Agrobacterial transformation of cell culture Thalictrum minus L

Developed a method of Agrobacterium-mediated transformation of callus culture cells of medicinal plants Thalictrum minus. Agrobacterium tumefaciens strain containing cointegrable vector pGV 3850 was used for the transformation. The plasmid contains the target ipt gene encoding the key cytokinin biosynthesis enzyme isopentenyltransferase, under the 35S promoter of the cauliflower mosaic virus, and the selective npt II gene encoding the neomycin phosphotransferase enzyme conferring kanamycin resistance under the control of the NOS promoter. Two strains, «B» and 233, which differ in their growth intensity and in the level of biosynthesis of the protoberberine alkaloids, were transformed. The effectiveness of the influence on the transformation of vacuum infiltration and tobacco extract was evaluated. Selection of transformed calli was carried out at a sublethal concentration of kanamycin, which is different for different strains, for the initial strain «B» - 50 mg / l, for strain 233 -20 mg / l. Further evaluation of the transformation was carried out by PCR. High callus stability after transformation on kanamycin in both strains is shown. A distant reaction to kanamycin was noted, that is, a decrease in the viability of calli in the absence of kanamycin occurred at the third passage. The lowest survivability of calli was noted in the version with tobacco extract. However, the PCR method showed that the most effective variant of transformation was only tobacco extract without vacuum infiltration. As a result, ipt-positive five lines from strain 233 and one line from strain «B» were isolated. The transformation efficiency (selection on kanamycin + PCR) for strain 233 was 14.6%, for strain «B» - 0.1%. Of the five ipt-positive lines from strain 233, two of them had a higher growth rate and a level of content of protoberberine alkaloids, the only ipt-positive line from strain «B» had, on the contrary, a lower growth rate and a low level of protberberine alkaloids. Among the ipt-positive lines, the variability in growth intensity and the level of biosynthesis of the protoberberine alkaloids.