Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella

Abstract

Two commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.