A simple and efficient Agrobacterium-mediated in planta transformation protocol for horse gram (Macrotyloma uniflorum Lam. Verdc.)

Abstract

Background: Recalcitrant 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, cocultivation, and sonication combined with vacuum infiltration duration which were optimized.Results: Germinated 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. T 1 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.Conclusions: It 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.

A comprehensive review of in planta stable transformation strategies

“…As such, multiple highly efficient in planta research articles performing callus regeneration under in vivo conditions have been published over the years [16][17][18][19][20]. Similarly, several effective in planta protocols using minimal in vitro steps have also been published [21][22][23][24][25]. Conceptually speaking, these published methods all fall within the scope of in planta transformation and were self-described as in planta by their authors; however, their methods do not strictly follow the definitions mentioned above.…”

Embryo and Ovary Culture of Important Legume Horse Gram, Macrotyloma uniflorum (Lam.) Verdc

“…To increase the number of viable embryos and ovaries via in vitro techniques, future work should entail various hormones and concentrations of the different embryo and ovary ages. Among other varieties of horse gram paiyur-2 variety exhibited a maximum germination percentage of 93% 26 . So, this study was carried out in the paiyur-2 variety.…”

“…Earlier works have been able to induce the horse gram plant regeneration and organogenesis of plantlets using shoot tip 25 , leaf 22 , immature cotyledon 23 , cotyledonary nodal 26 explants and recently 27 done the embryo culture. But no attempt was made to culture the embryo and ovary explants.…”

Comparative Evaluation of Transient Protein Expression Efficiency in Tissues across Soybean Varieties Using the Tsukuba System

“…Moreover, even when vacuum agroinfiltration is performed on the entire plant, observing the expression in the petioles and stem is often not possible [ 29 , 30 ]. However, studies focused on the seedling expression have potential relevance for future in planta transformation procedures [ 31 , 32 ]. An example of this potential was demonstrated in a study by Hu et al [ 33 ] with radishes, who obtained mutants through an in planta transformation procedure in seedlings.…”