Optimization of in vitro Tissues Growth of English Oak (Quercus Robur L.) Plants

Abstract

The development of an effective technology for the mass production of high quality planting material of English Oak plants (Quercus robur L.) is one of the urgent tasks of forestry in Ukraine nowadays. One approach to solving this problem is to use of in vitro the plant tissues culture method. This method, in contrast to conventional methods of reproduction, allows obtaining fungal and bacterial diseases-free, virus-free, genetically homogeneous plants in a minimum amount of donor material throughout the year, increasing the multiplication factor and obtaining new economically valuable variants and mutants due to the somaclonal variability.

A lot of number of biotechnological researches are focused on the development of effective microclonal propagation protocols for genotypes of plants of the Fagaceae Dumort. family. At the same time, the authors note that the regenerative ability of the tissues of woody plants of this family in vitro is rather low. 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", Separated Subdivision of NULES of Ukraine «Boyarka Forest Research Station»). The aim of the research was the optimization of growth of Q. robur in vitro shoots for mass microclonal propagation.

For the studies that were carried out in spring-summer period 2018–2019, the fragments shoots of 50–100-year-old donors plants of Q. robur were used. The sterilization of the plant material was carried out by using 0.1 % HgCl₂ (5‒10 min.), 1.0 % AgNO₃(10‒20 min.), 2.5 % NaClO (10‒20 min.) with preprocessing by means of 70 % ethanol (within 1 min.). The plants material (fragments shoots long 1.0−1.5 cm) were introduced into the culture in vitro on the hormoneless nutrient medium prescribed by MS (Murashige & Skoog) with the addition of  2.0 mg·L^-1 activated carbon. The regenerative ability of plant material in vitro was studied on a nutrient media MS, WPM (McCown & Lloyd, 1981) and DKW (Driver & Kuniyuki, 1984) on the 30th day of cultivation. The modified MS was made by introducing to the medium 100 mg·L^-1 myo-Inositol, 30 g·L^-1 sucrose, 7.0–7.3 g·L^-1 microbiological agar, as well as cytokinin type growth regulators (6-(Furfurylamino)purine (kinetin) 6-(ɣ,ɣ - Dimethylallylamino)purine (2-iP). For the optimization of  in vitro tissues growth of Q. robur plant a number of techniques were used, the effectiveness of which was determined by the following indicators: regenerative ability of shoots in vitro (%), length of shoots in vitro (cm), pigmentation. The following enterosorbents and antioxidants were used: polyvinylpyrrolidone (PVP, 2.0 mg·L^-1), activated carbon (2.0 g·L^-1), ascorbic acid (1.0 mg·L^-1). An index of acidity (pH) was adjusted to the level of 5.7–5.9. Plant material was incubated in a culture room at a temperature of 24±1 °C and lighting 2 000–3 000 Lux with 16 h light/8 h dark photoperiod and a relative humidity of 70–75 %. Biotechnological and statistical research methods were used. Statistical analysis of experimental data was performed using MS Excel analysis package.

It was found out that the effective sterilization (above 80 %) of Q. robur fragments shoots was obtained through the use of NaClO and AgNO₃. A significant regenerative ability (above 80%) had in vitro shoots cultured on WPM culture medium. An effective technique for optimization of growth of Q. robur tissues has been developed, which made it possible to obtain above 60% of regenerative plant material in vitro. A significant number of Q. robur plants was obtained on WPM culture medium with 2-iP, adenine, activated carbon and PVP (multiplication factor − 5.5 ± 0.7, cultivation cycle 45−50 days). Further studies are aimed microclonal propagation of shoots of Q. robur plants with various types of in vitro morphogenesis.