Дія регуляторів росту на регенераційну здатність експлантатів рослин Quercus Robur L. in vitro

Анотація

EFFECT OF GROWTH REGULATORS ON IN VITRO REGENERATIVE ABILITY OF EXPLANTS OF QUERCUS ROBUR L.

O. Yu. Chornobrov

Development of efficient in vitro mass production technology of uninfected planting material of valuable forest-forming woody plants, including English oak (Quercus robur L.), is one of the urgent tasks of today. The alternative to traditional propagation of the plants of species Q. robur is the use of biotechnological methods, in particular, the tools of in vitro plant tissue culture, that allows obtaining lot virus-free plant regenerants during the year. Today micropropagation of valuable plants of species Q. robur is carried in Poland (Institute of Dendrology Polish Academy of Sciences), Belarus (Institute of Forest of NAS of Belarus) and Ukraine (SI "Institute of Food biotechnology and genomics NAS of Ukraine"). Domestic and foreign authors note that getting stably growing in vitro culture explants of woody plants of the family Fagaceae Dumort. is quite difficult since the regenerative ability significantly slowed down with the aging. The purpose of the research was to determine the effect of growth regulators on in vitro regenerative ability of Q. robur explants.

For the studies that were carried in January and February 2016, the embryos with fragments of the endosperm of 120-year-old donor plant were used. The sterilization of the plant material was carried out by using 0,1 % HgCl₂ (5‒21 min.) with preprocessing by means of 70 % ethanol (within 1 min.). The embryos were administered into the culture in vitro on the hormoneless nutrient medium prescribed by T. Murashige & F. Skoog (MS). The regenerative ability of plant material in vitro was studied on a nutrient media MS with the addition of
2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthylacetic acid (NAA),
6-Benzyladenine (BAP), 6-(Furfurylamino)purine (kinetin) and
6-(ɣ,ɣ - Dimethylallylamino)purine (2-iP). The modified MS was made by introducing to the medium 100 mg·L^-1 myo-Inositol, 30 g·L^-1 sucrose and
7.0–7.3 g·L^-1 microbiological agar; 2 g·L^-1 activated carbon were added in the culture medium to neutralize secondary metabolites. An index of acidity (pH) was adjusted to the level of 5,7–5,9. Callus tissue was obtained from sterile leaf blades on which by using scalpel artificially were made notches. The intensity and frequency of explants callus formation were recorded on the thirtieth day of cultivation. Plant material was cultivated in a light room and a thermostat TS-80 with no light at a temperature of 25±1 °C and lighting 2 000–3 000 Lux with 16 h light/8 h dark photoperiod and a relative humidity of 70–75 %. The following study methods were exploited: biotechnological (plant tissue culture in vitro, microclonal propagation, callus culture), statistical. Statistical analysis of experimental data was performed using analysis package MS Excel.

Effective sterilization (above 80 %) of embryos of Q. robur that was obtained in January and February from 120-year-old donor plant was achieved through the use of 0.1 % HgCl₂ within 15–16 minutes. At the stage of introduction of Q. robur plant material to the culture in vitro should be done alternation of hormoneless culture medium (MS with 2.0 g·L^-1 activated carbon) and hormonal (MS with addition of 0.25 mg·L^-1 kinetin or 0.5 mg·L^-1 BAP) under cultivation cycle of 5–7 days. The influence of growth regulators of auxin and cytokinin types of action on in vitro regenerative ability of plant tissues of species Q. robur was studied. Shoots of Q. robur received by using the activation of growth of explants meristem and direct morphogenesis. The optimal conditions for induction of callus formation in leaf blade tissues of plants Q. robur with a frequency of more than 90 % and active growth were received on MS medium with the addition of 1.0 mg·L^-1 BAP and 0.5 mg·L^-1 NAA in an incubator and without light. An active shoots formation in plant explants in vitro was fixed on MS medium modified with 1.0 mg·L^-1 2-iP and 20 mg·L^-1 of adenine.

Therefore, as a result of the conducted studies we have investigated in vitro regenerative ability of plant tissues of species Q. robur under the influence of growth regulators and obtained shoots by activating the growth of existing explants meristem and direct morphogenesis. Further studies are aimed at obtaining stably growing in vitro cultures of species Q. robur on modified basic nutrient media for mass microclonal propagation with the following adaptation to the conditions in vivo.