Impact of different Agrobacterium rhizogenes strains on secondary metabolites accumulation in Nitraria schoberi L. hairy roots and antiviral activity of their extracts against influenza virus of subtypes A (H5N1) and A (H3N2)

To optimize protocol for obtaining hairy roots of Nitraria schoberi L. with high antiviral activities, factors such as four strain types of Agrobacterium rhizogenes (A4, ATCC15834, R-1601, 8196), two explant types, namely cotyledonous and primary leaves of seedlings, and different cultivation durations (30 and 90 d) were studied. The formation of hairy roots was observed after 2 to 4 wk of incubation, depending on the type of explant and the strain of A. rhizogenes used. The maximum transformation frequency (85.7%) was observed in the cotyledons genetically modified with the strain ATCC15834. The transgenic nature of hairy roots was revealed by PCR with primers to the Agrobacterium oncogenes rolB and rolC. The absence of contamination of the culture by A. rhizogenes was confirmed by primers to the virC and virD1 genes. Phytochemical analysis showed that accumulation of individual metabolites in the line samples exceeded their levels in the native Nitraria roots. Catechin content in the cultures of long-term cultivation (90 d) was found 1.4 to 2.2 times higher than the same samples of short cultivation (30 d) and 4.8 to 10.8 times higher in comparison with the native roots. The most productive in terms of catechin level were hairy roots of long-term cultivation obtained during the transformation of primary leaves of N. schoberi seedlings with ATCC15834 strain. These data were consistent with the highest antiviral activities against influenza viruses of A (H5N1) and A (H3N2) subtypes with neutralization indexes 6.5 to 6.75 log10, and selectivity index values were in the range 15.4 to 16.4.