Abstract
BackgroundRecalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. Therefore, an improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, co-cultivation, and sonication combined with vacuum infiltration duration which were optimized. ResultsGerminated seeds were sonicated and vacuum infiltrated with different densities of Agrobacterium culture and co-cultivated in half-strength MS medium with 100 μM of acetosyringone for 48 h. Seedlings were washed with cefotaxime and sowed in vermiculite soil for maturation. T1 plants were subjected to histochemical and molecular analysis to ensure transformation efficiency. Among various combinations analyzed, maximum transformation efficiency (20.8%) was attained with seeds of 5 min sonication combined with vacuum infiltration with 0.6 optical density of Agrobacterium culture. ConclusionsIt concludes that a different Agrobacterium cell density with sonication combined with vacuum infiltration has improved transgenic efficiency in horse gram plants. This simple and efficient method is feasible for the stable expression of foreign genes that could be beneficial for future food security.
Highlights
Recalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members
Further studies were carried out in the Paiyur 2 variety to assess different parameters influencing the in planta method
Agrobacterium cell density on survival rate and transgenic efficiency Agrobacterium optical cell density and infection time plays a significant role in the in planta Agrobacteriummediated transformation
Summary
Recalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. An improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, cocultivation, and sonication combined with vacuum infiltration duration which were optimized. Horse gram (Macrotyloma uniflorum Lam. Verdc.) belongs to the family member of Fabaceae, widely used as a food crop in Asian countries like Sri Lanka, Burma, and India [1]. The “in planta” transformation method was studied, where Agrobacterium is used to infect the seedlings. This method is simple and easy to perform in less time and does not require any skilled labor. Several reports on the in planta transformation methods are emerging more than in vitro culture techniques [20,21,22,23]
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