Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot

Abstract

A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T(0)) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T(0)) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.

Agrobacterium-mediated genetic transformation of peanut and the efficient recovery of transgenic plants

“…It was shorter than the 120Á150 d reported by Sharma and Anjaiah (2000), Sharma and Ortiz (2000). Although Agrobacteriummediated transformation of peanut was reported previously, these studies revealed a marked disparity between the conditions considered to enable optimal efficiency of transformation (Li et al 1997;Rohini and Sankara 2001;Anuradha et al 2006;Tiwari et al 2008;Yang et al 2011;Iqbal et al 2012). The present study demonstrated that factors such as the choice of genotype, source of explants, efficiency of the interaction of Agrobacterium with cells of the target tissue, and a reproducible complete plant regeneration protocol are crucial for successful transformation of peanut.…”

“…The regeneration percentage of shoots from cotyledons was slightly lower than that of the shoots from mesocotyl but a considerably higher transformation efficiency was obtained with mesocotyl-derived explants than the efficiency obtained with cotyledonderived explants. Most previous studies of Agrobacteriummediated transformation of peanut have used leaf discs, cotyledons, cotyledonary nodes, and embryo axes as target tissues (Eapen and George 1994;McKently et al 1995;Cheng et al 1996;Gelvin 2003;Anuradha et al 2006;Iqbal et al 2012). Cotyledon-derived explants are suitable for the transformation and regeneration of fertile plants, including crop species such as chickpea, rape, and peanut (Moloney et al 1989;Sharma and Anjaiah 2000;Sharma and Ortiz 2000;Bhattacharjee et al 2010;Iqbal et al 2012).…”

“…The pedigree selection process in peanut often lasts several years. Compared to an unexpected threat, a satisfactory solution could just be offered through plant genetic transformation (Jones et al 2005;Iqbal et al 2012). Agrobacterium-mediated transformation is a widely used and powerful tool to transfer foreign DNA into many plant species.…”

“…However, several constraints to peanut productivity cause considerable economic losses annually (Sharma and Anjaiah 2000;Sharma and Ortiz 2000). Genetic transformation could provide a powerful tool to identify genes that control important agronomical traits linked to disease resistance and/or abiotic stresses, and is rapidly becoming a key element in the process of varietal improvement (BhatnagarMathur et al 2007;Iqbal et al 2012;Sparks et al 2014).…”

“…Although several genes have been delivered into the peanut genome by using Agrobacterium-mediated transformation (Li et al 1997;Rohini and Sankara 2001;Anuradha et al 2006;Tiwari et al 2008;Yang et al 2011;Iqbal et al 2012), only a few peanut cultivars have a high transformation efficiency. Factors that affect the interaction of host cells with bacteria might limit the success of Agrobacterium-mediated transformation (Gelvin 2003).…”