Development of methods for regeneration and genetic transformation of the commercial plum variety Startovaya from somatic tissues

Abstract

An effi cient technique for regeneration of plants from somatic tissues of domestic plum (Prunus domestica L.) varieties, as well as selecting schemes using various reporter (uidA, gfp) and selective marker genes (nptII, hpt) have been developed. During the transformation of somatic tissues of the commercial plum variety, transgenic plants containing both reporter genes and genes of economically valuable traits were obtained. For the fi rst time, the eff ect of calcium pantothenate on the adventive regeneration of plum shoots was investigated. It was shown that the selective antibiotic hygromycin is more eff ective for the selection of plum transformants than kanamycin or geneticin, which was expressed in a higher frequency of transformation — 1.2-2.2 % versus 0.2-0.5 % in selection on kanamycin. When using geneticin, transgenic plants were not obtained. Optimization of conditions made it possible to increase the percentage of regeneration of shoots to 79.4 % for the variety Startovaya and up to 39.6 % for the variety Etude. Analysis of GFP expression in transgenic lines showed that the leader sequences of herbaceous plant species encoding signal peptides can be used to eff ectively cellular compartmentalization of heterologous gene expression products in the plum. Preliminary results also show a higher level of expression of the reticular form of GFP in comparison with the vacuolar and cytosolic forms. Nine transgenic lines containing the insertion of the target bar gene, which gives resistance to phosphinothricin-containing herbicides of the “Basta” type, were obtained, which was confi rmed by the presence in the genome of the sequence of the transferred gene and the resistance of the obtained transgenic plants to 3 times the fi eld concentration of the herbicide “Basta”.