Effects of photoperiod,mineral medium strength,inorganic ammonium,sucrose and cytokinin on root,shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.     

Next-generation sequencing based genotyping,cytometry and phenotyping for understanding diversity and evolution of guinea yams

Key message

Genotyping by sequencing (GBS) is used to understand the origin and domestication of guinea yams, including the contribution of wild relatives and polyploidy events to the cultivated guinea yams.

Abstract

Patterns of genetic diversity within and between two cultivated guinea yams (Dioscorea rotundata and D. cayenensis) and five wild relatives (D. praehensilis, D. mangenotiana, D. abyssinica, D. togoensis and D. burkilliana) were investigated using next-generation sequencing (genotyping by sequencing, GBS). Additionally, the two cultivated species were assessed for intra-specific morphological and ploidy variation. In guinea yams, ploidy level is correlated with species identity. Using flow cytometry a single ploidy level was inferred across D. cayenensis (3x, N = 21), D. praehensilis (2x, N = 7), and D. mangenotiana (3x, N = 5) accessions, whereas both diploid and triploid (or aneuploid) accessions were present in D. rotundata (N = 11 and N = 32, respectively). Multi-dimensional scaling and maximum parsimony analyses of 2,215 SNPs revealed that wild guinea yam populations form discrete genetic groupings according to species. D. togoensis and D. burkilliana were most distant from the two cultivated yam species, whereas D. abyssinica, D. mangenotiana, and D. praehensilis were closest to cultivated yams. In contrast, cultivated species were genetically less clearly defined at the intra-specific level. While D. cayenensis formed a single genetic group, D. rotundata comprised three separate groups consisting of; (1) a set of diploid individuals genetically similar to D. praehensilis, (2) a set of diploid individuals genetically similar to D. cayenensis, and (3) a set of triploid individuals. The current study demonstrates the utility of GBS for assessing yam genomic diversity. Combined with morphological and biological data, GBS provides a powerful tool for testing hypotheses regarding the evolution, domestication and breeding of guinea yams.     

Axillary proliferation and tuberisation of Dioscorea cayenensis–D. rotundata complex

Yams (Dioscorea spp) are tuber crops used as staple food in Africa because of their nutritional value. However agronomic constraints, phytosanitary problems and the lack of good healthy planting material restrict their production. In contrast to the inefficiency of traditional method of planting, tissue culture techniques allow to increase the multiplication and the rapid production of pathogen- free plant material. This work was undertaken to provide farmers in African countries with healthy microplants and microtubers as seeds. In vitro nodal segments of two varieties of local yams D. cayenensis–D. rotundata complex (cv. ‘Singo’, cv. ‘Singou’ and cv. ‘Gnidou’) were micropropagated on the modified medium of Murashige and Skoog. The morphogenesis, the growth of microplants and microtuber formation have been found to be controlled by external factors that act individually and synergistically. Addition of kinetin (2 mg l⁻¹) to the culture media could reduce multiplication rate (node number) of some clones. An increase of the sucrose concentration from 3% to 5% induced no change in the multiplication and tuberisation parameters. An important reduction of the multiplication (shoot number, height and node number) and the tuberisation (tuber number and length) was observed with 8% sucrose. Multiplication (shoot and node number) was increased in the presence of jasmonic acid (10 μM). JA also induced an increase of tuber number in the absence of Kin. Multiplication of yam by in vitro growth of nodal segments is a way for rapid clonal multiplication and could allow solving the problem of lack of seed material faced by farmers. This method could also be used for multiplication of elite cultivars, independently of the growing season.     

Inheritance pattern of tetraploid Dioscorea alata and evidence of double reduction using microsatellite marker segregation analysis

Recent studies have shown that the basic chromosome number of the three major edible yams, Dioscorea alata, Dioscorea rotundata and Dioscorea trifida, is x?=?20, and that the clones with 2n?=?40 chromosomes are diploids. D. alata breeding programmes were limited to the production of diploid hybrids until 2006, when the tetraploids (2n?=?80) were found to be fertile and polyploid hybrids were produced by conventional hybridisation. However, the nature of the polyploidy (autotetraploidy or allotetraploidy) was not known in D. alata tetraploid clones. In the present study, the inheritance pattern of simple sequence repeat markers was determined in a tetraploid progeny using a Bayesian approach and by examining double reduction events. Results obtained confirm the autotetraploid nature of the 2n?=?80 clones of D. alata.     

Tuber Germination and Early Growth in Four Edible Dioscorea Species

The gross morphology and the sequence of anatomical events duringtuber germination in D. alata, D. esculenta, D. rotundata andD. trifida yams are compared with particular reference to theorigin of the organ of renewed growth, the primary nodal complex.The significance of this organ in yam phylogeny and ontogenyis briefly discussed. Dioscorea species, yams, tuber germination, nodal complex     

Disease-induced changes in carotenoid content of edible yams (Dioscorea spp.) infected by Botryodiplodia theobromae and Aspergillus niger

When pieces of Dioscorea dumentorum, Dioscorea alata and Dioscorea cayenensis were inoculated with suspensions of Botryodiplodia theobromae and Aspergillus niger and incubated at room temperature, the quantities of carotenes and xanthophylls were decreased by infection. The quantities of carotenes and xanthophylls were highest in D. dumentorum followed by D. cayenensis; D. alata had the lowest amounts.     

Promiscuous arbuscular mycorrhizal symbiosis of yam (Dioscorea spp.), a key staple crop in West Africa

Yam (Dioscorea spp.) is a tuberous staple food crop of major importance in the sub-Saharan savannas of West Africa. Optimal yields commonly are obtained only in the first year following slash-and-burn in the shifting cultivation systems. It appears that the yield decline in subsequent years is not merely caused by soil nutrient depletion but might be due to a loss of the beneficial soil microflora, including arbuscular mycorrhizal fungi (AMF), associated with tropical “tree-aspect” savannas and dry forests that are the natural habitats of the wild relatives of yam. Our objective was to study the AMF communities of natural savannas and adjacent yam fields in the Southern Guinea savanna of Benin. AMF were identified by morphotyping spores in the soil from the field sites and in AMF trap cultures with Sorghum bicolor and yam (Dioscorea rotundata and Dioscorea cayenensis) as bait plants. AMF species richness was higher in the savanna than in the yam-field soils (18–25 vs. 11–16 spp.), but similar for both ecosystems (29–36 spp.) according to the observations in trap cultures. Inoculation of trap cultures with soil sampled during the dry season led to high AMF root colonization, spore production, and species richness (overall 45 spp.) whereas inoculation with wet-season soil was inefficient (two spp. only). The use of D. cayenensis and D. rotundata as baits yielded 28 and 29 AMF species, respectively, and S. bicolor 37 species. AMF root colonization, however, was higher in yam than in sorghum (70–95 vs. 11–20%). After 8 months of trap culturing, the mycorrhizal yam had a higher tuber biomass than the nonmycorrhizal controls. The AMF actually colonizing D. rotundata roots in the field were also studied using a novel field sampling procedure for molecular analyses. Multiple phylotaxa were detected that corresponded with the spore morphotypes observed. It is, therefore, likely that the legacy of indigenous AMF from the natural savanna plays a crucial role for yam productivity, particularly in the low-input traditional farming systems prevailing in West Africa.     

Evaluation of isolates of Trichoderma,Pseudomonas and Bacillus species as treatment for the control of post-harvest fungal rot disease of yam (Dioscorea spp.)

The efficacy of four biological control agents (BCAs): Trichoderma asperellum strain NGT158, T. longibrachiatum strain NGT167, Bacillus subtilis and Pseudomonas fluorescens for the management of post-harvest tuber rot among four yam species, Dioscorea rotundata, D. cayenensis, D. alata, and D. dumetorum was evaluated. Rotted yam tubers were collected across three agroecological zones in Nigeria to isolate six infecting fungal pathogens: Aspergillus niger, Lasiodiplodia theobromae, Rhizoctonia solani, Penicillium oxalicum, Fusarium oxysporum and Sclerotium rolfsii. The BCAs were isolated by serial dilution and rot inhibition of treated tubers was evaluated using destructive sampling method in vivo after six months of storage. Bacillus subtilis was generally most effective, especially when applied 24 h before the inoculation of test pathogens across the four yam species, with percent inhibition that ranged between 47.8 and 81.2%. However, the four BCAs showed good potential in the control of the fungal pathogens causing post-harvest yam rot.     

Plastid phylogenetics of Oceania yams (Dioscorea spp., Dioscoreaceae) reveals natural interspecific hybridization of the greater yam (D. alata)

Phylogenetic relationships of Oceanian staple yams (species of Dioscorea section Enantiophyllum) were investigated using plastid trnL‐F and rpl32‐trnL^(UAG) sequences and nine nuclear co‐dominant microsatellites. Analysis of herbarium specimens, used as taxonomic references, allowed the comparison with samples collected in the field. It appears that D. alata, D. transversa and D. hastifolia are closely related species. This study does not support a direct ancestry from D. nummularia to D. alata as previously hypothesized. The dichotomy in D. nummularia previously described by farmers in semi‐perennial and annual types was reflected by molecular markers, but the genetic structure of D. nummularia appears more complex. Dioscorea nummularia displayed two haplotypes, each corresponding to a different genetic group. One, including a D. nummularia voucher from New Guinea, is closer to D. tranversa, D. alata and D. hastifolia and encompasses only semi‐perennial types. The second group is composed of semi‐perennial and annual yams. However, some of these annual yams also displayed D. alata haplotypes. Nuclear markers revealed that some annual yams shared alleles with D. alata and semi‐perennial D. nummularia, suggesting a hybrid origin, which may explain their intermediate morphotypes and the difficulty met in classifying them.     

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01–10 μM MeJA, either under root-inductive conditions, i.e., on medium containing 10 μM indole-3-butyric acid (IBA) and 0.1 μM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1–4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 μM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 μM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.     

Effect of jasmonic acid on in vitro explant growth and microtuberization in potato

The shoot fresh mass, root length and root numbers of two potato (Solanum tuberosum L.) cultivars Favorita and Helanwuhua were increased significantly by the application of 0.2 – 2 mg dm⁻³ jasmonic acid (JA) in the Murashige and Skoog medium. However, the growth of potato explants was inhibited by JA at high concentrations (20 – 50 mg dm⁻³). Chlorophyll content in explant leaves decreased with an increase in the concentration of JA. In leaves treated with 0.2 mg dm⁻³ JA acid peroxidase activity increased, while in the leaves treated with more than 2 mg dm⁻³ JA peroxidase activity decreased. Under the dark, the microtuber numbers, fresh mass and percentage of big microtubers of two potato cultivars were not promoted by the application of 0.2 – 50 mg dm⁻³ JA.     

Screening of cassava and yam cultivars for resistance to anthracnose using toxic metabolites of colletotrichum species

Collectotrichum gloeosporioides f. sp. manihotis and C. gloeosporioides, causal agents of cassava (Manihot spp.) and yam (Dioscorea spp.) anthracnose diseases, respectively, produce toxic metabolites in culture that fluoresce at 254 nm and 366 nm, producing bands with Rf of 0.65 and 7.0, respectively. Symptoms induced on yam and cassava by the extracted metabolites were similar to those induced by the pathogens. Twenty-four clones of tropical D. rotundata (TDr), D. alata (TDa), D. esculenta (TDe), and D. cayenensis (TDc) were screened by applying toxic metabolites of C. gloeosporioides to their leaves and stems. Only TDr131, TDe 179 and TDc750 were resistant. Other clones were susceptible to varying degrees. Nineteen of the 45 clones of M. esculenta were resistant to varying degrees of toxic metabolites of C. gloeosporioides f. sp. manihotis. Results from in vitro screening of’ cassava and yam clones using toxic metabolites compared favourably with field screening based on natural epidemics. Using toxic metabolites appears to be a more effective technique for screeningfor disease resistance than conventional inoculation with plant. pathogens. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative Observations on the Polarity of Sprouting of Bulbils of Dioscorea bulbifera L. and Dioscorea alata L.

The sprouting process of aerial bulbils of D. bulbifera andD. alata was compared. In D. alata, sprouts originated normallyfrom the proximal pole, at or near to the point of abscision,and irrespective of moisture, light or orientation of the bulbil.In D. bulbifera, sprouts originated primarily in response tomoisture and were less influenced by polarity. Post-harvesttreatment with gibberellic acid delayed the sprouting of bulbilsof D. alata but not of D. bulbifera; treatment with 2-chloroethanolor ethanol promoted sprouting in both species. Physiologically,sprouting was characterized by a transient increase in respiratoryrate at the onset of, or just prior to, sprout emergence. Thepromotion of sprouting by chloroethanol was accompanied by asurge in respiratory activity. Dioscorea spp., yams, bulbil germination, polarity     

Tuber formation and development of <Emphasis Type="Italic">Dioscorea cayenensis–Dioscorea rotundata</Emphasis> complex <Emphasis Type="Italic">in vitro</Emphasis> effect of polyamines

Tuberisation was obtained in vitro on yam (Dioscorea cayenensis–Dioscorea rotundata complex). The effect of exogenous polyamines on tuber formation and development (length and weight of microtubers) was investigated and discussed in relation with changes in endogenous polyamines. Application of exogenous polyamines, inhibitors of their metabolism, and polyamines precursors in various concentrations positively affected microtuber formation by yam nodal cuttings and their further development. In control conditions, 3 wk are needed to obtain 100% of tuberisation. With low concentrations of putrescine (10⁻⁵ or 10⁻⁶ M), tuber formation occurred earlier. Polyamine endogenous level and metabolism can be significantly affected by exogenous polyamines, but modifications of endogenous free polyamines could not be directly correlated to the tuber formation process. Increases in endogenous putrescine and auxins were observed in tubers showing a better development in the presence of putrescine. These results can be used for optimising in vitro conditions for mass production of larger microtubers of the D. cayenensis–D. rotundata complex.     

A new species ofLiparis (Orchidaceae) from India

Liparis indiraii spec. nova from India is close toL. alata A. Rich. andL. atropurpurea Lindl.     

Investigating a new cryopreservation protocol for yams (<Emphasis Type="Italic">Dioscorea</Emphasis> spp.)

Experiments were initiated to develop protocols for four species of Dioscorea, namely D. bulbifera L., D. oppositifolia L., D. alata L. and D. cayenensis Lam., and to assess the effects of various factors on the survival and regrowth of explants after rewarming. The treatments consisted of three different methods—vitrification, droplet and a modified droplet method—as well as the following variables—cryoprotective solution, sucrose concentration in the preadaptation medium and cold acclimation. While most of the protocols resulted in low to zero survival and regrowth rates, maximum survival rates of 100% and 75% were obtained for D. oppositifolia L. and D. cayenensis Lam., respectively, using two different protocols of the modified droplet method. A higher average survival rate was obtained using the droplet and modified droplet techniques than for the original vitrification methods. When the average of all the experiments was taken, more than one-half (52%) of the surviving explants developed further within 1 month with the modified droplet method compared to zero regrowth with the two other methods. The optimum protocol for cryopreservation is specific for each genotype.Abbreviations AC Activated charcoal - BAP 6-Benzylaminopurine - DMSO Dimethylsulfoxide - EG Ethylene glycol - GA₃ Gibberellic acid - Gly Glycerol - IAA Indole-3-acetic acid - Kin Kinetin - NAA -Naphthaleneacetic acid - PVS Plant vitrification solutionCommunicat by H. Lörz     

Effect of jasmonic acid on developmental morphology during in vitro tuberization of Dioscorea alata (L.)

A developmental morphology study was performed during different stages of in vitro yam microtuber formation on both hormone-free medium (HF) and medium supplemented with 10 µM of jasmonic acid (JA). The axillary protuberance, considered as the first morphological evidence for microtuber formation, became visible after one-week of culture in JA-medium and after three weeks of culture in HF-medium. In addition, the formation of the axillary protuberance in JA-cultures preceded the stem elongation, whereas in HF-medium stem elongation was visible before the formation of the axillary protuberance. Tuberization improvement in medium supplemented with JA is likely to be the result of morphogenetic modifications occurring during the early stages of microtuber formation. At the molecular level, 2D-PAGE analysis of HF- and JA-cultures allowed the identification of four differentially expressed polypeptides, including two putative pathogenesis-related proteins and one putative glutathione S-transferase. For JA-cultures, three additional differentially expressed polypeptides were identified.     

Metabolic design in the amino-acid-producing bacteriumCorynebacterium glutamicum

The Gram-positive bacteriumCorynebacterium glutamicum is used for the industrial production of amino acids,e.g. ofl-glutamate andl-lysine. By cloning and expressing the various genes of thel-lysine pathway inC. glutamicum we could demonstrate that an increase of the flux ofl-4-aspartaldehydate tol-lysine could be obtained in strains with increased dihydro-dipicolinate synthase activity. Recently we detected that inC. glutamicum two pathways exist for the synthesis ofdl-2,6-diaminopimelate andl-lysine. Mutants defective in one pathway are still able to synthesize enoughl-lysine for growth but thel-lysine secretion is reduced to 50–70%. Using NMR-spectroscopy we could calculate how much of thel-lysine secreted into the medium is synthesizedvia the one and the other pathway. Amplification of the feedback-inhibition-insensitive-homoserine dehydrogenase and homoserine kinase in a highl-lysine-overproducing strain made it possible to channell of the carbon flow from the intermediate 4-aspartaldehydate toward homoserine, resulting in a high accumulation ofl-threonine. For a further flux froml-threonine tol-isoleucine the allosteric control of threonine dehydratase was eliminated. Dedicated to Dr. Z. Vaněk on the occasion of his 70th birthday Presented at theIUMS Congresses '94-7th International Congress of Bacteriology and Applied Microbiology Division, Prague, July 3–8, 1994 (Bacteriological Symposium BS-12Regulation of Microbial Product Overproduction).     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

Rapid assessment of resistance of tissue-cultured water yam (Dioscorea alata) and white guinea yam (Dioscorea rotundata) to anthracnose (Colletotrichum gloeosporioides Penz.)

Yam anthracnose is caused by the pathogen Colletotrichum gloeosporioides Penz. and has been identified as the most important biotic constraint to yam production worldwide. Rapid assessment of the disease is vital to its effective diagnosis and management. In this study, tissue-cultured yam plantlets of five lines of Dioscorea alata and nine of D. rotundata were rapidly assessed for their reactions to two isolates of yam anthracnose. The plantlets, obtained from meristem of the nodal cuttings, were grown for 8?weeks on Murashige and Skoog (MS) basal medium, acclimatised for 3?weeks, hardened for an additional 3?weeks, arranged in screen house in completely randomised design and sprayed with spore inocula prepared from 7?day-old culture of the two strains of Colletotrichum gloeosporioidies Penz. The relative resistance of the different Dioscorea spp. was evaluated using three disease indices – severity at seventh day after inoculation, SD7; area under disease progress curve, AUDPC; and disease severity rate, Rd. A modified rank-sum classification method put TDa 1425 and TDr 2040, with rank sum of 2.0 each, as resistant. TDr 2121, TDr 2287 and TDr 2048 were susceptible with rank sum of 27.50, 25.50 and 24.50, respectively. Dioscorea alata TDa 1425 and Dioscorea rotundata TDr 2040 were recommended in areas endemic with yam anthracnose, and also as parent lines while breeding for resistance to anthracnose.