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Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut ( Vigna subterranea L. Verdc.).

An efficient protocol has been developed for direct shoot organogenesis from embryo axes derived from mature seeds of two different landraces of Bambara groundnut. Multiple shoots were initiated on several media containing different concentrations and combinations of benzylaminopurine (BAP) or thidiazuron (TDZ). Efficient regeneration occurred when the embryo axes were first plated for 6 days on a medium containing high concentrations of BAP (1 mg/l) and alpha-naphthaleneacetic acid (NAA, 1 mg/l) and then cut transversely and transferred onto a medium containing 1.5 mg/l BAP. Shoot regeneration frequency was 100% and from five to eight shoots per explant were obtained. The importance of using embryo explants and cytokinins in the culture media, with respect to controlling the development of a highly organogenic system, was demonstrated. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants without an intermediate callus phase. The regenerated shoots were rooted on a medium containing 1 mg/l NAA and then transferred to the greenhouse. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. All were morphologically normal and fertile. The short duration, high efficiency and low frequency of somaclonal variation of this system make it well suited for wider biotechnological applications of Bambara groundnut-a neglected and under-utilized crop.

Culture Techniques↗

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny.

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.

Culture Media↗

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp].

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and L: -Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l(-1) 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l(-1) 2,4-D, 20 mg l(-1) L: -Proline (Pro), 5 muM Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l(-1) potassium nitrate, and 0.05 mg l(-1) thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.

Cells, Cultured↗

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp].

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anti-clinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of pro-embryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anti-clinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of pro-embryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient pro-cambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the pro-cambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of pro-cambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.

Cells, Cultured↗

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker.

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and alpha-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris alpha-amylase inhibitor-1 (alphaAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris alpha-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T(1) lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T(1) plants. Transgenic plants could be recovered after 8-10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.

Agrobacterium tumefaciens↗

Light differentially regulates the expression of two members of the auxin-induced 1-aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.) seedlings.

Auxin induces the expression of the two ethylene-biosynthetic genes VR-ACS6 and VR-ACS7 in etiolated mung bean hypocotyls. However, while it also enhances VR-ACS6 expression in light-grown tissues, it does not up-regulate VR-ACS7 expression in these tissues. Here we show that transfer of 3-day-old etiolated seedlings into light quickly reduced the auxin-induced expression of both genes. However, while auxin-induced VR-ACS6 expression recovered after 24 h of light, VR-ACS7 transcription continued to reduce and was almost completely absent at 36 h. Thus, light differentially modulates the expression of the auxin-inducible VR-ACS genes. In hormone-treated etiolated seedlings, VR-ACS7 was primarily induced in the rapidly elongating zones of hypocotyl and epicotyl tissues, while auxin-induced VR-ACS6 mRNA was evenly distributed throughout the whole seedling. VR-ACS7 promoter-driven beta-glucuronidase (GUS) activity in auxin-treated etiolated transgenic Arabidopsis seedlings was observed in the highly elongating zones of the hypocotyl. During de-etiolation, the GUS activity gradually declined to become confined to the uppermost region of hypocotyls. In situ mRNA localization studies showed that in etiolated mung bean hypocotyls, the auxin-dependent VR-ACS7 transcript was predominantly present in the epidermis, which is the driving site for auxin-mediated elongation. Thus, it appears that the modulation by light of auxin-induced VR-ACS7 expression may correlate closely with the elongation growth response in early seedling development.

Arabidopsis↗

Antennal electrophysiological responses of three parasitic wasps to caterpillar-induced volatiles from maize (Zea mays mays), cotton (Gossypium herbaceum), and cowpea (Vigna unguiculata).

Many parasitic wasps are attracted to volatiles that are released by plants when attacked by potential hosts. The attractiveness of these semiochemicals from damaged plants has been demonstrated in many tritrophic systems, but the physiological mechanisms underlying the insect responses are poorly understood. We recorded the antennal perception by three parasitoids (Cotesia marginiventris, Microplitis rufiventris, and Campoletis sonorensis) to volatiles emitted by maize, cowpea, and cotton plants after attack by the common caterpillar pest Spodoptera littoralis. Gas chromatography-electroantennography (GC-EAG) recordings showed that wasps responded to many, but not all, of the compounds present at the physiologically relevant levels tested. Interestingly, some minor compounds, still unidentified, elicited strong responses from the wasps. These results indicate that wasps are able to detect many odorant compounds released by the plants. It remains to be determined how this information is processed and leads to the specific behavior of the parasitoids.

Animals↗

Oligosaccharins of black gram (Vigna mungo L.) as affected by processing methods.

The oligosaccharide content was determined in 12 different cultivars of black gram. The effect of various treatments such as soaking, cooking, and enzyme treatment on the raffinose family oligosaccharides of dry seeds and flour was studied. Ajugose, a higher oligosaccharide (DP 6) found in trace quantities in seeds, was shown in black gram by HPLC. The percent reduction of raffinose, stachyose, verbascose, and ajugose after soaking for 16 hr was 41.66%, 47.61%, 28.48%, and 26.82%, respectively in Local-I variety and 43.75%, 20.58%, 23.60%, and 15.88%, respectively in Local-II variety. Cooking for 60 min resulted in decrease of 100% for raffinose, 76.19% for stachyose, 36.39% for verbascose, and 60.97% for ajugose in Local-I variety and 100% for raffinose, 55.88% for stachyose, 48.52% for verbascose, and 56.07% for ajugose in Local-II variety. Thin layer chromatographic analysis of 3 hr enzyme-treated samples revealed almost complete hydrolysis of raffinose family of oligosaccharides. Among the different methods employed, enzyme treatment was found to be the most effective for removing alpha-galactosides in black gram.

Chromatography, High Pressure Liquid↗

PCR cloning and expression analysis of a cDNA encoding a pectinacetylesterase from Vigna radiata L.

A cDNA clone encoding a pectinacetylesterase (PAE) was isolated from 3-day-old mung bean seedlings using PCR-based techniques. Degenerate oligonucleotide primers were designed according to the N-terminus and internal peptides from the purified PAE. The full-length clone of 1453 bp codes for a signal peptide of 24 amino acids and a mature protein of 375 amino acids. The Mr and the pI of the cDNA-deduced amino acid sequence agree with the values estimated for the purified enzyme. No significant sequence identity between the PAE and any known protein could be found in the databases. Northern analysis revealed developmentally regulated expression of the mRNA in mung been seedlings.

Amino Acid Sequence↗