Agrobacterium Mediated Foreign Gene Transfer in Pigeonpea Cajanus cajan L and Production of Fungal Resistant Plant

Abstract

With an aim to develop transgenic pigeonpea with resistance to fungal disease, the newlinetransfer of a rice chitinase gene to pigeonpea [Cajanus cajan (L.) Millsp.] is reported. The newlinegenetic transformation of pigeonpea was carried out using the Agrobacterium tumefaciens newlineLBA4404 harbouring the binary plasmid pCAMBAR.chi11 harbouring chitinase gene under the newlinecontrol of ubiquitin promoter and nos terminator at its 3 end. For the same establishment of newlineefficient protocol for the direct and multiple shoot induction from cotyledonary node explants newlinewas achieved in pigeonpea (Cajanus cajan (L.) cv H77216 (Manak). Seeds of pigeonpea were newlinesurface sterilized and soaked overnight. These sterilized seeds were decoated and inoculated on newlineMS medium fortified with different concentrations of BAP, KIN and ZEA. Among the various newlineconcentrations tested, 2.0 mg/L BAP was found to be the best for maximum number of shoots newlineper explants. For shoot elongation, MS medium supplemented with different concentrations of newlineGA3 were tested and 1 mg/L GA3 gave maximum elongation of multiple shoots. The elongated newlineshoots were successfully rooted on MS medium containing different concentrations of IBA. newlineAmong them, IBA at 1.0 mg/L induced maximum frequency of rooting. The regenerated newlineplantlets were hardened in soil + sand + compost (1:1:1) in Plant Growth Chamber under newlinecontrolled conditions (humidity 80%; temp. 25°C). After 3 weeks, plants were successfully newlinetransferred to field, where 90 to 95% of them have been developed into morphologically normal newlineand fertile plants. This protocol was advantageously applied in the production of transgenic newlinepigeonpea plants. newlineTo study the morphological and differential development pathway, histological analysis newlineof the explants undergoing morphogenesis was carried out to determine the developmental newlinepathway of the competent cells capable of morphogenesis, and to continue the adventitious newlinenature of the regenerated shoots. For ontogeny studies, 4 to 5-day old cotyledonary node explants newline(0 day) cultured on shoot induction medium for different periods (2, 4, 6, 8, 10, 12 and 14 day) newlinewere taken for tissue processing. The nodular structures resulting from the meristematic activity newlineproliferated and gave rise to well-defined shoot buds by 14 day-old cotyledonary node explant. newlineThe new shoot buds developed were either at the base or junction of already developed shoots. newlinexviii newlineThe anatomy of the developing shoot bud originating from compact mass of cells confirms the newlineorganogenetic pathway of regeneration. newlineFurther, an efficient transformation protocol was developed to produce putative newlinetransgenic plant via cotyledonary node explants followed by subsequent regeneration of newlinecomplete plants on selection media containing hygromycin. For determination of optimum newlinehygromycin concentration, explants were cultured on MS with different levels of hygromycin to newlineknow the sensitivity during shoot regeneration in which 7.5 mg/L proved to be most optimum for newlineselection of putative transgenic shoots. An experiment was conducted to identify the optimum newlineAgrobacterium cell density during co-cultivation. Explants were infected with Agrobacterium newlinecultures at different OD read at 600 nm and Agrobacterium culture at 0.6 OD proved to be most newlinesuitable by giving maximum number of putative transformants (surviving green shoots). In order newlineto increase the transformation frequency, explants were co-cultivated with Agrobacterium with newlinedifferent levels of acetosyringone and 100and#956;M was recorded for the maximum number of shoots newlineproduced on the selection medium. Putative transformed pigeonpea plants were recovered with newlinestringent selection pressure and confirmed using molecular techniques. Stable integration and newlineexpression of the chitinase gene has been confirmed in the T0 transgenics through molecular newlineanalysis.