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Standardization of Agrobacterium-mediated Genetic Transformation of Pigeonpea [Cajanus cajan (L.) Millsp.] Using Tissue Culture and Non Tissue Culture Based Methods
Pigeonpea (Cajanus cajan (L.) Millsp.) is an important high protein grain legume of the semi-arid tropics and caters to the protein requirement of the population in Indian sub-continent. The productivity of pigeonpea is constrained by several biotic and abiotic stresses. Since, pigeonpea is a self pollinated crop with narrow genetic base, conventional breeding has no conspicuous effects on the genetic improvement. Genetic engineering offers an attractive alternative at this juncture for several traits which cannot be improved through conventional breeding. Among the various approaches for genetic transformation of plants, Agrobacterium mediated method is most popular and studies on Agrobacterium tumefaciens have provided the basis that has made this soil bacterium the dominant customer for plant transformation. Development of transgenics in pigeonpea remains dogged by poor plant regeneration in vitro with low frequency of transformation. Hence, there is a need for more focused and consistent efforts to develop protocols for obtaining stable in vitro regeneration and genetic transformation. In the present study, attempts were made to standardize regeneration as well as transformation protocols for the both tissue culture based and non-tissue culture i.e., in planta based methods for Agrobacterium mediated transformation of pigeonpea cultivar, Asha (ICPL 87119). Regeneration from axillary meristem explants (AMEs) of pigeonpea cultivar Asha was attempted. Among the AMEs isolated from 7 day, 9 day and 11 day old seedlings, the 9th day old AMEs were most suitable for regeneration. These were inoculated on to four combinations of shoot induction media (SIM) of which MS with 0.5 mg l-1 BAP (SIM1) was found to be the best for maximum shoot bud induction after two sub-cultures in the same media. Elongation of multiple shoots was tested on MS media with three concentrations of gibberellic acid (GA3) and 0.3 mg l-1 (GA3) gave response to elongation. Only six shoots elongated and these were used for rooting on half strength MS medium supplemented with 0.2 mg l-1 IBA, however, only one shoot rooted, but could not survive upon transfer to sterile vermiculite for acclimatization. The factors for tissue culture based Agrobacterium mediated transformation were standardized. Ninth day old AMEs were found to be appropriate for Agrobacterium mediated transformation. Out of the various concentrations (0, 50, 100, 150 and 200 mg l-1) of kanamycin tested, SIM 1 supplemented with 150 mg l-1 kanamycin, was identified as most ideal concentration for selecting the transformants. Among the infection times tested, of few seconds, 15 min and 30 min, for co-cultivation with Agrobacterium, it was revealed that 30 min with the culture having optical density of 0.6 (OD600nm) (among 0.3, 0.5 and 0.6 tested) was optimum with maximum response for transformation (9.75%). A co-cultivation period of two days was found to be optimum as beyond this period, bacterial overgrowth was observed. Also addition of acetosyringone (100 μM), enhanced the response to transformation. The parameters for in planta based Agrobacterium mediated transformation were standardized. The germinated seedlings from pigeonpea cultivar Asha were observed from 30 h to 70 h after sowing and the germinated seedlings at 44 h to 48 h old (2 days) stage were identified as most optimum for in planta transformation. Agrobacterium culture with OD600nm values of 1.2 and 1.5 were used for infection of pricked seedlings at just emerging plumule for in planta method of transformation. Infection time of Agrobacterium culture 1 h was kept constant for in planta transformation. Attempts were made to develop transgenic plants through tissue culture based Agrobacterium mediated transformation, using EHA105
Molecular Biology and Biotechnology
Standardization of Agrobacterium-mediated Genetic Transformation of Pigeonpea [Cajanus cajan (L.) Millsp.] Using Tissue Culture and Non Tissue Culture Based Methods