledóneas, algodón y meristemas in vitro de banano. Un almacenamiento seguro de las suspensiones celulares de tipos normales y competentes, desde el punto de vista morfogénico, es muy importante, ya que este es el material de selección para la ingeniería genética. El procedimiento de crioconservación para las suspensiones celulares involucra la congelación lenta (1 °C/min) en presencia de una solución crioprotectora (dimetilsulfóxido). En vista de la conservación de germoplasma, la crioconservación de los meristemas in vitro mantiene una gran promesa, ya que no es necesario iniciar una suspensión para cada accesión. Los meristemas de banano pueden ser crioconservados utilizando un protocolo sencillo y rápido que no necesita de un equipo sofisticado. Este protocolo involucra un precultivo en el medio MS que contiene altas concentraciones en sacarosa.] Hide full abstract
Panis, B.; Totté, N.; Van Nimmen, K.; Withers, L.A.;
Swennen, R.;
Plant Science, 1996 |
Peer Reviewed
A simple cryopreservation method is described for the long term- conservation of banana meristem cultures. It involves preculturing the proliferating meristems for 2-4 weeks on MS medium, enriched with 0.3-0.5 M sucrose. Surviving meristematic clumps are then excised and transferred to cryotubes… Show full abstract and plunged directly into liquid nitrogen for storage. The protocol was initially optimized for meristem cultures cf the cultivar 'Bluggoe' (Musa spp., ABB group) and gave a viability rate of up to 42.5 percent. A reduction of the moisture content by exposure of the meristematic clumps to the sterile air flow of a laminar air flow bench did not result in an increased survival rate. This cryopreservation method was tested on seven banana cultivars belonging to different genomic groups and resulted in viability rates between 12 and 72 percent depending on the cultivar. Microscopical observations revealed that only the most meristematic parts cf the proliferating clumps survived freezing (Author's abstract).
[Une méthode simple de cryoconservation est décrite pour la conservation à long terme des cultures méristématiques de bananier. Elle implique de précultiver les méristèmes en prolifération active pendant 2-4 semaines sur un milieu MS enrichi de sucrose à 0,3-0,5 M. Les amas méristématiques survivants sont ensuite excisés et transférés dans des cryotubes et plongés directement dans l'azote liquide pour le stockage. Le protocole a été, au début, optimisé pour des cultures méristématiques du cultivar 'Bluggoe' ( Musa spp., groupe ABB) et a donné un taux de viabilité jusqu'à 42,5 pourcent. Une réduction de la teneur hydrique par l'exposition des amas méristématiques au courant d'air stérile d'une hotte à flux laminaire n'a pas accru le taux de survie. Cette méthode de cryoconservation a été appliquée sur sept cultivars de bananier appartenant à différents groupes génomiques et a donné des taux de viabilité de 12 pourcent à 72 pourcent, selon le cultivar. Les observations microscopiques ont indiqué que seules les régions les plus méristématiques des amas en prolifération active avaient survécu à la congélation (Résumé d'auteur).]
[Se describe un sencillo método de crioconservación para una conservación a largo plazo de los cultivos de meristemas de banano. Este método involucra el precultivo de meristemas proliferantes durante 2-4 semanas en el medio MS, enriquecido con 0.3-0.5 M de sacarosa. Luego se extraen los agregados meristemáticos sobrevivientes, los cuales se transfieren a los criotubos y se sumergen directamente en nitrógeno líquido para su almacenamiento. El protocolo fue optimizado inicialmente para los cultivos de meristemas del cultivar 'Bluggoe' (Musa spp., grupo ABB) y dio una tasa de viabilidad de hasta 42.5 porciento. Una reducción del contenido de humedad mediante la exposición de los agregados meristemáticos a una corriente de aire esterilizado proveniente de una banca de flujo de aire laminar, no resultó en una tasa de supervivencia más alta. Este método de crioconservación fue examinado en siete cultivares de banano pertenecientes a diferentes grupos genómicos y dio como resultados tasas de viabilidad entre 12 y 72 porciento dependiendo del cultivar. Las observaciones microscópicas revelaron que sólo la mayoría de las partes meristemáticas de los agregados proliferantes sobrevivieron a la congelación (Resumen del autor).] Hide full abstract View article on publisher's site
Panis, B.;
Côte, F.; Escalant, J.V.;
Sági, L.In: Frison, E.A. (ed.), Horry, J.P. (ed.), De Waele, D. (ed.). New frontiers in resistance breeding for nematode, #Fusarium# and Sigatoka.
Workshop held in Kuala Lumpur, Malaysia, 2-5 October 1995
INIBAP, 1996
The availability of a good in vitro system is essential for genetic transformation. The main characteristic of such a system is that a normal true-to-type plant can be regenerated from the explant material at a high frequency. The following banana tissues possess this potential: meristems,… Show full abstract embryogenic cell suspensions, somatic embryos and protoplast cultures. Two categories of transformation systems can be distinguished. The first comprises the indirect transformation systems of which Agrobacterium-mediated transformation is the most important one. Direct gene transfer techniques fall into the second group which comprises, among other techniques, particle bombardment and protoplast electroporation. The DNA fragments (plasmids) used for genetic transformation contain the following sequences: a reporter gene for visual detection of transient expression, a selectable marker gene coding for antibiotic or herbicide resistance, and agronomically useful gene(s). These genes need to be preceded by a promoter sequence which regulates their expression. Useful genes will give an additional value to banana for pests and diseases control.
[La disponibilité d'un bon système in vitro est essentielle pour la transformation génétique. La principale caractéristique d'un tel système est qu'un plant normal puisse être régénéré à partir d'explants à un taux élevé. Les tissus de bananier suivants possèdent ce potentiel: les méristèmes, les suspensions cellulaires embryogènes, les embryons somatiques et les cultures de protoplastes. Deux catégories de systèmes de transformation peuvent être distingués. Le premier comprend les systèmes de transformation indirecte parmi lesquels la transformation à l'aide d'Agrobacterium est le plus important. Les techniques de transfert direct de gènes font partie du second groupe qui comprend, parmi d'autres techniques, le bombardement de particules et l'électroporation du protoplaste. Les fragments d'ADN (plasmides) utilisés pour la transformation génétique contiennent les séquences suivantes: un gène signal pour la détection visuelle de l'expression transitoire, un gène marqueur sélectionnable codant pour une résistance à un antibiotique ou à un herbicide, et un ou des gènes agronomiquement utiles. Ces gènes doivent être précédés par une séquence promoteur qui régule leur expression. Des gènes utiles donneront une valeur additionnelle aux bananiers pour la lutte contre les ravageurs et les maladies.]
[La disponibilidad de un buen sistema in vitro es esencial para la transformación genética. La principal característica de tal sistema consiste en que una planta normal puede ser regenerada a partir del explante a una alta frecuencia. Los siguientes tejidos de banano poseen este potencial: meristemas, suspenciones de células embriogénicas, embriones somáticos y cultivos de protoplastos. Se puede distinguir dos categorías de sistemas de transformación. La primera de estas dos categorías comprende los sistemas de transformación indirecta, de los cuales la transformación con la ayuda de Agrobacterium es la más importante. Las técnicas de transferencia directa de genes caen en el segundo grupo que comprende, entre otras técnicas, el bombardeo de partículas y electroporación de protoplastos. Los fragmentos del ADN (plasmidos) utilizados para la transformación genética contienen las siguientes secuencias: un gen reportero para la detección visual de la expresión transitoria, un gen marcador seleccionable codificador para un antibiótico o resistencia a los herbicidas y los genes agronómicamente útiles. Estos genes deben ser precedidos por una secuencia promotora que regule su expresión. Los genes útiles darán un valor adicional al banano respecto al control de plagas y enfermedades.] Hide full abstract View PDF
Côte, F.;
Bakry, F.; Grapin, A.;
Teisson, C.; Escalant, J.V.; Buin Trang, V.; Haïcour, R.; Rossignol, L.;
Panis, B.; Schoofs, H.;
Swennen, R.In: Soto, M.V. (ed.). .
XI ACORBAT meeting, San José, Costa Rica, 1994/02/13-18
ACORBAT, 1995
As complement of the genetic breeding programs, biotechnologies open two great perspectives: the production of hybrids via protoplasts fusion and the obtention of transgenic plants. Two main methods are being studied in order to establish cell suspensions. The first one utilizes as initial material… Show full abstract male flowers and the second one proliferating apexes. Embryogenic callus were obtained by bath methods. At the present time, domain of suspensions and plants regeneration is attained totally or in part: in the case of cv. 'French Sombre', 'Grand Naine', and in the Musa acuminata malaccensis diploid, through the first method, and for cv. 'Bluggoe', 'Cardaba', 'Saba', 'Three Hand Planty', 'Mysore' and Musa balbisiana TANI, utilizing the second method. The reaction with either method is slow (6 months or more) and varies according to the cultivars. Plants obtained are being evaluated in several sites for true-to-type characteristics. The isolation of protoplasts suitable for division, was obtained from diploid (cv. 'Long Tavoy', M. acuminata banksii, M. acuminata malaccensis) and triploid bananas (cv. 'Matavia'). The employment of nursing cells and the protoplasts density are determinant elements for protoplasts development. It was possible to regenerate plants from the cv. 'Matavia'. The protoplasts fusion between diploids and triploids leads to callus and organized structures formation. The introduction of foreign genes by particles acceleration on protoplasts and cell suspensions was also studied. It was possible to obtain a transitory expression of the GUS gene on several cultivars: 'French Sombre', 'Matavia', 'Long Tavoy' and M. acuminata banksii.
[En complément des programmes d'amélioration génétique, les biotechnologies ouvrent deux grandes perspectives: la production d'hybrides somatiques par fusion de protoplastes et celle de plantes transgéniques. Deux principales méthodes d'établissement des suspensions cellulaires sont étudiées. La première utilise comme matériel initial les fleurs mâles, la seconde des apex en prolifération. Des cals embryogènes de plusieurs cultivars diploïdes et triploïdes ont été obtenus par les deux méthodes. La maîtrise des suspensions et la régénération de plantes sont actuellement acquises totalement ou en partie chez les cultivars 'French Sombre', 'Grande Naine' et le diploïde Musa acuminata malaccensis par la première méthode et pour les cultivars 'Bluggoe', 'Cardaba', 'Saba', 'Three Hand Planty', Musa balbisiana 'TANI', 'Mysore' par la seconde méthode. Dans les deux méthodes il a été mis en évidence que la réactivité du matériel est très différente d'un cultivar à un autre et que le délai d'établissement des suspensions est long (6 mois et plus). Plusieurs essais concernant la conformité du matériel produit sont en cours de réalisation. L'isolement des protoplastes aptes à se diviser a été obtenu à partir des bananiers diploïdes (cv. 'Long Tavoy', M. acuminata banksii, M. acuminata malaccensis) et triploïdes (cv. 'Matavia'). L'utilisation de cellules nourricières et d'une forte densité de protoplastes en culture sont des éléments déterminants du développement des protoplastes. La régénération de plantes a été obtenue chez le cv. 'Matavia'. Des essais de fusion ont été réalisés entre protoplastes diploïdes et entre di- et triploïdes. Ils ont conduit à la formation de cals et de structures organisées. L'introduction de gènes exogènes par accélération de particules a été étudiée sur deux types de matériels: les suspensions cellulaires et les protoplastes. Des expressions transitoires du gène GUS ont été obtenues sur plusieurs cultivars.]
[Suspensiones embriogénicas y protoplastos de bananos: resultados y perspectivas de utilización para el mejoramiento genético]
[Como complemento a los programas de mejoramiento genético, las biotecnologías abren dos grandes perspectivas: la producción de híbridos por fusión de protoplastos y la obtención de plantas transgénicas. Se están estudiando dos principales métodos para el establecimiento de suspensiones celulares. El primera utiliza como material inicial las flores masculinas, el segundo, ápices en proliferación. Se obtuvieron callos embriogénicas par ambos métodos. En la actualidad el dominio de suspensiones y la regeneración de plantas son dominados totalmente o en parte: por el primer método en los cv. 'French Sombre', 'Grande Naine' y en el diploide Musa acuminata malaccensis y por el segundo método en los cv. 'Bluggoe', 'Cardaba', 'Saba', 'Three Hand Planty', 'Mysore' y por Musa balbisiana TANI. Por ambos métodos la reacción es lenta (6 meses y más) y varia según los cultivares. La conformidad de las plantas obtenidas se está evaluando en varios sitios. El aislamiento de los protoplastos, aptas a dividirse, se obtuvo a partir de bananos diploides (cv. 'Long Tavoy', M. acuminata banksii M. acuminata malaccensis) y a partir de triploides (cv. 'Matavia'). El usa de células nodrizas y la densidad de protoplastos son elementos determinantes en el desarrollo de los protoplastos. Se logró regenerar plantas a partir del cv. 'Matavia'. La fusión de protoplastos entre diploides y triploides conduce a la formación de callos y estructuras organizadas. Se estudió la introducción de genes foráneos por aceleración de partículas sobre protoplastos y suspensiones de células. Se pudo obtener una expresión transitoria del gen GUS sobre varios cultivares: 'French Sombre', 'Matavia', 'Long Tavoy', M. acuminata banksii.] Hide full abstract View PDF
Sági, L.;
Panis, B.; Remy, S.; Cammue, B.P.A.;
Swennen, R.In: Soto, M.V. (ed.). .
XI ACORBAT meeting, San José, Costa Rica, 1994/02/13-18
ACORBAT, 1995
A simple protocol was developed to allow the production of transgenic banana. Embryogenic cell suspensions (Musa spp. cv. 'Bluggoe') were bombarded with accelerated particles coated with DNA of foreign genes. Bombarded cells were proliferated under the selection of hygromycin and regenerated into… Show full abstract plants. Histochemical and molecular characterization of transformants demonstrated that foreign genes were present and expressed in the plants. Bombardment parameters were partiality epitomized for a modified flowing helium gun resulting in high levels of transient expression of the uidA gene (up to 1000 blue foci/25 mg cells/shot in cv. 'Bluggoe') in banana and also plantain cells. Electroporation conditions were also established for transient expression of introduced DNA in banana protoplasts isolated from regenerable embryogenic cell suspensions of cv. 'Bluggoe'. The following parameters were found to be important: electroporation buffer, polyethylene glycol treatment, use of a heat shock, and chimaeric gene constructs. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8 percent of total protoplasts. Since plants have recently been regenerated from banana protoplasts at a highly frequency, these results may contribute to the production of transgenic banana also from protoplasts.
[Un protocole simple permettant l'obtention de bananiers transgéniques a été développé. Pour cela, des suspensions cellulaires embryogènes (Musa spp. cv. 'Bluggoe') sont bombardées avec des particules accélérées recouvertes d'ADN de gènes exogènes. Les cellules bombardées sont sélectionnées avec de l'hygromycine et régénérées en plantes. La caractérisation moléculaire et histochimique des transformants permet de confirmer une intégration stable des gènes étrangers. Les paramètres de bombardement ont été partiellement optimisés pour un pistolet à particules modifié, permettant d'obtenir un renforcement de l'expression transitoire du gène uidA (jusqu'à 400 focus/point bleu) sur des cellules de bananier et bananier plantain. Les conditions d'électroporation ont été établies pour l'expression transitoire de l'ADN dans des protoplastes de bananier isolés de suspensions cellulaires embryogènes du cv. 'Bluggoe'. Les paramètres suivants se sont révélés important: le tampon de l'électroporation, le traitement au polyéthylène glycol et sa durée avant l'électroporation, l'utilisation d'un choc thermique et les constructions de gènes chimériques. La fréquence maximum d'introduction de l'ADN, détectée à travers un essai in situ de l'expression transitoire du gène uidA, atteint 1.8 pourcent du total des protoplastes. Du fait que ces plantes venaient d'être régénérées de protoplastes de bananier, ces résultats peuvent contribuer à la production des bananiers transgéniques à l'aide des protoplastes.]
[Se ha desarrollado un protocolo simple para permitir la producción de banano transgénico. Para tal fin se bombardearon suspensiones celulares embriogénicas (Musa spp. cv. 'Bluggoe') con partículas aceleradas cubiertas con ADN de genes externos. Las células bombardeadas fueron seleccionados con higroquímicina y regeneradas en plantas. La caracterización molecular e histoquímica de los transformantes reveló que los genes externos se integraron establemente. Los parámetros de bombardea fueron optimizados parcialmente para una pistola de flujo interna de partículas modificadas, dando como resultado niveles más altos de expresión transitoria del gen uidA (hasta 400 foci/shot azules) en células de banano y plátano. Las condiciones de electroporación fueron establecidas para la expresión transitoria del ADN introducida en los protoplastos de banano, aislados de suspensiones celulares embriogénicas regenerables del cv. 'Bluggoe'. Se encontró que los parámetros siguientes son importantes: el buffer de electroporación, el tratamiento de glicol polietileno y su duración antes de la electroporación, el usa de shock de calor y las construcciones de genes quiméricos. La frecuencia máxima de introducción de ADN detectada mediante un ensayo in situ de la expresión transitoria del gen uidA, alcanzó 1.8 porciento del total de protoplastos. Debido a que las plantas habían sido regeneradas recientemente de los protoplastos de banano a una alta frecuencia, estas resultados pueden contribuir también a la producción de banano transgénico de protoplastos.] Hide full abstract View PDF
Panis, B.; De Smet, K.;
Van Den Houwe, I.;
Swennen, R.In: Soto, M.V. (ed.). .
XI ACORBAT meeting, San José, Costa Rica, 1994/02/13-18
ACORBAT, 1995
The largest in vitro Musa germplasm collection (more than 1000 accessions) is located at the INIBAP (International Network for the Improvement of Banana and Plantain) Transit Centre at K.U. Leuven. Proliferating meristems are maintained under minimal growth conditions necessitating on average about… Show full abstract one annual subculture. Cryopreservation would avoid the occurrence of somaclonal variation and eliminate the need for routine subculture. The cryopreservation of embryogenic cell suspension cultures of five cultivars is reported. This is of outermost importance since true-to-type suspension cultures are the material of choice for genetic manipulation. Also preliminary results on the cryopreservation of in vitro meristems using various methods (slow freezing, vitrification and encapsulation-dehydration) are described.
[La plus grande collection de germplasm in vitro de Musa (plus de 1000 introductions), se trouve au centre de transit de l'INIBAP à l'Université Catholique de Leuven. Les méristèmes en prolifération sont maintenus en conditions de croissance minimale ; cela exige au moins une sub-culture annuelle selon les cultivars. La cryoconservation de cultures embryogéniques a été étudiée sur des suspensions cellulaires de cinq cultivars. Les résultats montrent que la cryoconservation permet d'éviter l'apparition de variations somaclonales et d'éliminer la sub-culture routinière. Ces résultats sont d'une grande importance puisque les cultures conformes de suspensions constituent le matériel idéal pour les manipulations génétiques. Les résultats préliminaires obtenus par la cryoconservation de méristèmes in vitro en utilisant diverses méthodes (congélation lente, vitrification et encapsulation-déshydratation) sont décrits.]
[La colección más grande de germoplasma in vitro de Musa (más de 1000 introducciones), se encuentra ubicada en el centra de tránsito de INIBAP en la U.C. de Lovaina. Los meristemos en proliferación son mantenidos baja condiciones de crecimiento mínimas, requiriendo de un subcultivo anual, dependiendo de cultivar. La críoconservación detendría la aparición de variaciones somaclónales y eliminaría la necesidad del subcultivo rutinaria. Se informa acerca de la críoconservación de cultivo embriogénicas de suspensiones celulares en cinco cultivares. Lo anterior es de gran importancia puesto que las cultivas de suspensiones conformes san el material que se escoge para su manipulación genética. Además se describen los resultados preliminares sobre la críoconservación de meristemos in vitro utilizando diversos métodos (congelamiento lento, vitrificación y encapsulamiento-deshidratación).] Hide full abstract View PDF
Panis, B.;
Katholieke Universiteit Leuven, 1995
An efficient cryopreservation protocol for embryogenic banana suspensions was developed for the cooking banana 'Bluggoe' (ABB group). The older, and thus the more vacuolated the cells are, the lower the chance for recovering after thawing. Different viability rates were observed. It is concluded… Show full abstract that the capacity of a banana cell suspension to be cryopreserved in liquid nitrogen probably does not depend on its genotype but rather on the quality and the amount of embryogenic cells in suspension. To obtain survival of organized structures like proembryos, highly concentrated plant vitrification solutions were tested. Post-thaw survival was limited due to the toxicity of this solution and osmotic injury. Meristems excised from in vitro proliferating clumps were subjected to a slow freezing protocol similar to the one for cell suspensions. Regrowth capacity was totally lost and vitrifying solutions were inappropriate. The encapsulation-dehydration method resulted in a maximum post-thaw recovery of 8.1 percent. A simple cryopreservation method was applied to meristems of five banana cultivars belonging to distinct Musa groups and resulted in survival rates ranging from 6 percent to 42.5 percent.
[Un protocole efficace de cryoconservation de suspensions embryogènes de bananier a été développé pour le bananier de type à cuire 'Bluggoe' (groupe ABB). Plus les cellules sont âgées et donc plus vacuolées, moins les chances de récupération après décongélation sont élevées. La méthode optimisée de cryoconservation a été appliquée à des suspensions d'autres cultivars. Différents taux de viabilité ont été observés. La conclusion est que la capacité d'une suspension cellulaire de bananier à être cryoconservée dans de l'azote liquide ne dépend probablement pas de son génotype mais plutôt de la qualité et du taux de cellules embryogènes en suspension. Afin d'obtenir la survie de structures organisées comme des pré-embryons, des solutions de vitrification végétale fortement concentrées ont été testées. La survie post-décongélation est limitée à cause de la toxicité de ces solutions et des dégâts osmotiques. Des méristèmes excisés d'amas cellulaires proliférant in vitro ont été soumis à un protocole de congélation lente similaire à celui utilisé pour les suspensions cellulaires. La capacité de croissance est perdue et les solutions vitrifiantes sont inappropriées. La méthode d'encapsulation-déshydratation a permi une récupération maximale post-décongélation de 8,1 pour cent. Une méthode simple de cryoconservation a été appliquée à des méristèmes de cinq cultivars bananiers appartenant à des groupes distincts de Musa, et a donné des taux de survie allant de 6 à 42,5 pour cent.]
[Un protocolo eficaz de crioconservación de suspensiones embriogénicas de banano fue desarrollado para el banano de cocción 'Bluggoe' (grupo ABB). Mientras más viejas y más vacuolizadas son las células, la probabilidad de recuperación después de la descongelación es más baja. Se observaron diferentes tasas de viabilidad. La conclusión es que la capacidad de una suspensión celular de banano para ser crioconservada en nitrógeno líquido no depende de su genotipo, sino más bien de la calidad y de la cantidad de células embriogénicas en la suspensión. Para conseguir la supervivencia de las estructuras organizadas, como los proembriones, fueron examinadas soluciones de vitrificación vegetal altamente concentradas. La sobrevivencia después de la descongelación fue limitada debido a la toxicidad de esta solución y al daño osmótico. Los meristemos extraídos de los grupos celulares proliferados in vitro, se sometieron a un protocolo de congelación lenta, similar al protocolo desarrollado para las suspensiones celulares. La capacidad de crecimiento fue totalmente perdida y las soluciones vitrificantes resultaron inapropiadas. El método de encapsulación-deshidratación resultó en una recuperación máxima después de la descongelación del 8.1 por ciento. Un método de crioconservación simple fue aplicado a los meristemos de cinco cultivares de banano pertenecientes a diferentes grupos de Musa que dio como resultado tasas de sobrevivencia entre 6 y 42.5 por ciento.] Hide full abstract View article on publisher's site
Sági, L.;
Panis, B.; Remy, S.; Schoofs, H.; De Smet, K.;
Swennen, R.; Cammue, B.P.A.;
Nature Biotechnology, 1995 |
Peer Reviewed
The authors developed a simple protocol to allow the production of transgenic banana plants. Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA. Bombardment parameters were optimized for a modified particle gun resulting in high levels of… Show full abstract transient expression of the bêta-glucuronidase gene in both banana and plantain cells. Bombarded banana cells were selected with hygromycin and regenerated into plants. Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.
[Transformation génétique des bananiers et bananiers plantain (Musa spp.) par bombardement de particules]
[Les auteurs ont développé un protocole simple afin de permettre la production de plants de bananier transgéniques. Des gènes étrangers ont été intégrés dans une suspension de cellules embryogènes en utilisant des particules accélérées recouvertes d'ADN. Les paramètres de bombardement ont été optimisés pour un canon à particules modifié, ce qui aboutit à des niveaux élevés d'expression transitoire du gène de la bêta-glucuronidase dans les cellules de bananier et de bananier plantain. Les cellules bombardées de bananier ont été sélectionnées à l'hygromycine et régénérées en plants. La caractérisation moléculaire et histochimique des transformants a révélé une intégration stable des gènes transférés dans le génome bananier.]
[Transformación genética de bananos y plátanos (Musa spp.) vía bombardeo de partículas]
[Los autores desarrollaron un protocolo simple para permitir la producción de plantas transgénicas de banano. Los genes foráneos fueron introducidos en las suspensiones de células embriogénicas utilizando partículas aceleradas recubiertas con ADN. Los parámetros de bombardeo fueron optimizados para un cañón de partículas modificado, lo que dio como resultado altos niveles de expresión transitoria del gen beta-glucuronidasa en las células de bananos y plátanos. Las células de banano bombardeadas fueron seleccionadas con higromicina y regeneradas en plantas. La caracterización molecular e histoquímica de los transformantes reveló la integración estable de los genes transferidos en el genoma de bananos.] Hide full abstract View article on publisher's site
Sági, L.; Remy, S.; Verelst, B.;
Panis, B.; Cammue, B.P.A.; Volckaert, G.;
Swennen, R.;
Euphytica, 1995 |
Peer Reviewed
In order to introduce currently-available genes with agronomical value into banana, two genetic transformation protocols have been optimized. Firstly, regenerable protoplasts isolated from embryogenic cell suspensions of the cultivar 'Bluggoe' have been used for the introduction of several… Show full abstract chimaeric uidA gene constructs by electroporation. With the inclusion of polyethylene glycol and heat shock, the frequency of transiently expressing protoplasts reached 1.8 percent. A duplicated 35S promoter with an alfalfa mosaic virus leader sequence induced the highest expression rate among the constructs tested. Embryogenic cell suspensions of cultivar 'Bluggoe' have also been bombarded with accelerated particles coated with a high expression uidA gene construct using a biolistic gun. After a partial optimization of the procedure, transient beta-glucuronidase assays reproducibly demonstrated the presence of 400 blue foci in 30 microlitres of settled cell volume (approximately 25 mg cells). Selection and characterization of antibiotic-resistant transformed cultures is in progress.
[Expression génétique transitoire chez des protoplastes et des suspensions de cellules embryogènes de bananier (Musa cv. Bluggoe) transformés.]
[Afin d'introduire dans le bananier des gènes à valeur agronomique actuellement disponibles, deux protocoles de transformation génétique ont été optimisés. Tout d'abord, des protoplastes régénérables isolés de suspensions cellulaires embryogènes du cultivar 'Bluggoe' ont été utilisés pour l'introduction de plusieurs gènes chimères uidA par électroporation. Avec l'inclusion de polyéthylène glycol et un choc thermique, la fréquence de protoplastes s'exprimant transitoirement atteint 1,8 pour cent. Un promoteur 35S dupliqué avec une séquence de tête de virus alfalfa de la mosaïque induit le taux d'expression le plus élevé parmi les chimères testées. Des suspensions de cellules embryogènes du cultivar 'Bluggoe' ont aussi été bombardées avec un canon biolistique par des particules accélérées recouvertes d'un gène chimère uidA à expression élevée. Après optimisation partielle de la procédure, les dosages de la bêta-glucuronidase révèlent de manière reproductible la présence de 400 foci bleu dans 30 microlitres de volume cellulaire formant un dépôt (environ 25 mg de cellules). La sélection et la caractérisation de cultures transformées résistant aux antibiotiques sont en cours.]
[Expresión genética transitoria en los protoplastos y suspensiones de células embriogénicas de banano (Musa cv. Bluggoe) transformados.]
[Para introducir genes de valor agronómico actualmente disponibles en los bananos, fueron optimizados dos protocolos de transformación genética. En primer lugar, se utilizaron los protoplastos regenerables, aislados de las suspensiones de células embriogénicas del cultivar 'Bluggoe', para introducir varios genes quimeras uidA mediante electroporación. Con la inclusión del polietileno glicol y choque eléctrico, la frecuencia de los protoplastos con expresión transitoria alcanzó un 1.8 por ciento. Un promotor 35S duplicado con una secuencia líder del virus del mosaico de alfalfa indujo la mayor tasa de expresión entre las quimeras examinadas. Las suspensiones de células embriogénicas del cultivar 'Bluggoe' también han sido bombardeadas a través de un cañon biolístico por partículas aceleradas, recubiertas con un gen quimera uidA de alta expresión. Después de una optimización parcial del procedimiento, las dosis de la beta-gluconasa revelaron de manera reproducible la presencia de 400 foci azules en 30 microlitros de volumen celular formado en un recipiente (aproximadamente, 25 mg de células). Actualmente, se realiza la selección y la caracterización de los cultivos transformados resistentes a los antibióticos.] Hide full abstract View article on publisher's site
Sági, L.; Remy, S.; Verelst, B.;
Swennen, R.;
Panis, B.In: Bajaj, Y.P.S. (ed.). Plant protoplasts and genetic engineering VI .
Springer, 1995
A simple protocol was developed to allow the production of transgenic banana. Embryogenic cell suspensions (Musa spp. cv. 'Bluggoe') were bombarded with particles coated with DNA of foreign genes. Transformed cells were selected with hygromycin and regenerated into plants. Histochemical and… Show full abstract molecular characterization of transformants demonstrated that foreign genes were present and expressed in the plants. Bombardment parameters were partially optimized for a modified flowing helium gun resulting in high levels of transient expression of the uidA gene in banana cells. Electroporation conditions were also established for transient expression of introduced DNA in banana protoplasts isolated from regenerable embryogenic cell suspensions of cv. 'Bluggoe'. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8 percent of total protoplasts. The present method directly provides a basis for functional analysis of promoters in banana.
[Transformation génétique chez des espèces de Musa (bananier)]
[Un protocole simple a été développé afin de permettre la production de bananiers transgéniques. Des suspensions de cellules embryogènes (Musa spp. cv. "Bluggoe") ont été bombardées avec des particules recouvertes d'ADN de gènes étrangers. Des cellules transformées ont été sélectionnées avec de l'hygromycine et régénérées en plantes. La caractérisation histochimique et moléculaire des transformants démontre que des gènes étrangers sont présents et s'expriment dans les plantes. Des paramètres de bombardement ont été partiellement optimisés pour un canon à flux d'hélium modifié résultant en des niveaux élevés d'expression transitoire du gène uidA dans les cellules de bananier. Des conditions d'électroporation ont aussi été établies pour l'expression transitoire d'ADN introduit dans des protoplastes de bananier isolés de suspensions de cellules embryogènes régénérables du cultivar "Bluggoe". La fréquence maximale d'introduction d'ADN, détectée par un dosage in situ de l'expression transitoire du gène uidA, atteint 1,8 pour cent du total des protoplastes. La méthode présente fournit directement une base pour l'analyse fonctionnelle des promoteurs chez le bananier.]
[Transformación genética en especies de Musa (banano)]
[Un simple protocolo fue desarrollado con el fin de permitir la producción de bananos transgénicos. Las suspensiones de células embriogénicas (Musa spp. cv. 'Bluggoe') fueron bombardeadas con partículas revestidas con ADN de genes foráneos. Las células transformadas fueron seleccionadas con la ayuda de higromicina y regeneradas en plantas. La caracterización histoquímica y molecular de las transformaciones demostró la presencia y expresión de los genes foráneos en las plantas. Los parámetros de bombardeo fueron parcialmente optimizados para un cañón de flujo de helio modificado, lo que dio como resultado niveles altos de expresión transitoria del gen uidA en las células de banano. Las condiciones de electroporación también fueron establecidas para la expresión transitoria del ADN introducido en los protoplastos de banano aislados de las suspensiones de células embriogénicas regenerables del cv. 'Bluggoe'. La frecuencia máxima de introducción de ADN detectada por una dosis in situ de la expresión transitoria del gene uidA, estuvo alrededor de 1.8 por ciento del total de los protoplastos. Este método brinda directamente una base para el análisis funcional de los promotores en los bananos.] Hide full abstract View article on publisher's site
Panis, B.;
Swennen, R.In: Bajaj, Y.P.S. (ed.). Cryopreservation of plant germplasm I.
Springer, 1995
A cryopreservation protocol was developed for embryogenic cell suspension cultures of Musa. Slow freezing (1 degree C/min) in the presence of 7.5 percent dimethylsulfoxide (DMSO) followed by storage in liquid nitrogen and rapid thawing results in high regrowth rates. The cells surviving the… Show full abstract cryopreservation protocol are able to regenerate into normal plants through somatic embryogenesis. The applicability of this method depends on the quality of the embryogenic cell suspension. The slow freezing method in the presence of DMSO proved to be unsuccessful for in vitro banana meristems. The encapsulation-dehydration method is a novel approach in which the material is frozen at ultra-rapid cooling rates and stored in the vitrified state. To date, results using the encapsulation-dehydration method are very poor since only 5 percent of the frozen banana meristems survived. A more promising technique, resulting in 50 percent survival, involves a 2-week preculture on high sucrose-containing media.
[Cryoconservation du matériel génétique de bananier et de bananier plantain (Musa species)]
[Un protocole de cryoconservation a été développé pour des suspensions de culture de cellules de Musa. Une lente congélation (1 degré C/min) en présence de 7,5 pour cent de diméthylsulfoxyde (DMSO) suivie par un stockage en azote liquide et une décongélation rapide a permis d'obtenir des taux élevés de reprise de croissance. Les cellules ayant survécu au protocole de cryoconservation sont capables de régénérer des plants normaux par embryogénèse somatique. L'application de cette méthode dépend de la qualité de la suspension de cellules embryogènes. Cette méthode de lente congélation en présence de DMSO s'est révélée être infructueuse pour des méristèmes in vitro de bananier. La méthode d'encapsulation-déshydratation est une nouvelle approche dans laquelle le matériel est congelé à un taux de refroidissement ultra-rapide et stocké au stade vitrifié. A ce jour, les résultats de la méthode d'encapsulation-déshydratation sont très faibles puisque seulement 5 pour cent des méristèmes congelés de bananier survivent. Une technique plus prometteuse, permettant d'obtenir 50 pour cent de survie, comporte une pré-culture de deux semaines sur milieux à taux de saccharose élevé.]
[Crioconservación de germoplasma de banano y plátano (Musa species)]
[Un protocolo de crioconservación fue desarrollado para las suspensiones de cultivos de células embriogénicas de Musa. Una congelación lenta (1 grado C/min) en presencia de 7.5 por ciento de dimetilsulfóxido (DMSO), seguida por un almacenamiento en nitrógeno líquido y una descongelación rápida, permiten obtener altas tasas de recuperación. Las células que sobreviven la crioconservación, son capaces de generarse en plantas normales a través de embriogénesis somática. La aplicación de este método depende de la calidad de la suspensión de células embriogénicas. El método de congelamiento lento en presencia del DMSO fue infructuoso para los meristemas in vitro de banano. El método de encapsulacióndeshidratación es un nuevo enfoque, donde el material se congela a tasas de enfriamiento ultrarrápidas y se almacena en estado vitrificado. Hasta la fecha, los resultados con la utilización del método de encapsulación-deshidratación han sido muy pobres, ya que sólo un 5 por ciento de los meristemas congelados de banano sobrevivieron. Una técnica más prometedora, que permite obtener un 50 por ciento de sobrevivencia, incluye un cultivo previo de dos semanas en un medio con alto contenido de sacarosa.] Hide full abstract View article on publisher's site
Panis, B.;
IPGRI Newsletter for Europe (ITA), 1995
The collection of Musa germplasm held at the INIBAP Transit Centre at K.U. Leuven (Katholieke Universiteit Leuven), Belgium, is currently stored as in vitro meristems under slow growth conditions. Initially, a technique was developed for cryopreservation of embryogenic banana cell suspensions of… Show full abstract different cultivars. More recently, cryopreservation research on banana has focused on meristem cultures since embryogenic cell suspension cultures are time consuming to initiate and are still difficult for some cultivars. A simple technique developed in the K.U. Leuven laboratory resulted in post-thaw recovery for all the cultivars tested. This involves a preculture step on solid media containing high sucrose levels, followed by plunging the material enclosed in cryotubes in liquid nitrogen (-196 degrees C). Survival rates ranged from 7 to 72 percent, depending on the cultivar.
[La collection de matériel génétique de Musa maintenue au Centre de transit de l'INIBAP à l'Université Catholique de Leuven (KUL) en Belgique, est actuellement stockée sous forme de méristèmes in vitro sous conditions de croissance lente. Initialement, une technique a été développée pour la cryoconservation de suspensions de cellules embryogènes de différents cultivars de bananiers. Plus récemment, les recherches sur la cryoconservation du bananier se sont concentrées sur les cultures de méristème puisque les cultures de suspensions de cellules sont longues à initier et encore difficiles pour certains cultivars. Une technique simple développée au laboratoire de KUL a permis d'obtenir une récupération post-décongélation pour tous les cultivars testés. Elle implique une étape de pré-culture sur milieux solides contenant des taux élevés de saccharose, suivie par l'immersion dans l'azote liquide (-196 degrés C) du matériel enfermé dans des cryotubes. Les taux de survie s'échelonnent entre 7 et 72 pour cent selon le cultivar.]
[La colección del germoplasma de Musa, que se conserva en el Centro de Tránsito de INIBAP en la Universidad Católica de Lovaina, Bélgica, actualmente es almacenada en forma de meristemas in vitro bajo condiciones de crecimiento lento. Inicialmente, fue desarrollada una técnica para crioconservación de las suspensiones de células embriogénicas de diferentes cultivares de banano. Más recientemente, la investigación en el área de crioconservación se enfocó en los cultivos de meristemas, ya que el inicio de los cultivos de suspensiones de células embriogénicas consume mucho tiempo y es todavía difícil de realizar para algunos cultivares. Una técnica simple, desarrollada en el laboratorio de la Universidad Católica de Lovaina, dio como resultado la recuperación después del descongelamiento de todos los cultivares investigados. Esto implica un paso de cultivo previo en un medio sólido con altos niveles de sacarosa, seguido por la inmersión del material en criotubos en nitrógeno líquido (196 grados C). Las tasas de sobrevivencia variaron entre 7 y 72 por ciento, dependiendo del cultivar.] Hide full abstract View PDF
Sági, L.; Remy, S.;
Panis, B.;
Swennen, R.; Volckaert, G.;
Plant Cell Reports, 1994 |
Peer Reviewed
Study of the parameters for electroporation treatment of a protoplast suspension. Those with the most marked effect were the electroporation buffer medium, treatment with polyethylene glycol (PEG) and its duration and the thermal shock. The maximum frequency of introduction of the DNA detected… Show full abstract during the in situ experiment by transitory expression of the uidA gene of Escherichia coli coding the bêta-glucuronidase enzyme (GUS) was 1.8 percent of total protoplasts. As protoplast regeneration is possible, there might be a pathway for the production of transgenic bananas.
[Expression transitoire d'un gène dans des protoplastes isolés de suspensions de cellules embryogéniques régénérables de bananier (Musa spp., cv. 'Bluggoe', groupe ABB) et soumises à l'électroporation]
[Etude des paramètres du traitement par électroporation d'une suspension de protoplastes. Les plus influents sont les suivants : le milieu tampon d'électroporation, le traitement au polyéthylène glycol (PEG) et sa durée, et le choc thermique. La fréquence maximale de l'introduction de l'ADN détectée pendant l'expérimentation in situ par l'expression transitoire du gène uidA d'Escherichia coli codant pour l'enzyme bêta-glucuronidase (GUS) a été de 1.8 pourcent du total des protoplastes. La régénération des protoplastes étant possible, une voie pourrait s'ouvrir à la production de bananiers transgéniques.]
[Expresión transitoria de un gen en los protoplastos aislados de suspensiones de células embriogénicas regenerables de banano (Musa spp., cv. 'Bluggoe', grupo ABB) y sometidos a la electroporación]
[Estudio de los parámetros de una suspensión de protoplastos para el tratamiento con electroporación. Los más influyentes fueron: el medio búfer de electroporación, tratamiento con glicol de polietileno (PEG) y su duración y el choque terminal. La frecuencia máxima de introducción del ADN, detectado durante el experimento in situ mediante la expresión transitoria del gen uidA de Escherichia coli, codificando la enzima beta-glucuronidasa (GUS), fue de 1.8 por ciento de total de los protoplastos. Como la regeneración de los protoplastos es posible, podría existir una vía para la producción de bananos transgénicos.] Hide full abstract View article on publisher's site
De Smet, K.;
Panis, B.;
Sági, L.; Cammue, B.P.A.;
Swennen, R.;
African Crop Science Journal, 1994
Bananas are a staple food in eastern Africa, with 25.3 percent of the total world production. the production is, however, threatened by the presence of several diseases, of which the fungal diseases black sigatoka and panama disease are the most important. With the development of embryogenic cell… Show full abstract suspension cultures, the isolation of protoplasts therefrom and their successful regeneration, an invaluable vegetative material for genetic manipulation of banans became available. The discovery of new types of antifungal proteins (AFP's) and the cloning of their encoding genes provide a source of resistance to fungal diseases that can be induced into plants cells by gentic engeneering. Transformation techniques are currently under investigarion. Electroporation of banana protoplasts have resulted in transient expression frequencies of more than 1 percent as visualized by the action of the B-glucuronidase (GUS) marker gene. experiments with particle bombardment on cell suspensions have resulted in transient expression frequencies up to 3300 blue spots/0.5 ml cells, and stable transformants have also been selected. Optimisation of the cell suspension culture technique and transformation methodology is in progress (Author's abstract).
[Les bananes constituent l'alimentation de base en Afrique de l'Est avec 25,3 pourcent de la production totale mondiale. Les contraintes de production sont constituées par de nombreuses maladies parmi lesquelles la cercosporiose noire et la maladie de Panama sont les plus importantes. Le développement des techniques de culture de suspension de cellules embryogéniques, l'isolement des protoplastes et la réussite de leur régénération permettent d'obtenir un matériel végétal pour la manipulation génétique du bananier. La découverte de nouveaux types de protéines antifongiques (PAF) et le clonage des gènes correspondants offrent des sources de résistance aux maladies fongiques pouvant être incorporées dans les cellules par génie génétique. Les techniques de transformation sont en cours d'évaluation. L'électroporation des protoplastes a abouti à des fréquences d'expression transitoire de plus de 1 pourcent pourvant être visualisées par l'activité du gène marqueur B-glucoronidase (GUS). Des essais de bombardement de particules sur les suspensions de cellules ont abouti à des fréquences d'expression transitoire de plus de 3300 points bleus par 0,5 ml de cellules; des transformants stables ont aussi été sélectionnés. L'optimisation de la technique de culture de suspension cellulaire et de la méthodologie de transformation est en cours d'études (Résumé d'auteur).] Hide full abstract View article on publisher's site
Sági, L.; Remy, S.;
Panis, B.; De Smet, K.; Cammue, B.P.A.; Volckaert, G.;
Swennen, R.In: Ganry, J. (ed.). Breeding banana and plantain for resistance to diseases and pests.
International Symposium on Genetic Improvement of Bananas for Resistance to Diseases and Pests, Montpellier, France, 1992/09/7-9
CIRAD-FLHOR, 1993
The recent development of embryogenic cell suspensions forms significant progress in tissue culture of banana. It makes it possible to obtain material for cryopreservation and to perform genetic manipulations on banana. The transitory expression of genes in banana cells was studied. Protoplasts… Show full abstract were isolated and purified from cell suspensions of 'Bluggoe' (ABB). Transformation was performed by electroporation using the plasmid pBI221 containing a chimeric gene uidA. Strong betaglucuronidase (GUS) expression was easily observed after 24 h of culture and blue protoplasts were easily detected by an in situ test. The average frequency of expression of the GUS gene varied from 10 to the minus 4 and 5 10 to the minus 5. The technique is being optimised. Successful application of the technique of particle bombardment applied to intact cells in suspension to induce expression of the GUS gene at higher frequencies. The results encourage the regeneration of stable transformants in banana.
[Expression transitoire de gènes dans les cellules et protoplastes de bananier]
[La récente mise au point de la culture de suspensions cellulaires embryogènes représente une avancée significative pour la culture in vitro du bananier. Elle permet d'obtenir du matériel pour la cryoconservation et d'effectuer des manipulations génétiques sur le bananier. L'expression transitoire de gènes dans les cellules de bananier a été étudiée. Des protoplastes ont été isolés et purifiés à partir de suspensions de cellules embryogènes du cv. 'Bluggoe' (ABB). La transformation a été effectuée par électroporation avec le plasmide pBI221 contenant un gène chimérique uidA. Une forte expression de la bêtaglucuronidase (GUS) a été aisément observée après 24 h de culture et des protoplastes bleus ont pu être facilement détectés par un essai in situ. La fréquence moyenne d'expression transitoire du gène GUS varie entre 10*-4 et 5.10*-5. Technique en voie d'optimisation. Application avec succès de la technique de bombardement de particules à des cellules intactes en suspension, pour induire l'expression du gène GUS à des fréquences plus élevées. Ces résultats encouragent à régénérer des transformants stables chez le bananier.]
[Manifestación transitoria de genes en protoplastos y células de bananos]
[El reciente desarrollo de las suspensiones de células embriogénicas representa un progreso significativo en el cultivo de tejidos de bananos. Se estudió la manifestación transitoria de los genes en las células de banano. Los protoplastos fueron aislados y purificados a partir de las suspensiones celulares del 'Bluggoe' (ABB). La transformación se realizó mediante electroporación utilizando el plásmido pBI221, que contenía un gene quimera uidA. Una fuerte manifestación de la betaglucoronidasa (GUS) fue observada con facilidad después de 24 h de cultivo y los protoplastos azules fueron detectados fácilmente mediante una prueba in situ. La frecuencia promedio de la manifestación del gene GUS varió de 10 exp.-4 y 5.10 exp.-5. La técnica está siendo optimizada. Aplicación exitosa de la técnica de bombardeo con partículas a las células intactas en la suspensión para inducir la manifestación del gene GUS a frecuencias más altas. Los resultados fomentan la regeneración de transformantes estables en banano.] Hide full abstract View PDF
Panis, B.;
Dhed'a, D.B.; De Smet, K.;
Sági, L.; Cammue, B.P.A.;
Swennen, R.In: Ganry, J. (ed.). Breeding banana and plantain for resistance to diseases and pests.
International Symposium on Genetic Improvement of Bananas for Resistance to Diseases and Pests, Montpellier, France, 1992/09/7-9
CIRAD-FLHOR, 1993
Embryogenic cell suspensions of Musa produced by in vitro meristem proliferation regenerate readily and seem to be excellent material for biotechnological applications. 1) Mass clone propagation: the multiplication of suspensions and their regeneration by somatic embryogenesis requires less work… Show full abstract than meristem proliferation for a much higher multiplication rate. 2) Genetic transformation: the unicellular origin of regenerated plants prevents the appearance of chimeras. Embryogenic cells or isolated protoplasts with proved regeneration capacities form material for different transformation methods. The first electroporation trials have shown the transitory expression of beta-glucuronidase. New techniques such as particle bombardment and ultrasonication are being studied. 3) Cryopreservation: Musa germplasm collections kept at very low temperatures (-196 degrees C) may solve the problem of somaclonal variation. It has been proved that the cells of 5 Musa cultivars can be kept in liquid nitrogen and then regenerated by somatic embryogenesis.
[Les suspensions de cellules de tissus somatiques de Musa : applications et perspectives] Les suspensions de cellules embryogéniques de Musa, issues de prolifération de méristèmes in vitro régénèrent facilement et semblent être le matériel de choix pour des applications biotechnologiques. 1) Propagation clonale de masse : la multiplication des suspensions et leur régénération par l'embryogénèse somatique demande par rapport à la prolifération de méristèmes un travail moins important pour 1 taux de multiplication très supérieur. 2) Transformation génétique : l'origine unicellulaire des plantes régénérées évite l'apparition de chimères. Les cellules embryogènes ou les protoplastes isolés, avec des capacités de régénération prouvées constituent un support pour différentes méthodes de transformation. Les premiers essais d'électroporation ont montré l'expression transitoire de la bêta-glucuronidase. De nouvelles techniques comme le bombardement de particules ou l'ultrasonication sont en cours d'étude. 3) Cryoconservation : les collections de matériel génétique de Musa à très basses températures (-196 degrés C) peuvent résoudre le problème de la variation somaclonale. Il a été prouvé que des cellules de 5 cv. de Musa peuvent être conservées dans l'azote liquide, puis régénérées par embryogénèse somatique.
[Suspensiones celulares de tejido somático de Musa: aplicaciones y perspectivas] Las suspensiones de células embriogénicas de Musa, producidas mediante la propagación de meristemas in vitro, regeneran fácilmente y parecen ser un material excelente para las aplicaciones biotecnológicas. 1) Propagación masiva de clones: la multiplicación de suspensiones y su regeneración mediante embriogénesis somática requiere menos trabajo que la propagación de meristemas y tiene una mayor tasa de propagación. 2) Transformación genética: El origen unicelular de las plantas regeneradas previene la aparición de quimeras. Las células embriogénicas o protoplastos aislados con capacidades probadas de regeneración, constituyen un soporte para diferentes métodos de transformación. Los primeros ensayos de electroporación han demostrado la expresión transitoria del gene de la beta-glucoronidasis. Se estudian nuevas técnicas, tales como el bombardeo de partículas y aplicación de ultrasonido. 3) Crioconservación: las colecciones del germoplasma de Musa, mantenidas a muy bajas temperaturas (-196 grados C), pueden resolver el problema de la variación somaclonal. Se estableció que las células de 5 cultivares de Musa pueden mantenerse en nitrógeno líquido y luego regenerarse mediante la embriogénesis somática. Hide full abstract View PDF
Panis, B.; Van Wauwe, A.;
Swennen, R.;
Plant Cell Reports, 1993 |
Peer Reviewed
Isolation and regeneration of protoplasts from a banana embryogenic cell suspension from in vitro proliferating meristem. Description of the method, the material used and the experimental conditions enabling the formation of microcolonies with a frequency of 20-40 percent. They developed directly… Show full abstract into somatic embryos without intervention of the callus phase. The somatic embryos germinated and grew into plants.
[Isolement et régénération de protoplastes issus d'une suspension de culture de cellules embryogéniques de bananier provenant de méristèmes en prolifération in vitro. Description de la méthode, du matériel utilisé et des conditions de l'expérimentation qui permettent d'obtenir la formation de microcolonies à la fréquence de 20-40 pour cent. Elles se développent directement sans intervention de la phase cal, en embryons somatiques. Ceux-ci germent et donnent une plante.] Hide full abstract View article on publisher's site
Sági, L.; Remy, S.; Volckaert, G.;
Panis, B.;
Swennen, R.;
Banana Newsletter, 1992
A study performed on protoplasts isolated from embryonic cell suspensions of 'Bluggoe' (ABB). Technique and results.
[Etude faite à partir de protoplastes isolés de suspensions de cellules embryonnaires du cv. "Bluggoe" (ABB).Technique et résultats.] View PDF
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