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, co-cultivation, 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. 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.

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.

Keywords: GUS expression; Horse gram; In planta transformation; Optical cell density; Sonication; Vacuum infiltration.

Fig. 1

T-DNA regions of binary vector pCAMBIA2301 used for the in planta transformation have the NPTII gene as plant selectable marker in the left border (LB) driven by CaMV35S2 promoter and CaMV35S terminator and Intron–GUS A as a reporter gene in the right border (RB) driven by CaMV35S promoter and NOS-terminator

Fig. 2

In planta Agrobacterium-mediated genetic transformation of horse gram (var. Paiyur 2). a Germinated horse gram seeds used for the infection of Agrobacterium tumefaciens strain. b 7-day-old acclimatized putatively transformed plant. c 14-day-old acclimatized putatively transformed plant. d 50-day-old acclimatized putatively transformed plant by seedlings co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA2301 binary plasmid. e Putatively transformed plants shows the flowering. f Pod formation at 55 days. g GUS histochemical staining was performed in T₁ horse gram seedlings. h Leaves show GUS expression. i Non-transferred (control) plant leaves show no GUS expression at all

Fig. 3

Flow chart of Agrobacterium-mediated in planta transformation protocol for the development of transgenic horse gram plants

Fig. 4

Effect of Agrobacterium optical cell density, infection period, and sonication combined with vacuum infiltration on influencing the in planta transformation of horse gram. a Effect of Agrobacterium cell density and infection time on transformation efficiency. b Effect of Agrobacterium cell density and sonication combined with vacuum infiltration on influencing the in planta transformation efficiency

Fig. 5

Molecular analysis of in planta T₁ transgenic horse gram plants. a, b Genomic DNA was isolated from control (CN) and nine putative T₁ transgenic horse gram leaf samples and the PCR analysis performed using NPTII (a) and GUS (b) gene-specific primers, respectively. c, d RT-PCR analysis of transgenic horse gram plants using NPTII c and GUSd gene-specific primers, respectively. Total RNA was isolated from control and putative transgenic lines and then converted to the single-stranded cDNA and performed the RT-PCR to study the expression of NPTII and GUS transcript. Note: 796 bp and 1.0 kb PCR products were observed in all transgenic plants except control (CN) using NPTII and GUS gene-specific primer, respectively. M 1.0 kb DNA ladder (Fermentas, Thermo Scientific), CN control plant, H1–H9 different transgenic lines, BP E. coli binary plasmid pCAMBIA2301