Interspecific hybridization between Vigna radiata (L.) Wilczek and V. glabrescens

Summary Interspecific hybrids of the mungbean, Vigna radiata (L.) Wilczek (2n=22) and V. glabrescens (2n=44) were generated with the aid of embryo culture. V. glabrescens x V. radiata hybrids were recovered via germination of the immature embryos. Reciprocal hybrids were obtained via shoot formation from embryonic callus. The authenticity of the hybrids was determined by morphological characteristics, chromosome number, and isozyme patterns. The hybrids were highly sterile upon selfing, but backcrossing to the diploid parent yielded viable seeds. Some of the plants resembled the diploid parent morphologically while others resembled neither parent. The backcross plants were sufficiently fertile to give a large number of mature, selfed seeds. Plants obtained differed morphologically and in their isozyme patterns from either parent, indicating introgression. These progeny populations will be used as bridging materials to transfer pest resistance from the wild tetraploid to the cultivated mungbean.     

Comparative efficiency of four selection methods for deriving high-yielding lines in mungbean [Vigna radiata (L.) Wilczek]

The comparative efficiency of four selection methods, viz., honeycomb (HC), pedigree selection (PS), single-seed descent (SSD) and the bulk method (BM), was assessed in three crosses of mungbean. The lines derived by each method, along with check varieties, were yield-tested in a compact family block design in F₅ and F₆ generations during summer and kharif of 1990. On the basis of the mean of the lines, the range, the number of superior lines over the best check, and the proportion of the top 10% lines in all the crosses and generations, the honeycomb method exhibited superiority over PS, SSD and BM for yield per plant and its component traits. PS, SSD and BM did not differ from each other. The honeycomb and SSD methods were found suitable for deriving superior lines for seed yield and pods per plant in mungbean.     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

Comparative efficacy of pedigree selection and selective intermating in greengram (Vigna radiata (L.) Wilczek)

Summary Selective intermating and pedigree selection methods were applied simultaneously to highly heterogeneous and heterozygous base populations of greengram in order to compare their relative efficacy in terms of evolving the number of productive lines as well as their production potential. Selection after two cycles of selective intermating was found to be a better method than traditional pedigree selection. The demerits of pedigree selection and merits of selective intermating are discussed. It is suggested that selective intermating replace the widely adopted but less effective pedigree selection for generating promising new material in such autogamous crops as greengram.Part of Ph.D. Thesis submitted by the senior author to the Haryana Agricultural University, Hisar, India     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l^–1 TDZ followed by three cycles of selection on medium with 0.5 mg l^–1 BA and increasing concentrations of hygromycin (20–40–60 mg l^–1). Selected shoot clusters were transferred to medium with 0.5 mg l^–1 BA for proliferation and 0.2 mg l^–1 BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l^–1 NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.     

Root initiation in Vigna radiata (L.) Wilczek hypocotyl cuttings is stimulated by smoke-derived extracts

Aqueous extracts of smoke, derived from Themeda triandra, a fire-climax grass, and Passerina vulgaris, a fynbos plant, stimulated root initiation and development in the hypocotyls of mung beans. Immersion time and the dilution of the smoke extracts were important in optimising the rooting response.     

Inheritance of powdery mildew (Erysiphe polygoni DC) resistance in mungbean (Vigna radiata L. Wilczek)

Detached mungbean (Vigna radiata L.Wilczek) leaves were inoculated with a conidial suspension of a local isolate (TI-1) of the powdery mildew pathogen (Erysiphe polygoni DC) under controlled environment conditions. Based on the latent period and severity of the infection, a rating scale of 0–5 was used to classify the host pathogen interactions. Reactions 0, 1 and 2 were considered resistant and referred to as R0, R1 and R2 while 3, 4 and 5 were classified as susceptible (S). RUM lines (resistant to powdery mildew) and their derivatives are crossed with several susceptible (reaction types 3–5) genotypes and the inheritance of the resistance was studied in the F₁, F₂ and F₃ generations. The results showed that powdery mildew resistance in mungbean is governed by two dominant genes designated as Pm-1 and Pm-2. When both Pm-1 and Pm-2 were present, an R0 reaction was observed after inoculation with TI-1. The resistant reaction was R1 when only Pm-1 was present and R2 in the presence of Pm-2. In the absence of both Pm-1 and Pm-2, susceptible reactions 3, 4 and 5 were observed.     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

Comparison of single seed descent,selective intermating and mass selection for seed size in greengram (Vigna radiata (L.) Wilczek)

Summary Three selection methods (single seed descent (SSD), mass selection and selective intermating) were applied simultaneously to a highly heterogeneous and broadly based population of greengram. Progeny developing after two cycles of selection were evaluated for yield and seven other economic characters. The relative efficacy of each selection method was judged on the basis of the number of high yielding progeny, mean yield of top 10% progeny, and mean of the highest yielding progeny. Selection after two cycles of selective intermating was found to be the best method for generating productive progeny although mass selection favouring smaller seeds was an equally efficient method. Both of these were found superior to SSD selection.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Phylogenetic diversity and symbiotic functioning in mungbean (Vigna radiata L. Wilczek) bradyrhizobia from contrast agro-ecological regions of Nepal

Nepal consists wide range of climatic and topographical variations. Here, we explored the phylogeny of native mungbean bradyrhizobia isolated from different agro-ecological regions of Nepal and accessed their nodulation and nitrogen fixation characteristics. Soil samples were collected from three agro-ecological regions with contrasting climate and topography. A local mungbean cultivar, Kalyan, was used as a trap plant. We characterized isolates based on the full nucleotide sequence of the 16S rRNA, ITS region, and nodA genes; and partial sequences of nodD1 and nifD genes. We found 50% of isolates phylogenetically related to B. yuanmingense, 13% to B. japonicum, 8% to B. elkanii, and 29% to novel phylogenetic origin. Results of the inoculation test suggested that expression of different symbiotic genes in isolates resulted in different degrees of symbiotic functioning. Our results indicate B. yuanmingense and novel strains are more efficient symbiotic partners than B. elkanii for the local mungbean cv. Kalyan. We also found most mungbean rhizobial genotypes were conserved across agro-ecological regions. All the strains from tropical Terai region belonged to B. yuanmingense or a novel lineage of B. yuanmingense, and dominance of B. japonicum related strains was observed in the Hill region. Higher genetic diversity of Bradyrhizobium strains was observed in temperate and sub-tropical region than in the tropical region.     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

Random amplified polymorphic DNA (RAPD) analysis in Indian mung bean (Vigna radiata (L.) Wilczek) cultivars

Greengram [Vigna radiata (L.) Wilczek], also known as mung bean, widely cultivated in a large number of countries, is an important pulse crop of Asia and is considered one of the ancestral species of the genus Vigna. Since yields of greengram have remained low across subtropical and tropical Asia, it is important to estimate genetic diversity in existing cultivars in order to see if the lack of genetic variability might be a constraining factor. In this study, 32 Indian cultivars of greengram were subjected to random amplified polymorphic DNA (RAPD) analysis using 21 decamer primers. A total of 267 amplification products were formed at an average of 12.71 per primer with an overall polymorphism of 64%. The extent of polymorphism was moderate to low. Jaccard similarity coefficient values ranged from 0.65 to 0.92. The cluster analysis resulted in mainly three clusters revealing greater homology between cultivars released from the same source. The results of principal components analysis also substantiated this conclusion. The close genetic similarity between the cultivars could be explained due to the high degree of commonness in their pedigrees. The narrow genetic base of the greengram cultivars revealed in the present analysis emphasises the need to exploit the large germplasm collections having diverse morphoagronomic traits in cultivar improvement programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker

Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.     

Early developmental and stress responsive ESTs from mungbean, <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek,seedlings

Although mungbean (Vigna radiata (L.) Wilczek) is commonly used as human food; the genomic resources of this species available in databases are limited. This study aims to develop expressed sequence tag (EST) resources for mungbean genes informative to early seedling development and chilling response. Two mungbean varieties that differ in disease resistance were found to also differ in their susceptibility to chilling temperatures. A total of 1,198 ESTs were obtained from one cDNA library and four PCR-select cDNA subtraction libraries; among these 523 were clustered into 136 contigs and 675 were singletons. The 811 non-redundant uniESTs were compared to GenBank using the Basic Local Alignment Search Tool (BLAST) and WU-BLAST algorithms, of these only 489 uniESTs had significant sequence homology, which may be involved in resuming the metabolic activity of seedlings, switching on photomorphogenesis, fuelling photosynthesis and/or initiating the unique developmental programs. Their encoded proteins may associate with regulatory proteins to trigger a direct stress response or participate in acclimation to environmental stressors. The uniEST platform reported will enrich the genomic resources of mungbean for functional genomic research on seedling development and chilling response of tropical crops and provide targets for improving the chilling tolerance of the tropical crops. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata,L. Wilczek)

Summary Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F₂ progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F₂ population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.