மூலக்கூறு உயிரியல்: திறந்த அணுகல்

Improved Protocol for Agrobacterium-Mediated Transformation of Pea (Pisum sativum)

Abstract

Mitra Aftabi, Alemayehu Teressa Negawo and Fathi Hassan

Pea is one of the most important legume crops whose production is constantly threatened by field and storage pests and diseases. Developing insect resistant transgenic pea plants through Agrobacterium-mediated transformation is a promising solution to maintain crop yield. However, the transformation efficiency is still low. Therefore, there was an attempt to enhance transformation efficiency by optimizing infection time, co-cultivation period and in vitro regeneration system. Transformation was performed using segments of embryonic axes from mature pea seeds (Pisum sativum L. cv. Sponsor). The segments were inoculated with the hyper virulent EHA105 strain of Agrobacterium tumefaciens. The Agrobacterium strain harboured a binary vector pGII35S containing the bar gene which confers resistance to phosphinotricin. The selection medium contained P2 medium with increasing concentrations of phosphinothricin. To improve the regeneration efficiency, 4.5 μM zeatin was added to the selection medium. The highest transformation efficiency (7.89%) was achieved with infection time of 90 min and co-cultivation period of 2 days. The shoots elongated well and the number of shoots/explants was increased (6 folds) after addition of zeatin. Resistant shoots were grafted onto rootstocks in soil and grafting success rate was 100%. The integration of cry1Ac gene in T0 transgenic plants was confirmed primarily by Polymerase Chain Reaction and further analysed by Southern blotting.