Agrobacterium tumefaciens-mediated transformation is an efficient method for incorporating genes and recovering stable transgenic plants in cowpea because this method offers several advantages such as the defined integration of transgenes, potentially low copy number, and preferential integration into transcriptional active regions of the chromosome. Cotyledonary node explants of cowpea present an attractive target for T-DNA delivery followed by regeneration of shoots via axillary proliferation without involvement of a de novo regeneration pathway. In this chapter, we describe a detailed protocol for Agrobacterium-mediated transformation of the cowpea variety Pusa Komal. The seedling cotyledonary node explants are used for cocultivation with an Agrobacterium strain EHA105 harboring standard binary vector, pCAMBIA2301 or pNOV2819, and putative transformed plants are selected using aminoglycoside antibiotic or mannose as sole carbon source, respectively. The entire process includes explant infection to transgenic seed generation in greenhouse.

An efficient plant regeneration system from embryogenic callus of two elite indica rice (Oryza sativa spp. indica) varieties of Northeast India, Ketokijoha and Monoharsali is established. The effect of auxin, 2,4-dicholorophenoxy acetic acid (2,4-D) on callus induction was optimized. Friable, nodular and creamish-white embryogenic calli were induced from mature seeds on NB medium supplemented with 2.5 mg/l 2,4-D. Plants were regenerated from 40-50 days old embryogenic callus on NB medium containing 0.5 mg/l BAP (6-benzylaminopurine) and 0.25 mg/l ABA (abscisic acid). Regenerated plants with multiple tillers were rooted on half strength MS medium and rooted plants were acclimatized with 94% survival rate. Higher frequency of callus induction as well as plant regeneration was recorded in Ketokijoha as compared to Monoharsali. The calli of both the varieties were found amenable to Agrobacterium-mediated transformation as evident from strong GUS (β-glucuronidase) expression. The results may find wide application for genetic improvement for valuable traits these elite indica rice varieties of Northeast India. Int J Appl Sci Biotechnol, Vol 3(4): 680-686

Salinity is a major threat to sustainable agriculture worldwide. Plant NHX exchangers play an important role in conferring salt tolerance under salinity stress. In this study, a vacuolar Na+/H+ antiporter gene VrNHX1 (Genbank Accession No. JN656211.1) from mungbean (Vigna radiata) was introduced into cowpea (Vigna unguiculata) by the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot hybridization confirmed the stable integration of VrNHX1 into the cowpea genome. Comparative expression analysis by semi-quantitative RT-PCR revealed higher expression of VrNHX1 in transgenic cowpea plants than wild-type. Under salt stress conditions, T2 transgenic 35S:VrNHX1 cowpea lines exhibited higher tolerance to 200 mM NaCl treatment than wild-type. Furthermore, T2 transgenic 35S:VrNHX1 lines maintained a higher K+/Na+ ratio in the aerial parts under salt stress and accumulated higher [Na+] in roots than wild-type. Physiological analysis revealed lower levels of lipid peroxidation, hydrogen peroxide and oxygen radical production but higher levels of relative water content and proline, ascorbate and chlorophyll contents in T2 transgenic 35S:VrNHX1 lines.

Arsenic (As) contamination is a serious hazard to human health and agriculture. It has emerged as an important threat for rice cultivation mainly in South Asian countries. In this study, we investigated the effect of iron (Fe) supplementation on arsenic (As(V)) induced oxidative stress responses in rice (Oryza sativa L.). Rice seedlings treated with As(V) for 24 and 48 h in presence or absence of 2.5 mM Fe after which the root and shoot tissues were harvested for analysis. The results indicate significant (p ≤ 0.05) reduction in root and shoot length/dry biomass. Supplementation of Fe showed improved growth responses under stress as compared to As(V) alone. The scanning electron microscopy (SEM) analysis of roots under As(V) treatment for 48 h showed major alterations in root structure and integrity, although no noticeable changes were observed in Fe - supplemented seedlings. Significantly high (p ≤ 0.05) accumulation of As(V) was observed in root and shoot after 24 and 48 h of stress. However, under Fe - supplementation As accumulation in root and shoot were considerably low after 24 and 48 h of As(V) treatment. The hydrogen peroxide (H2O2) and malondialdehyde (MDA) content in both root and shoot increased significantly (p ≤ 0.05) after 24 and 48 h of As(V) treatment. In Fe - supplemented seedlings, the levels of H2O2 and MDA were considerably low as compared to As(V) alone. Ascorbate (AsA) and glutathione (GSH) levels also increased significantly (p ≤ 0.05) under As(V) stress as compared to control and Fe-supplemented seedlings. Activities of catalase (CAT) and superoxide dismutase (SOD) were significantly (p ≤ 0.05) high after 24 and 48 h of As(V) treatment as compared to Fe-supplemented seedlings. The gene expression analysis revealed up-regulation of metallothionein (MT1, MT2) and nodulin 26-like intrinsic protein (NIP2;1) genes after 5d of As treatment, while their expressions were repressed under Fe-supplementation. Our results indicate that Fe regulates oxidative stress and promotes growth under As stress.

Agrobacterium-mediated transformation of indica rice has been established in only a limited number of cultivars because the regeneration of plants from transformed embryogenic calli is highly cultivar-specific. Establishment of a highly efficient plant regeneration system from shoot apex explants applicable to many cultivars of indica rice will accelerate the application of transformation technology in breeding programs and functional genomics study. We established an efficient shoot multiplication and plant regeneration system from shoot apices of indica rice using thidiazuron (TDZ) as a plant growth regulator. Shoot apices cultured on MS basal medium devoid of plant growth regulators formed solitary shoots in 90% of cultures. Addition of TDZ or benzylaminopurine to regeneration medium significantly influenced formation of multiple shoots directly from shoot apex explants without an intervening callus stage. Best shoot proliferation response (10.3 shoots per explant) was recorded when shoot apices were cultured on media supplemented with 4 mg/l TDZ. No synergistic effect on shoot proliferation was observed when indole-3-acetic acid and indole-3-butyric acid were supplemented to media containing 4 mg/l TDZ. The regeneration system was efficient in evoking multiple shoot proliferation in eight different cultivars of indica rice. Shoots were rooted in MS basal medium and plantlets were acclimatized with 100% survival rate. The shoot apex explants of all the eight cultivars of indica rice were found competent to Agrobacterium-mediated transformation while explants from IR-64 showed highest transient GUS expression. This variety-independent transformation amenable regeneration system from shoot apices may widely be applicable for genetic transformation of indica varieties.