Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation–dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation–dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1–3) and seven to nine (L7–9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1–3 and L7–9 cells. Nuclear protein content increased, particularly in L1 and L1–3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7–9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1–3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1–3 and L7–9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.     

The wild yam question: How well could independent hunter-gatherers live in a tropical rain forest ecosystem?

It has been generally assumed until recently that tropical rain forests are food-rich biomes for human foragers, and that prehistoric hunter-gatherers once lived completely independent of cultivated foods in such environments. An alternative hypothesis that such forests are actually food-poor for humans is proposed here. Specifically, that wild starch foods such as yams were so scarce and so hard to extract that human foragers could not have lived in such biomes without recourse to cultivated foods. The symbiotic relationship found today between tropical forest hunter-gatherers and farmers is not a recent phenomenon, but evolved long ago as an adaptive strategy for successfully exploiting the tropical forest.     

Effect of activated charcoal on multiplication of African yam (Dioscorea cayenensis-rotundata) nodal segments using a temporary immersion bioreactor (RITA®)

In this study, we compared the growth of Dioscorea cayenensis-rotundata (African yam) nodal segments, using semisolid medium in test tubes and liquid medium in 1-L Recipient for Automated Temporary Immersion (RITA^®) temporary immersion bioreactors (TIB), and the application of various culture parameters. The addition of activated charcoal (AC) had a positive effect on the growth of nodal segments, both in semisolid medium and in liquid medium in RITA^® bioreactors. After 2 mo culture in the presence of AC, plantlets were 6.4–6.6 cm long compared to 3.2–3.8 cm in absence of AC, with no significant difference observed between the culture systems. In the range of inoculation densities tested (5–20 nodal segments per RITA^® bioreactor), there was no effect on the number of buds produced per nodal segment, the moisture content of plantlets (fresh weight basis), or on net fresh weight gain. By contrast, the individual leaf surface area of plantlets decreased in line with increasing inoculation density. Among the range of benzylaminopurine (BAP) concentrations tested (0–17.6 μM), 0.44 μM induced the highest number of buds (3.8 buds per nodal segment) in the TIB. However, comparable numbers of buds could be produced with media devoid of BAP, either by increasing the frequency of 1-min daily immersion cycles in RITA^® bioreactors from one every 12 h to one every 4 h or by using semisolid medium containing AC.     

Phylogenetic relationship of wild and cultivated yam species (Dioscorea spp.) of India inferred from PCR–RFLP analysis of two cpDNA loci

The genus Dioscorea comprises approximately 600 species worldwide and about 50 in India. Dioscorea is economically very useful because of its food and medicinal value. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was employed to study interspecific relationship between four cultivated and thirteen wild species belonging to seven taxonomical sections found in India. Restriction digestion analysis was performed for the amplified products of chloroplast DNA (cpDNA) loci psbA–trnH and trnL-F regions. Out of 17 restriction endonucleases tested, 5 endonucleases had restriction sites in psbA–trnH region and 6 endonucleases had restriction sites in trnL-F region. Digestion of amplified products with these restriction endonucleases showed 70 fragments of which 62 fragments (88 %) were polymorphic. Dendrogram obtained from UPGMA cluster analysis of two combined loci shows D. bulbifera of section Opsophyton to be closely placed with species of sect. Enantiophyllum. High similarity values obtained in this study between D. pentaphylla (sect. Botrysicyos) and D. esculenta (sect. Combilium) support their common origin. The average of genetic similarities between the ten species of Enantiophyllum section was 82.3 % showing their closeness. The present study suggests that the less studied New World species, D. floribunda (sect. Heterostemon) could have evolved from old world section, Lasiophyton. The principal component analysis (PCA) obtained from data of the two loci shows D. deltoidea of sect. Stenophora to be an outgroup. Interspecific genetic similarities showed D. deltoidea with low similarity values ranging from 27.5 to 37.5 % compared with other species and was, therefore, the most divergent species. D. deltoidea of section Stenophora shows distinctness because of its primitive characters. This study, therefore, establishes species relationships between wild and cultivated yams of India which would help in yam crop improvement programmes.     

Effects of storage conditions on sprouting of microtubers of yam (Dioscorea cayenensis–D. rotundata complex)

The control of field tuber dormancy in the yam (Dioscorea cayenensis–D. rotundata complex) is poorly understood. Although studies have examined single environmental factors and chemical treatments that might prolong tuber dormancy and storage, only a few were focused on further tuber sprouting. The present study concerns microtubers obtained by in vitro culture. When microtubers were harvested (after 9 months of culture) and directly transferred on a new medium without hormones, the tubers rapidly sprouted in in vitro conditions. No dormancy was observed in this case. Harvested microtubers were also stored dry in jars in sterile conditions during 2 to 18 weeks before in vitro sprouting. In this case, microtubers stored during 18 weeks sprouted more rapidly than those stored 8 weeks. A constant “dormancy-like period” (storage duration + sprouting delay) was observed, between 20 and 28 weeks respectively for the more rapid and the slower microtubers. The size of the tubers used for the storage had great influence on further sprouting. The larger they were, the better they sprouted. Light during storage had no effect on the sprouting delay while a temperature of 25 °C permit a quicker sprouting than 18 °C. The medium used to obtain microtubers could also have an effect on sprouting rate.     

Effects of reducing sugar concentration on in vitro tuber formation and sprouting in yam (<Emphasis Type="Italic">Dioscorea cayenensis–D. rotundata</Emphasis> complex)

The effects of reducing sucrose level on tuber formation (% of cultures with microtubers), development (length and fresh weight of microtubers) and sprouting in yam Dioscorea cayenensis–D. rotundata complex in vitro were investigated. Only 29% of the explants showed tuber formation after 3 weeks in the presence of 1% sucrose in contrast to 100% with 3%. After 120 days of culture, the length and the weight of the tubers obtained in the presence of 1% sucrose were less than with 3% sucrose. Addition of sorbitol to keep osmolarity at the same level did not restore normal rate of tuber formation. Similar results were obtained with the use of reduced fructose or glucose level. Microtuber sprouting was also affected by sucrose level incorporated into the tuberisation medium. Tubers obtained on reduced sucrose level sprouted later and the increase of osmolarity with sorbitol did not restore normal sprouting. The bigger tubers obtained on high sucrose media could contain more carbohydrate reserves that could partially explain a higher sprouting rate. These results can be used for optimising in vitro conditions for mass production of microtubers in yam and especially in Dioscorea cayenensis–D. rotundata complex, a very important species in West Africa. They specially showed the importance of tuberisation conditions on precocity of tuberisation, on tuber length and weight and on their further sprouting.