Maximizing genetic representation in seed collections from populations of self and cross-pollinated banana wild relatives.

BACKGROUND: 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 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. We assessed genetic representation of seed collections compared to source populations for three wild relatives of bananas and plantains. Focal species and sampling regions were M. acuminata subsp. banksii (Papua New Guinea), M. balbisiana (Viet Nam) and M. maclayi s.l. (Bougainville, Papua New Guinea). We sequenced 445 samples using suites of 16-20 existing and newly developed taxon-specific polymorphic microsatellite markers. Samples of each species were from five populations in a region; 15 leaf samples from different individuals and 16 seed samples from one infructescence ('bunch') were analysed for each population. RESULTS: Allelic richness of seeds compared to populations was 51, 81 and 93% (M. acuminata, M. balbisiana and M. maclayi respectively). Seed samples represented all common alleles in populations but omitted some rarer alleles. The number of collections required to achieve the 70% target of the Global Strategy for Plant Conservation was species dependent, relating to mating systems. Musa acuminata populations had low heterozygosity and diversity, indicating self-fertilization; many bunches were needed (> 15) to represent regional alleles to 70%; over 90% of the alleles from a bunch are included in only two seeds. Musa maclayi was characteristically cross-fertilizing; only three bunches were needed to represent regional alleles; within a bunch, 16 seeds represent alleles. Musa balbisiana, considered cross-fertilized, had low genetic diversity; seeds of four bunches are needed to represent regional alleles; only two seeds represent alleles in a bunch. CONCLUSIONS: We demonstrate empirical measurement of representation of genetic material in seeds collections in ex situ conservation towards conservation targets. Species mating systems profoundly affected genetic representation in seed collections and therefore should be a primary consideration to maximize genetic representation. Results are applicable to sampling strategies for other wild species.

Ecological divergence of wild strawberry DNA methylation patterns at distinct spatial scales.

Epigenetic change is considered relatively unstable and short-lived, raising questions of its contribution to long-term adaptive potential. However, epigenetic modifications can accumulate in the presence of environmental stress, resulting in beneficial epigenetic memories where environments are challenging. Diverging epigenetic memories have been observed across large spatial scales, and can persist through multiple generations. It is unknown, however, to what extent epigenetic variation contributes to fine-scale population structure and evolution. We compared DNA methylation patterns between a steep, altitudinal gradient (<2 km) and a wide spatial gradient (>500 km) using whole genome bisulphite sequencing data from 30 Fragaria vesca plants germinated and grown in controlled conditions. To assess the stability of spatial epigenetic variation in the presence of an environmental stressor, we applied acute drought stress to part of the plants and quantified drought-induced changes in DNA methylation signatures. We find that epigenetic memories and genomic islands of epigenetic divergence arise even at fine spatial scale, and that distinct spatial scales are featured by distinct epigenetic patterns. For example, demethylation of transposable elements consistently occurred at the large but not the fine spatial scale, while methylation differentiation for most biological processes were shared between spatial scales. Acute drought stress did not result in significant epigenetic differentiation. Our results indicate that population history, rather than short-term environmental stress, plays a dominant role in shaping epigenetic signatures. Specifically, repeated historical stress levels associated with heterogeneous environmental conditions may be required for acquiring a stable epigenetic memory and for coping with future environmental change.

The proteome profile of embryogenic cell suspensions of Coffea arabica L.

Somatic embryogenesis, is a process by which new viable embryos are produced from somatic tissues. Somatic embryogenesis is not only a useful biotechnological tool for the massive clonal propagation and genetic engineering but it also allows to obtain fundamental knowledge about the molecular changes that take place during embryogenesis. We present the proteome profile of two embryogenic cell suspensions. We identified 1052 non-redundant proteins. We present their known GO annotations and show two protein networks sharing the GO annotations related to stress and embryogenic capacity via the free program Cytoscape. To our knowledge these results give the first high-throughput proteome description of embryogenic cell suspensions and provide new information about somatic embryos for the whole plant community. The published proteome is a first step toward understanding somatic embryogenesis in coffee and toward a better annotation of proteins in an important non-model crop. All data are available via ProteomeXchange with identifier PXD002963.

Cryopreservation of Bituminaria bituminosa varieties and hybrids.

Bituminaria bituminosa (L.) C.H. Stirton is a drought tolerant, perennial legume pasture species and a source of pharmaceutical compounds. Bituminaria breeding programs aim to develop and conserve hybrids with desirable traits such as high forage quality, tolerance to biotic or abiotic stresses, and high contents of furanocoumarins. In this work we present a cryopreservation study of different B. bituminosa accessions: two varieties and eight intervarietal hybrids resulting from crosses between the three botanical varieties: var. bituminosa, var. crassiuscula, and var. albomarginata. No previous work on cryopreservation of Bituminaria species has been reported. We applied the ultra-fast cooling method, using droplet vitrification on aluminum foil strips. First, we investigated the PVS2 toxicity and cryopreservation damage in two genotypes, comparing three PVS2 treatments and two culture media. An incubation of 30 min in PVS2 resulted in regeneration rates after cryopreservation higher than 80%. The MS medium was selected for optimal meristem outgrowth, in order to avoid the prominent callus formation that was observed in the presence of BAP. These conditions were subsequently used to cryopreserve eight other genotypes. The results were highly variable; 45 days after cryopreservation, survival ranged between 22% and 98% while regeneration ranged between 0% and 96%, depending on the accession. A significant and positive correlation was observed between survival and regeneration. At 90 days post culture plantlets could be recovered from cryopreserved explants of all genotypes. This study shows that the droplet vitrification method is promising for the cryopreservation of eight of the 10 genotypes assayed and the method can thus be applied to develop a cryobank of B. bituminosa.

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics.

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.

Plant Cryopreservation: Principles, Applications, and Challenges of Banking Plant Diversity at Ultralow Temperatures.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

The use of 2D-DIGE to understand the regeneration of somatic embryos in avocado.

Avocado embryogenic cell cultures can be classified into two groups based on their morphology when cultured on a medium containing auxin: somatic embryo (SE) and proembryonic masses (PEM) type cultures. The calli of SE-type cell lines are able to go through the maturation process, whereas the calli of PEM cell lines rarely mature. We have investigated four independent avocado cell cultures (two SE and two PEM). The aim of this study was to link the differential regeneration capacity of the four cell cultures to a proteomic pattern and to gain insight into the regeneration capacity. A 2D-DIGE analysis followed by a blind multivariate analysis was able to separate the two SE lines from the PEM lines indicating that the protein profiles of SE and PEM calli are different. Based on the variable importance, that is, the differential protein pattern, we hypothesize that the regeneration capacity in avocado is correlated to the ability to overcome the physicochemical stress stimuli associated with the in vitro culture. Our identical culture conditions do not seem to trigger an appropriate response in PEM lines.

Differential Protein Expression in Response to Abiotic Stress in Two Potato Species: Solanum commersonii Dun and Solanum tuberosum L.

Better knowledge on responses to dehydration stress could help to improve the existing cryopreservation protocols for potato, since plant tissues processed for cryopreservation are often submitted to similar in vitro stress conditions. Cryopreservation (the best method of conservation for vegetatively propagated plants) of potato still needs to be standardized to make it available and to conserve the wide diversity of this crop. In the present work, the response to osmotic stress and chilling temperature was investigated in two potato species, Solanum tuberosum and its relative, frost-tolerant S. commersonii. After 14 days of exposure, different growth parameters, such as shoot length and number of leaves, were measured. Furthermore, differentially abundant proteins were identified after performing 2-fluorescence difference gel electrophoresis (2-DIGE) experiments, and soluble carbohydrates were analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). The results show different responses in both species depending on the stress treatment. Focusing on the differences in growth parameters during the treatments, Solanum commersonii seems to be more affected than S. tuberosum cv. Désirée. At the molecular level, there are some differences and similarities between the two potato species studied that are dependent on the type of stressor.

Cryopreservation of Neottopteris nidus prothallus and green globular bodies by droplet-vitrification.

Neottopteris nidus prothalli and green globular bodies (GGBs) were successfully cryopreserved by droplet-vitrification. Prothalli were subjected to different treatments. The following parameters were studied: the age of in vitro mother plants from which prothalli were originated (30 to 90-day old), length of exposure to loading solution (LS) (20 to 40 min) and length of exposure to the plant vitrification solution (PVS2) (10 to 55 min). N. nidus GGBs and GGBs in suspension were subjected to PVS2 for 20, 30 and 40 min before liquid nitrogen exposure. The highest prothalli regrowth (92%) occurred when they were exposed for 40 min to LS, followed by 20 min to PVS2 and when they originated from non-preconditioned 45-day old mother plants. The highest GGB (100%) and GGB suspension regrowth (100%) after cryopreservation occurred when they were exposed to PVS2 for either 20 min or 40 min.

Arbuscular mycorrhizal fungi affect both penetration and further life stage development of root-knot nematodes in tomato.

The root-knot nematode Meloidogyne incognita poses a worldwide threat to agriculture, with an increasing demand for alternative control options since most common nematicides are being withdrawn due to environmental concerns. The biocontrol potential of arbuscular mycorrhizal fungi (AMF) against plant-parasitic nematodes has been demonstrated, but the modes of action remain to be unraveled. In this study, M. incognita penetration of second-stage juveniles at 4, 8 and 12 days after inoculation was compared in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized or not by the AMF Glomus mosseae. Further life stage development of the juveniles was also observed in both control and mycorrhizal roots at 12 days, 3 weeks and 4 weeks after inoculation by means of acid fuchsin staining. Penetration was significantly lower in mycorrhizal roots, with a reduction up to 32%. Significantly lower numbers of third- and fourth-stage juveniles and females accumulated in mycorrhizal roots, at a slower rate than in control roots. The results show for the first time that G. mosseae continuously suppresses root-knot nematodes throughout their entire early infection phase of root penetration and subsequent life stage development.

Banana seed genetic resources for food security: Status, constraints, and future priorities.

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 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.

Phylogeography and conservation gaps of Musa balbisiana Colla genetic diversity revealed by microsatellite markers.

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 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. Supplementary Information: The online version contains supplementary material available at 10.1007/s10722-022-01389-4.

Drying banana seeds for ex situ conservation.

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 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.

Corrigendum to: Drying banana seeds for ex situ conservation.

[This corrects the article DOI: 10.1093/conphys/coab099.].

Diversity of Fusarium associated banana wilt in northern Viet Nam.

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 Fusariumoxysporumf.sp.cubense (Foc) directly affect the income, subsistence, and nourishment of thousands of farmers worldwide. For Viet Nam, 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. Fusariumcugenangense - 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 Fusariumfujikuroi 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 Musalutea, showing the importance of wild bananas as a possible sink for Foc.

Structure and regulation of the Asr gene family in banana.

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 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.

Palm cryobanking.

We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety 'Barhee'. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg per liter 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3 percent when PEMs were treated with PVS2 for 30 min at 0 degree C and 56.7 percent when PVS2 treatment lasted for 15 min at 25 degree C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 +/- 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation.

Development of the first axillary in vitro shoot multiplication protocol for coconut palms.

The coconut palm or "tree of life" is one of nature's most useful plants and the demand for its fruit is increasing. However, coconut production is threatened by ageing plantations, pests and diseases. Currently, the palm is exclusively propagated via seeds, limiting the amount of planting material. A novel micropropagation method is presented, based on axillary shoot formation. Apical meristems of in vitro coconut seedlings are cultured onto Y3 medium containing 1 µM TDZ. This induces the apical meristem to proliferate through axillary shoots in ~ 27% of the initiated explants. These axillary shoots are seen as white clumps of proliferating tissue and can be multiplied at a large scale or regenerated into rooted in vitro plantlets. This innovative micropropagation method will enable the production of disease-free, high quality in vitro plantlets, which will solve the worldwide scarcity of coconut planting material.

Using seminatural and simulated habitats for seed germination ecology of banana wild relatives.

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 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.

Seed Banks as Incidental Fungi Banks: Fungal Endophyte Diversity in Stored Seeds of Banana Wild Relatives.

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 (CWRs) (e.g., by the Millennium Seed Bank (MSB), Royal Botanic Gardens Kew). CWRs have been recognised as potential 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 CWRs 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 and 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.