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 GA 3 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.

High-efficiency Regeneration of Seedlings from Hypocotyl Explants of Tung Tree (Vernicia fordii)

“…According to Chen (2013), the multiplication index could increase to 16.4 when GA3 was added into MS medium. In the process of shoot elongation and strengthening culture, increasing gibberellins and reducing cytokinins would be helpful for shoot growth, which is in accordance with results obtained from other plant species including Acca sellowiana (Berg) Burret (CangahualaInocente et al, 2007), Capsicum annuum L. (Kumar et al, 2012), and Mytilaria laosensis Lecomte (Qiu et al, 2013). Culture of strong and healthy shoots is the key step for plant rooting.…”

Plant Regeneration from Hypocotyl Explants in Capsicum (Capsicum annuum L.)

“…Optimum root induction was reported by Kim et al, (2008) by sub-culturing the capsicum shoots on MS medium with NAA 0.3 mg/l. Kumar et al, (2012) observed 100% rooting when elongated shoots were transferred to MS medium supplemented with NAA at 0.5mg/l. Rooting of the chilli shoots were the highest in MS medium with 2 mg/l IBA compared to other hormones (Manamohan et al, 2016).…”

“…Valadez et al, (2009) developed separate in vitro regeneration protocol for four different chilli genotypes. Kumar et al, (2012) The better regeneration was observed in chilli genotype G4 compared to LCA334 from cotyledonary and hypocotyl explants on MS medium supplemented with 0.25 mg/l zeatin and 2 mg/l phenyl acetic acid (PAA). Among the explants, cotyledonary leaf exhibited a higher regeneration response compared to that of hypocotyls (Manamohan et al, 2016).…”

In Vitro Regeneration of Capsicum (Capsicum annuum L.) from Cotyledon Explants

“…Kumar and Tata (2010) obtained better in vitro plant regeneration from variety X-235. Kumar et al, (2012) studied regeneration of red pepper cultivars and observed variable degree of regeneration. Marta and Pawel (2015) observed organogenesis in the 3 genotypes representing C. annuum L. was similar and considerably lower for line SF9 derived from an interspecific hybrid (C. frutescens L. × C. annuum).…”