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. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.

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.

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.

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.

Corrigendum to: Drying banana seeds for ex situ conservation.

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

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.

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.

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.

Genetic diversity and structure of Musa balbisiana populations in Vietnam and its implications for the conservation of banana crop wild relatives.

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 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.

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.

Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and/or in Liquid Nitrogen.

The conservation of crop genetic resources, including their wild relatives, is of utmost importance for the future of mankind. Most crops produce orthodox seeds and can, therefore, be stored in seed genebanks. However, this is not an option for crops and species that produce recalcitrant (non-storable) seeds such as cacao, coffee and avocado, for crops that do not produce seeds at all; therefore, they are inevitably vegetatively propagated such as bananas, or crops that are predominantly clonally propagated as their seeds are not true to type, such as potato, cassava and many fruit trees. Field, in vitro and cryopreserved collections provide an alternative in such cases. In this paper, an overview is given on how to manage and setup a field, in vitro and cryopreserved collections, as well as advantages and associated problems taking into account the practical, financial and safety issues in the long-term. In addition, the need for identification of unique accessions and elimination of duplicates is discussed. The different conservation methods are illustrated with practical examples and experiences from national and international genebanks. Finally, the importance of establishing safe and long-term conservation methods and associated backup possibilities is highlighted in the frame of the global COVID-19 pandemic.

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.

Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources.

Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3-9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.

Challenges for Ex Situ Conservation of Wild Bananas: Seeds Collected in Papua New Guinea Have Variable Levels of Desiccation Tolerance.

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 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.

Strategies to revise agrosystems and breeding to control Fusarium wilt of banana.

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 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.

Is the bacterial leaf nodule symbiosis obligate for Psychotria umbellata? The development of a Burkholderia-free host plant.

BACKGROUND & AIMS: The bacterial leaf nodule symbiosis is an interaction where bacteria are housed in specialised structures in the leaves of their plant host. In the Rubiaceae plant family, host plants interact with Burkholderia bacteria. This interaction might play a role in the host plant defence system. It is unique due to its high specificity; the vertical transmission of the endophyte to the next generation of the host plant; and its supposedly obligatory character. Although previous attempts have been made to investigate this obligatory character by developing Burkholderia-free plants, none have succeeded and nodulating plants were still produced. In order to investigate the obligatory character of this endosymbiosis, our aims were to develop Burkholderia-free Psychotria umbellata plants and to investigate the effect of the absence of the endophytes on the host in a controlled environment. METHODS: The Burkholderia-free plants were obtained via embryo culture, a plant cultivation technique. In order to analyse the endophyte-free status, we screened the plants morphologically, microscopically and molecularly over a period of three years. To characterise the phenotype and growth of the in vitro aposymbiotic plants, we compared the growth of the Burkholderia-free plants to the nodulating plants under the same in vitro conditions. KEY RESULTS: All the developed plants were Burkholderia-free and survived in a sterile in vitro environment. The growth analysis showed that plants without endophytes had a slower development. CONCLUSIONS: Embryo culture is a cultivation technique with a high success rate for the development of Burkholderia-free plants of P. umbellata. The increased growth rate in vitro when the specific endophyte is present cannot be explained by possible benefits put forward in previous studies. This might indicate that the benefits of the endosymbiosis are not yet completely understood.

The cryoprotectant PVS2 plays a crucial role in germinating Passiflora ligularis embryos after cryopreservation by influencing the mobilization of lipids and the antioxidant metabolism.

Cryopreservation is a process whereby biological structures are preserved in liquid nitrogen (-196 °C) without losing their viability. Many cryopreservation techniques use the Plant Vitrification Solution 2 (PVS2) for cryoprotection. This study will therefore evaluate the influence of different exposure times to the cryoprotectant PVS2 and discuss the importance of the mobilization of reserves and the antioxidant metabolism during the germination of cryopreserved Passiflora ligularis embryos. The composition of P. ligularis seeds was analytically determined. We tested the germination capacity and the Germination Speed Index (GSI) of embryos (that is, seeds without external tegument) which were exposed to different PVS2 exposure times (0, 30, 60 and 120 min) at 30 days after thawing. Proline content, hydrogen peroxide, activity of isocitrate lyase (ICL), malate synthase (MSy), lipid peroxidation and antioxidant enzyme activities (SOD, CAT, APX) were measured at 7, 14 and 21 days after cryopreservation. The germination from cryopreserved embryos was maximal (85%) after 60 min PVS2 exposure with a GSI of 0.6. At 60 min, the highest activity of the enzymes involved in the glyoxylate cycle, ICL and MSy were recorded. We hypothesize that a 60 min exposure to PVS2 accelerates the reserve mobilization which correlates positively with germination. Until 60 min, there was a positive correlation between the PVS2 exposure time and the proline content, as well as the activity of antioxidant enzymes (SOD, CAT, APX), and a negative correlation with the lipid peroxidation. This study enables us to optimize the long-term conservation of this species. In conclusion, fundamental research is necessary to optimize the cryopreservation procedure, and this study offers an effective and efficient workflow which can be extrapolated to other (oil-rich) species.

Detection of Burkholderia in the seeds of Psychotria punctata (Rubiaceae) - Microscopic evidence for vertical transmission in the leaf nodule symbiosis.

BACKGROUND AND AIMS: The bacterial leaf nodule symbiosis is a close interaction between endophytes and their plant hosts, mainly within the coffee family. The interaction between Rubiaceae species and Burkholderia bacteria is unique due to its obligate nature, high specificity, and predominantly vertical transmission of the endophytes to the next generation of host plants. This vertical transmission is intriguing since it is the basis for the uniqueness of the symbiosis. However, unequivocal evidence of the location of the endophytes in the seeds is lacking. The aim of this paper is therefore to demonstrate the presence of the host specific endophyte in the seeds of Psychotria punctata and confirm its precise location. In addition, the suggested location of the endophyte in other parts of the host plant is investigated. METHODS: To identify and locate the endophyte in Psychotria punctata, a two-level approach was adopted using both a molecular screening method and fluorescent in situ hybridisation microscopy. KEY RESULTS: The endophytes, molecularly identified as Candidatus Burkholderia kirkii, were detected in the leaves, vegetative and flower buds, anthers, gynoecium, embryos, and young twigs. In addition, they were in situ localised in leaves, flowers and shoot apical meristems, and, for the first time, in between the cotyledons of the embryos. CONCLUSIONS: Both independent techniques detected the host specific endophyte in close proximity to the shoot apical meristem of the embryo, which confirms for the first time the exact location of the endophytes in the seeds. This study provides reliable proof that the endophytes are maintained throughout the growth and development of the host plant and are transmitted vertically to the offspring.

Somatic Embryogenesis in Coffee: The Evolution of Biotechnology and the Integration of Omics Technologies Offer Great Opportunities.

One of the most important crops cultivated around the world is coffee. There are two main cultivated species, Coffea arabica and C. canephora. Both species are difficult to improve through conventional breeding, taking at least 20 years to produce a new cultivar. Biotechnological tools such as genetic transformation, micropropagation and somatic embryogenesis (SE) have been extensively studied in order to provide practical results for coffee improvement. While genetic transformation got many attention in the past and is booming with the CRISPR technology, micropropagation and SE are still the major bottle neck and urgently need more attention. The methodologies to induce SE and the further development of the embryos are genotype-dependent, what leads to an almost empirical development of specific protocols for each cultivar or clone. This is a serious limitation and excludes a general comprehensive understanding of the process as a whole. The aim of this review is to provide an overview of which achievements and molecular insights have been gained in (coffee) somatic embryogenesis and encourage researchers to invest further in the in vitro technology and combine it with the latest omics techniques (genomics, transcriptomics, proteomics, metabolomics, and phenomics). We conclude that the evolution of biotechnology and the integration of omics technologies offer great opportunities to (i) optimize the production process of SE and the subsequent conversion into rooted plantlets and (ii) to screen for possible somaclonal variation. However, currently the usage of the latest biotechnology did not pass the stage beyond proof of potential and needs to further improve.