Pushkarova N. Establishment of microclonal propagation methods and study of in vitro cultivation effect on biochemical properties and genetic variability of endangered Crambe species

Optimal protocols of aseptic plants culture establishment were shown for five endangered species - Crambe koktebelica, C. tataria, C. aspera, C. steveniana, C. maritima. Ways of direct and indirect regeneration from lateral buds initiation were shown and its morphogenic potential was established. The highest rates of direct organogenesis were gained as a result of lateral bud cultivation on the medium containing 0.6 mg/L of BA for C. aspera and C. steveniana, 1 mg/L of kinetin for С. koktebelica and 1.5 mg/L of kinetin for C. tataria. Different ways of morphogenesis on three types of explants (root, leaf and petiole) was shown for all studied species. Petiole's highest regeneration potential was shown as well. Optimal growth regulators composition for successful de novo regeneration on three types of explants was established for all studied species. Regeneration from petiole and leaf explants was dependant on NAA concentration in the medium - cultivation of explants with 0.1-0.5 mg/L of NAA resulted in the higher propagation rates for all studied species. Petiole and leaf explants had the ability to form roots all over the explant. Also, leaves of all the studied species were more frequent to form roots on the medium with kinetin and NAA. Thus, for the studied Crambe species a determinacy of different explants for a certain type of morphogenesis was found: roots initialized callus tissue (more than 100 mm wide), leaves formed roots across the entire explant and petioles - plantlets. The highest propagation rates were noted for C. maritimа (for all types of explants studied) and the lowest propagation rates - for C. koktebelica, though petiole explants are advisable for its microclonal multiplication. The rooting of plantlets was studied on hormone-free MS medium or with twice reduced sucrose, macro- and microelements content (MS/2). C. steveniana plantlets had the highest rooting frequency and C. maritima had the lowest. Slight decrease of rooting frequency was observed as a result of cultivation on MS/2 medium. The in vivo adaptation of plantlets with nicely developed roots was conducted with different substratum: peat and sand blend (3:1), peat and perlite blend (2:1) indoor with fluorescent lamps and at +23 ± 2°С. The changes in biochemical properties of in vitro and in vivo cultured plants were shown to be due to the difference in the cultivation conditions. Also, no changes in the genotype following in vitro cultivation for all studied species except for C. steveniana (genetic polymorphism in aseptic plants of which was demonstrated) were shown. Therefore, biotechnology methods for conservation of five endangered Crambe species were established. Key words: Crambe koktebelica, C. tataria, C. aspera, C. steveniana, C. maritima, biodiversity, in vitro culture, genetic polymorphism.