An improved plant regeneration and Agrobacterium - mediated transformation of red pepper (Capsicum annuum L.)

Abstract

Capsicum annuum (red pepper) is an important spice cum vegetable crop in tropical and subtropical countries. Here, we report an effective and reproducible auxin free regeneration method for six different red pepper cultivars (ACA-10, Kashi Anmol, LCA-235, PBC-535, Pusa Jwala and Supper) using hypocotyl explants and an efficient Agrobacterium-mediated transformation protocol. The explants (hypocotyls, cotyledonary leaves and leaf discs) collected from axenic seedlings of six red pepper cultivars were cultured on either hormone free MS medium or MS medium supplemented with BAP alone or in combination with IAA. Inclusion of IAA in the regeneration medium resulted in callus formation at the cut ends of explants, formation of rosette leaves and ill defined shoot buds. Regeneration of shoot buds could be achieved from hypocotyls grown in MS medium supplemented with different concentrations of BAP unlike other explants which failed to respond. Incorporation of GA3 in shoot elongation medium at 0.5 mg/l concentration enhanced the elongation in two cultivars, LCA-235 and Supper, while other cultivars showed no significant response. Chilli cultivar, Pusa Jwala was transformed with βC1 ORF of satellite DNA β molecule associated with Chilli leaf curl Joydebpur virus through Agrobacterium tumefaciens. Transgene integration in putative transformants was confirmed by PCR and Southern hybridization analysis.

Keywords: Agrobacterium tumefaciens; Capsicum annuum; Chilli leaf curl virus; Genetic transformation; Plant regeneration; Transgenic plants.

Fig. 1

The regeneration potential (in per cent) of six different cultivars of Indian red pepper cultured on different hormonal combinations. The experiment was repeated thrice and the values were representative of average regeneration response after 14 days on shoot bud induction medium and expressed as means ± SE (n = 10). The concentration of BAP was used as mg/L

Fig. 2

Different stages of plant regeneration using hypocotyls explants of Capsicum annuum; a Induction of shoot bud after 1 week culture, b- Shoot bud elongation and rooting after 3 weeks culture, c Shoot elongation on MS basal medium, d Shoot elongation on MS + 1 mg/L GA₃ medium, e Well elongated plantlet after hardening on agropeat, f Fully grown regenerated plantlet in growth chamber

Fig. 3

T-DNA construct of binary vector, pBINAR harboring viral βC1 ORF of Chilli leaf curl Joydebpur virus. Viral gene was cloned into BamH1 and SalI restriction sites. LB Left border, RB Right border, CaMV 35S Cauliflower mosaic virus 35S promoter, MCS Multiple cloning site, OCS Octapine synthase, NOS Nopaline synthase

Fig. 4

Molecular analysis of transgenic C. annuum var Pusa Jwala by PCR (a,b) and Southern hybridization (c); a detection of βC1 ORF of Chilli leaf curl Joydebpur virus where pBINARβC1 ORF represents positive control (P); non-transgenic lines as negative control (NT); transgenic lines 2, 3, 7, 21 and 25 (Transformants); marker (M); b PCR analysis using vir-D1 gene specific primers with plasmid DNA from Agrobacterium tumefaciens as positive control (P); c Integration of βC1 fragment in red pepper. Total DNAs from plantlets were digested with Hind III and 380 bp viral ORF was used both as probe and as well as positive control (P)