Combination of RNA-Seq transcriptomics and iTRAQ proteomics reveal the mechanism involved in fresh-cut yam yellowing.

The aim of this study was to examine the regulation of transcriptomics and proteomics related to the yellowing of fresh-cut yams after storage. The comparison of yellow fresh-cut yam (YFY) vs. white fresh-cut yam (control) revealed 6894 upregulated and 6800 downregulated differentially expressed genes along with 1277 upregulated and 677 downregulated differentially expressed proteins. The results showed that the total carotenoids, flavonoids, and bisdemethoxycurcumin in YFY were higher than in the control due to the significant up-regulation of critical genes in the carotenoid biosynthesis pathway, flavonoid biosynthesis pathway, and stilbenoid, diarylheptanoid, and gingerol biosynthesis pathway. In addition, the tricarboxylic acid cycle and phenylpropanoid biosynthesis were both enhanced in YFY compared to the control, providing energy and precursors for the formation of yellow pigments. The results suggest that the synthesis of yellow pigments is regulated by critical genes, which might explain the yellowing of fresh-cut yam after storage.

Agro-ecological suitability assessment of Chinese Medicinal Yam under future climate change.

Chinese Medicinal Yam (CMY) has been prescribed as medicinal food for thousand years in China by Traditional Chinese Medicine (TCM) practitioners. Its medical benefits include nourishing the stomach and spleen to improve digestion, replenishing lung and kidney, etc., according to the TCM literature. As living standard rises and public health awareness improves in recent years, the potential medicinal benefits of CMY have attracted increasing attention in China. It has been found that the observed climate change in last several decades, together with the change in economic structure, has driven significant shift in the pattern of the traditional CMY planting areas. To identify suitable planting area for CMY in the near future is critical for ensuring the quality and supply quantity of CMY, guiding the layout of CMY industry, and safeguarding the sustainable development of CMY resources for public health. In this study, we first collect 30-year records of CMY varieties and their corresponding phenology and agro-meteorological observations. We then consolidate these data and use them to enrich and update the eco-physiological parameters of CMY in the agro-ecological zone (AEZ) model. The updated CMY varieties and AEZ model are validated using the historical planting area and production under observed climate conditions. After the successful validation, we use the updated AEZ model to simulate the potential yield of CMY and identify the suitable planting regions under future climate projections in China. This study shows that regions with high ecological similarity to the genuine and core producing areas of CMY mainly distribute in eastern Henan, southeastern Hebei, and western Shandong. The climate suitability of these areas will be improved due to global warming in the next 50 years, and therefore, they will continue to be the most suitable CMY planting regions.

Cloning and overexpression of the ascorbate peroxidase gene from the yam (Dioscorea alata) enhances chilling and flood tolerance in transgenic Arabidopsis.

Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar with high tolerance to flooding but sensitivity to chilling. MH1 responded differently to chilling and flooding according to various physiological parameters and antioxidant enzymes. Flooding led to an increase in ascorbate peroxidase (APX) activity in both roots and leaves, while chilling did not affect APX activity. The full length DaAPX ORF sequence from MH1 (750 bp) was then cloned. Phylogenetic analysis showed that plant cytosolic APXs into four major clusters and DaAPX was closely related to Oncidium. The DaAPX gene driven by a 35S promoter was transferred into Arabidopsis. The gene expression and enzyme activity of APX in the DaAPX transgenic lines 1-3 were significantly higher than in wild type (WT) plants. Compared to WT plants, seedling growth characteristics were significantly better in all transgenic lines under chilling, flooding, and oxidative stresses, indicating that the overexpression of DaAPX in Arabidopsis enhanced tolerance to several abiotic stresses. MH1 plants supplied with H2O2 presented an increase in the activity of APX leading to enhanced tolerance to chilling. Functional characterization of the APX gene should improve our understanding of the chilling- and flood-response mechanism in the yam.

Agromorphologic, genetic and methylation profiling of Dioscorea and Musa species multiplied under three micropropagation systems.

Plant in vitro vegetative propagation using classical semi-solid culture medium is limited due to the low degree of automation, suboptimal nutrient availability and induced physiological stress which often reduce its efficiency. Temporary Immersion System (TIS) emerged as an innovative approach to optimize and eliminate the drawbacks associated with the conventional system of micropropagation. In this study, both Dioscorea and Musa spp. were subjected to conventional semi-solid culture media, complete immersion in shaking liquid culture media and TIS using RITA bioreactor. In vitro grown plantlets were screened for possible vegetative changes using agro-morphological descriptors while genetic and methylation differences were assessed using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). In vitro results showed that the number of shoots produced in Musa spp. varied significantly (P≤0.001) with the type of culture system. The highest mean shoot produced was observed with TIS (28.40) and the least using semi-solid culture medium (1.13). For Dioscorea spp., there was no significant interaction between the hormone combination and the culture system. However, the lowest mean shoot value (1.55) was observed in the semi-solid culture medium. Genetic analysis via AFLP using 15 primer pair combinations revealed that the 3 culture systems maintained genetic variation for Musa and Dioscorea spp. under in vitro and field conditions. Results showed 99% and 91% of the total bands were polymorphic under in vitro and field conditions respectively for Musa and 100% polymorphism for Dioscorea under in vitro and field conditions. Methylation investigation via MSAP using 12 primer pair combinations showed 25% and 46% polymorphic methylated-sensitive loci, 100% and 78% of non-methylated loci of the total bands generated under in vitro and field conditions respectively. Unmethylated (HPA+/MSP+) levels were highest in TIS (0.0842) as compared to CI (0.0227) and SS (0.0161) while full methylation or absence of target (HPA-/MSP-) was lowest in TIS (0.5890) and highest in SS (0.7138). For Dioscorea, 52% and 53% methylated sensitive loci and 100% non-methylated loci were polymorphic under in vitro and field conditions respectively. Although in vitro plant tissue culture techniques led to methylation at some loci of both species, there were no observable changes in the phenotype of both crops under field conditions. This also confirmed that not all methylation events lead to phenotypic changes.

Morphological and functional seed traits of the wild medicinal plant Dioscorea strydomiana, the most threatened yam in the world.

Morphological and functional seed traits have important roles in characterising the species regeneration niche and help to understand the reproductive biology of rare and threatened plants, which can thus support appropriate plant conservation measures. Seed morphometric and dispersal kinetics of the critically endangered Dioscorea strydomiana were measured and compared with those of four other Dioscorea species, and seed germination response under constant temperatures (5-35 °C) was compared with that of the congeneric and widespread D. sylvatica. Seed mass of D. strydomiana (ca. 14 mg) was twice that of D. sylvatica, but similar to or smaller than the other species examined. Seeds of D. strydomiana have the lowest speed of descent and lowest variability in most of the morphological traits considered, suggesting lower phenotypic plasticity but higher variance in the wing-loading value. Seeds of D. strydomiana reached maximum germination at 15 °C (ca. 47%), which decreased slightly to ca. 37% at 25 °C and was completely inhibited at 35 °C. D. sylvatica seeds started to germinate at 10 °C (ca. 3%), reached 75-80% germination at 15-20 °C and maximum (ca. 90%) at 25-30 °C. Base temperatures for germination (Tb ) were 9.3 and 5.7 °C, for D. strydomiana and D. sylvatica, respectively. Due to the higher germination percentages of D. sylvatica, ceiling and optimum temperatures could also be modelled for this species, suggesting higher sensitivity to high temperature for seeds of D. strydomiana. The detected poor seed lot quality of D. strydomiana suggests difficulties in reproduction from seed, highlighting the need for further investigation and conservation actions for this threatened yam species.

Integrated Metabolomics and Proteomics Approach To Identify Metabolic Abnormalities in Rats with Dioscorea bulbifera Rhizome-Induced Hepatotoxicity.

Previous studies have shown that Dioscorea bulbifera rhizome (DBR) can induce hepatotoxicity in clinical practice. However, its underlying mechanisms remain largely unexplored. In the present study, we investigated the global effect of DBR exposure on the proteomic and metabolomic profiles in rats over a 12-week administration using an integrated proteomics and metabolomics approach. The abundance of 1366 proteins and 58 metabolites in the liver of rats after subchronic exposure to DBR was dose-dependently altered. The results indicated that DBR mainly damaged hepatic cells through the aberrant regulation of multiple systems mainly including purine metabolism, pyrimidine metabolism, taurine and hypotaurine metabolism, and bile acid metabolism. Notably, the deregulated proteins including Pnp, Dpyd, Upp1, and Tymp and the differential metabolites including uridine, uracil, cytidine, thymine, adenine, adenosine, adenosine 3'-monophosphate, and deoxycytidine were well correlated to purine and pyrimidine metabolism, which might be novel pathways involved in metabolic abnormalities in rats with DBR-induced liver damage. Collectively, these findings not only contributed to understanding the mechanisms underlying the hepatotoxicity of DBR, but also illustrated the power of integrated proteomics and metabolomics approaches to improve the identification of metabolic pathways and biomarkers indicative of herb-induced liver injury.

Characterization of the Dioscorin Gene Family in Dioscorea alata Reveals a Role in Tuber Development and Environmental Response.

Dioscorin is one of the major soluble proteins in yam tubers. Unlike other well-known plant storage proteins, such as patatin and sporamin, dioscorin is argued for its function as storage proteins, and the molecular mechanisms underlying its expressional complexity are little understood. In this study, we isolated five dioscorin genes from Dioscorea alata L., comprising three class A (Da-dio1, -3 and -4) and two class B (Da-dio2 and -5) isoforms. Expressions of all dioscorin genes gradually decreased in mother tubers during yam sprouting and regrowth. On the other hand, all dioscorin genes accumulated transcripts progressively with tuber development in new tubers, with Da-dio5 being the most prominent isoform. In yam leaves, the expressions of Da-dio5 were up-regulated by the treatments of five phytohormones (gibberellic acid, salicylic acid, indole-3-acetic acid, abscisic acid, and ethylene), and three abiotic stresses (high-temperature, low-temperature and drought). To further elucidate the regulatory mechanisms of Da-dio5 expressions, transgenic Arabidopsis plants harboring the Da-dio5 promoter-ß-glucuronidase (GUS) fusion were generated. GUS staining showed that expressions of the Da-dio5 promoter were detected mainly in the shoot apical meristem (SAM) and hypocotyls, and enhanced by the treatments of the five hormones, and the three abiotic stresses mentioned above. These results suggest diverse roles of Da-dio5 in yam sprouting, regrowth, and tuberization, as well as in response to enviromental cues.

Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins.

In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins.

Diversification into novel habitats in the Africa clade of Dioscorea (Dioscoreaceae): erect habit and elephant's foot tubers.

BACKGROUND: Dioscorea is a widely distributed and highly diversified genus in tropical regions where it is represented by ten main clades, one of which diversified exclusively in Africa. In southern Africa it is characterised by a distinct group of species with a pachycaul or "elephant's foot" structure that is partially to fully exposed above the substrate. In contrast to African representatives of the genus from other clades, occurring mainly in forest or woodland, the pachycaul taxa and their southern African relatives occur in diverse habitats ranging from woodland to open vegetation. Here we investigate patterns of diversification in the African clade, time of transition from forest to more open habitat, and morphological traits associated with each habitat and evaluate if such transitions have led to modification of reproductive organs and mode of dispersal. RESULTS: The Africa clade originated in the Oligocene and comprises four subclades. The Dioscorea buchananii subclade (southeastern tropical Africa and South Africa) is sister to the East African subclade, which is respectively sister to the recently evolved sister South African (e. g., Cape and Pachycaul) subclades. The Cape and Pachycaul subclades diversified in the east of the Cape Peninsula in the mid Miocene, in an area with complex geomorphology and climate, where the fynbos, thicket, succulent karoo and forest biomes meet. CONCLUSIONS: Diversification out of forest is associated with major shifts in morphology of the perennial tuber (specifically an increase in size and orientation which presumably led them to become pachycaul) and rotation of stem (from twining to non-twining). The iconic elephant's foot morphology, observed in grasslands and thicket biomes, where its corky bark may offer protection against fire and herbivory, evolved since mid Miocene. A shift in pollination trait is observed within the forest, but entry into open habitat does not show association with reproductive morphology, except in the seed wing, which has switched from winged all round the seed margin to just at the base or at the apex of it, or has been even replaced by an elaiosome.

Melatonin-loaded alginate beads improve cryopreservation of yam (Dioscorea alata and D. cayenensis).

BACKGROUND: The cryopreservation of yam is constrained with many challenges. OBJECTIVE: This study tested the effects of melatonin on shoot tips of D. alata and D. cayenensis accessions exposed to water and liquid nitrogen (LN) stresses. MATERIALS AND METHODS: Sucrose pretreatment (0.3 M) was applied for 48 h before cryopreservation. Shoot tips were encapsulated in beads loaded with 0.75 M sucrose, with and without melatonin and desiccated over sterile dry silica gel for 0 - 9 h. RESULTS: The beads moisture content declined from 100% to ~ 13% after 9 h. The 3 h desiccation period without melatonin produced a significantly higher regeneration compared to 6 h and 9 h. Shoot tips with melatonin had significantly higher regeneration after 3 - 6 h desiccation compared to 9 h and the regeneration of all accessions after 6 h was >80%. Regeneration following 6 h desiccation and LN was significantly greater for melatonin-treated shoot tips compared to non-treated ones. CONCLUSION: The results indicate that melatonin significantly increased regeneration from 15% to 35%.

Evolution and Phylogenetic Diversity of Yam Species (Dioscorea spp.): Implication for Conservation and Agricultural Practices.

Yams (Dioscorea spp.) consist of approximately 600 species. Presently, these species are threatened by genetic erosion due to many factors such as pest attacks and farming practices. In parallel, complex taxonomic boundaries in this genus makes it more challenging to properly address the genetic diversity of yam and manage its germplasm. As a first step toward evaluating and preserving the genetic diversity yam species, we use a phylogenetic diversity (PD) approach that has the advantage to investigate phylogenetic relationships and test hypotheses of species monophyly while alleviating to the problem of ploidy variation within and among species. The Bayesian phylogenetic analysis of 62 accessions from 7 species from three regions of Cameroon showed that most Dioscorea sections were monophyletic, but species within sections were generally non-monophyletic. The wild species D. praehensilis and cultivated D. cayenensis were the species with the highest PD. At the opposite, D. esculenta has a low PD and future studies should focus on this species to properly address its conservation status. We also show that wild species show a stronger genetic structure than cultivated species, which potentially reflects the management of the yam germplasm by farmers. These findings show that phylogenetic diversity is a promising approach for an initial investigation of genetic diversity in a crop consisting of closely related species.

Genetic and functional characterization of culturable plant-beneficial actinobacteria associated with yam rhizosphere.

Actinobacteria were isolated from the rhizosphere of yam plants from agricultural fields from Yeoju, South Korea and analyzed for their genetic and plant-beneficial functional diversity. A total of 29 highly occurring actinobacterial isolates from the yam rhizosphere were screened for various plant-beneficial traits such as antimicrobial activity on fungi and bacteria; biocontrol traits such as production of siderophore, protease, chitinase, endo-cellulase, and ß-glucanase. The isolates were also screened for plant growth-promoting (PGP) traits such as auxin production, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and in vitro Arabidopsis growth promotion. 16S rDNA sequence-based phylogenetic analysis was carried out on the actinobacterial isolates to determine their genetic relatedness to known actinobacteria. BOX-PCR analysis revealed high genetic diversity among the isolates. Several isolates were identified to belong to the genus Streptomyces and a few to Kitasatospora. The actinobacterial strains exhibited high diversity in their functionality and were identified as novel and promising candidates for future development into biocontrol and PGP agents.

Production of yam mosaic virus (ymv)-free Dioscorea opposita plants by cryotherapy of shoot-tips.

In the present study, Yam mosaic virus (YMV) could be efficiently eliminated by cryotherapy in Dioscorea opposita. Shoot apices were precultured for 16 h with 0.3 M sucrose, encapsulated in sodium alginate and dehydrated for 4 h prior to direct immersion in liquid nitrogen. Up to 90 percent of the plants regenerated from cryopreserved shoot tips were YMV-free, whereas only 40% of those regenerated using meristem culture were YMV-free. YMV-free yam plantlets could be propagated in vitro through nodal stem culture, with sequential subculturing at 6-week intervals on medium containing 0.5 mg per liter kinetin. The microtubers formed at the bottom and axil of the explants, incubated at 30 degreeC after being chilled (4 degree C) for 3 months, could be sprouted successfully under in vivo conditions. Healthy plants were established without any damaging symptoms of the virus. Thus, cryotherapy provides an alternative method for efficient elimination of yam viruses, and could be simultaneously used for long-term storage of yam germplasm and for the production of virus-free plants.

Cryopreservation for the 'in perpetuity' conservation of yam and cassava genetic resources.

Cryopreservation via droplet vitrification showed high efficiency for cassava meristems (79 pecent average recovery) when these were excised from in vitro seedlings. The efficiency of the process dropped considerably (to > 23 percent) when meristems were excised from field-grown plants, thus precluding the use of such explants for routine cryobanking. In yam, large disparities were observed in the ability of meristems to produce a shoot after cryopreservation ranging from 0 to 60 percent, depending on the accession. Overall, better recovery was observed for Dioscorea rotundata than for D. alata, the two main species tested. Using a probabilistic decision support tool and taking into consideration our cryoprocessing capacity, we conclude that processing 100 meristems per accession and retrieving 30 to estimate the recovery rate of the batch are a good compromise for the cryobanking routine.

Genetic structure of farmer-managed varieties in clonally-propagated crops.

The relative role of sexual reproduction and mutation in shaping the diversity of clonally propagated crops is largely unknown. We analyzed the genetic diversity of yam-a vegetatively-propagated crop-to gain insight into how these two factors shape its diversity in relation with farmers' classifications. Using 15 microsatellite loci, we analyzed 485 samples of 10 different yam varieties. We identified 33 different genotypes organized in lineages supported by high bootstrap values. We computed the probability that these genotypes appeared by sexual reproduction or mutation within and between each lineage. This allowed us to interpret each lineage as a product of sexual reproduction that has evolved by mutation. Moreover, we clearly noted a similarity between the genetic structure and farmers' classifications. Each variety could thus be interpreted as being the product of sexual reproduction having evolved by mutation. This highly structured diversity of farmer-managed varieties has consequences for the preservation of yam diversity.

Nonlinear relationships between vital rates and state variables in demographic models.

To accurately estimate population dynamics and viability, structured population models account for among-individual differences in demographic parameters that are related to individual state. In the widely used matrix models, such differences are incorporated in terms of discrete state categories, whereas integral projection models (IPMs) use continuous state variables to avoid artificial classes. In IPMs, and sometimes also in matrix models, parameterization is based on regressions that do not always model nonlinear relationships between demographic parameters and state variables. We stress the importance of testing for nonlinearity and propose using restricted cubic splines in order to allow for a wide variety of relationships in regressions and demographic models. For the plant Borderea pyrenaica, we found that vital rate relationships with size and age were nonlinear and that the parameterization method had large effects on predicted population growth rates, X (linear IPM, 0.95; nonlinear IPMs, 1.00; matrix model, 0.96). Our results suggest that restricted cubic spline models are more reliable than linear or polynomial models. Because even weak nonlinearity in relationships between vital rates and state variables can have large effects on model predictions, we suggest that restricted cubic regression splines should be considered for parameterizing models of population dynamics whenever linearity cannot be assumed.

Understanding the germination of bulbils from an ecological perspective: a case study on Chinese yam (Dioscorea polystachya).

BACKGROUND AND AIMS: Bulbils serve as a means of vegetative reproduction and of dispersal for many plants; this latter aspect making them analogous to seeds. However, germination of bulbils may differ considerably from seeds due to dissimilar anatomical structures and perhaps environmental cue perception. The few laboratory studies done on bulbils suggest that their germination is similar to that of seeds in the same habitats and to vegetative buds of winter-dormant plants. The present study is the first to examine how bulbil germination is controlled in nature in relation to dispersal (before vs. after winter of the same cohort) and to ambient temperatures. METHODS: Under laboratory conditions, temperature and light requirements for root and shoot emergences from bulbils of Dioscorea polystachya collected in September, 2005, February, 2006 (produced in 2005) and July, 2006 were determined. Effects of cold stratification and dry storage for releasing dormancy were tested on September and July bulbils. The phenology of dormancy release and of root and shoot emergences and the persistence of bulbils in soil were followed over time under field conditions. KEY RESULTS: Although a low percentage of bulbils collected in July or in September produced roots, but no shoots, in the laboratory and field, these roots died within approx. 1 month. Regardless of collection date, cold stratification markedly increased root and shoot emergences. Bulbils sown outdoors in October produced roots and shoots the following March and April, respectively. The soil bulbil bank is short lived. CONCLUSIONS: Bulbils of D. polystachya are similar to seeds of many temperate plants being mostly dormant when dispersed in summer or autumn and overcoming dormancy with cold stratification during winter. Adaptively, bulbil germination primarily occurs in spring at the beginning of a favourable period for survivorship and growth.

Differential effect of hot water treatment on whole tubers versus cut setts of yam (Dioscorea spp.).

BACKGROUND: The use of thermotherapy or hot water treatment (HWT) is recommended for the management of plant-parasitic nematodes and other pathogens for a range of planting material, especially vegetatively propagated crops including yams, Dioscorea spp. The sprouting (germination) and consequent viability of yam following HWT, however, appear to be influenced by the post-treatment method of planting (whole or cut setts) and cultivar. The present study was established to evaluate the sensitivity of the most popular yam cultivars in Benin and Nigeria, West Africa, to HWT at 50-53 degrees C for 20 min. RESULTS: Sprouting of both setts and whole tubers of most cultivars was affected by HWT. Across experiments, 47% of HWT material, compared with 61% of non-HWT material, sprouted over 8 weeks. When cut into setts, 41% of HWT or untreated tubers sprouted, compared with 72% of whole tubers. Whole, untreated tubers had highest sprouting rates (84%), and setts following HWT had the lowest (38%). Yam planting material was also not completely free of parasitic nematodes following HWT. The reaction to HWT or cutting was highly cultivar specific. CONCLUSION: Yam cultivars vary in their sensitivity to hot water therapy. Care is therefore advised in selecting yam cultivars for HWT, especially when using cut setts.

[Seedling botanical characters and determination of effective components on Dioscorea zingerbrensis by space mutation].

OBJECTIVE: In order to find the new varieties with different horticultural characters, and investigate the mutation effects of seeds of Dioscorea zingerbrensis. METHOD: The seeds were carried by a satellite into space and recovered. The space mutation effect on the germination, seedling growth, chromosomes and rhizome diosgenin content of SP, populations of D. zingerbrensis were investigated. RESULT: Stimulated by space environment, the seed possessed the fast germinating characteristics. Germination rate showed no change. It was also found that a few plants were aneuploid or tetraploid. Fresh rhizome weight and rhizome diosgenin content in the second year plant were remarkably higher than those of the control. However, the increasing of third year plant was slow down, and rhizome diosgenin content in the third year plant declined simultaneously. CONCLUSION: The space environment showed stimulating effects on seed germination, fresh rhizome weight and rhizome diosgenin content.

Sucrose treatment and explant water content: critical factors to consider in development of successful cryopreservation protocols for shoot tip explants of the tropical species Dioscorea rotundata (YAM).

A study was conducted to determine the optimum methods for conditioning explants to be used in the development of a simple protocol for long-term conservation of the germplasm of Dioscorea rotundata via cryopreservation. Shoot tips from cultures maintained in vitro were exposed to high concentrations of sucrose prior to silica gel-based dehydration and vitrification solution-based cryopreservation protocols. Explant water contents were determined, and ultrastructural studies were also carried out. Initially, culturing explants on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g/g dry mass to between 4.8 and 5.5 g/g dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for D. rotundata shoot tips treated with MPVS2 vitrification solution, unloaded with 1.0 M sucrose medium and cooled to -7 degree C, was 16 percent for 15 min treatment and 44 percent for 40 min. After the 40 min MPVS2 treatment the TTZ test indicated 88 percent viability retention of explants cooled to -70 degree C, and 44 percent at -196 degree C. Plantlet development was obtained for -70 degree C-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with air-dehydration techniques. It was concluded that vitrification-solution based cryopreservation presently offers the best option for conservation of this Dioscorea species.