Effects of different plant growth regulators on rapid propagation of three types of grape rootstocks based on quadratic regression orthogonal rotation

Abstract:【Objective】Xinjiang is the largest grape producing area in our country, and grape industry has developed rapidly, but the problems of soil drought and salinization in some areas have become the factors restricting its industrial development. Using resistant rootstocks has become one of the most effective measures for grape cultivation in the world. However, in actual production, grape rootstock breeding quantity is limited, because most rootstocks are susceptible to virus. Therefore, using plant tissue culture to cultivate strong seedlings has become one of the important ways. This study aimed to establish a high-efficiency stem section rapid propagation system by screening different media and plant growth regulators concentration for 5BB, 1103P and SO4 grape rootstock varieties in the process of primary culture, subculture, and rooting culture, so as to provide reference for rapid propagation and production of grape rootstocks.【Methods】The test materials were 5BB, 1103P and SO4 annual rootstockseedlings from the Sanping Teaching Practice Base of Xinjiang Agricultural University, which were planted in the Characteristic Fruit Tree Research Center, College of Horticulture, Xinjiang Agricultural University for potted seedling cultivation. After about 60 days, the diameters of 1-2 mm thick semi-lignified canes were taken for experimental treatment. Explant disinfection was as below: cutting new shoots into 2-3 cm long stem segment with a single bud, putting into the beaker, covering with gauze, rinsing 1 hour, and removing surface stains. Then, the explants were soaked in 75% ethanol for 30 s on the ultra-clean bench, disinfected with 0.10% HgCl2 solution for 8 minutes, and rinsed with sterile water for 3-5 times to remove the residual disinfectant on the surface of the explants. Finally, the stems were placed in the sterile inoculation plate for further use. In primary culture, B5, MS and WPM medium were used as the basic medium, and 2.00 mg·L-1 6-BA, 0.20 mg·L^-1 IBA, 0.20 mg·L^-1 GA3, 30 g·L^-1 sucrose, and 7 g ·L^-1 agar were added at the same time, pH was adjusted to 5.80, 1 stem segment per bottle, 10 bottles for each medium, and the experiment was repeated 3 times. After 30 days of culture, the pollution rate, mortality rate and axillary bud germination rate were counted. The subculture adopted a quadratic regression orthogonal rotation combined experimental design, and different concentrations of 6-BA (concentration of 1-3.00 mg·L^-1 ), IBA (concentration of 0-0.50 mg·L^-1 ), and GA3 (concentration of 0-1.00 mg·L^-1 ) were added, and its effect on the proliferation coefficient was explored. In rooting culture, 1/2MS medium was used as the basic medium, and different concentrations of IBA (0, 0.50 mg·L^-1 , 1.00 mg·L^-1 , 1.50 mg·L^-1 ) and Ac (0, 0.10 g·L^-1 0.20 g·L^-1 ) were added, and its effect on rooting coefficient was explored. During the whole culture process, the culture conditions were as follows: the culture temperature was (25±1) ℃, the light intensity was 2000-2500 lx, and the lighting time was 12 h·d-1 . 【Results】During primary culture, by screening three different media, it was found that compared with B5 medium and WPM medium, the primary medium suitable for the three types of grape rootstocks were MS medium, supplemented with 2.00 mg ·L^-1 6-BA, 0.20 mg ·L^-1 IBA, and 0.20 mg ·L^-1 GA3, and the germination rate could reach 100%. During subculture, the effects of different concentrations of 6- BA, IBA and GA3 on the proliferation coefficient of grape rootstocks were explored through quadratic regression orthogonal rotation combined experimental design. It was found that the primary and secondary order of the three plant growth regulators affecting the proliferation and culture of 5BB and 1103P grape rootstocks was: IBA＞GA3＞6-BA, and the most suitable proliferation culture formula was MS+ 2.00 mg ·L^-1 6-BA+0.25 mg ·L^-1 IBA+0.50 mg ·L^-1 GA3; the primary and secondary order of the three plant growth regulators affecting the proliferation and culture of SO4 grape rootstocks was: 6- BA＞ GA3＞IBA, and the most suitable proliferation culture formula was MS+1.52 mg·L^-1 6-BA+0.25 mg·L^-1 IBA+0.36 mg·L-1 GA3. During rooting culture, by adding different concentrations of IBA and Ac, under different dark treatment conditions (0 d, 3 d and 7 d), the optimal concentration and dark treatment time of plant growth regulators for rooting culture were explored. It was found that the most suitable culture formula for rooting culture of 5BB and SO4 rootstocks was 1/2MS+1.50 mg · L^-1 IBA+0.10 g · L^-1 Ac, and dark-treated for 7 days, the rooting coefficient of 5BB rootstocks could reach 2.60, and that of SO4 rootstocks up to 1.47. The most suitable culture formula for rooting culture of 1103P rootstock was 1/ 2MS+1.00 mg · L^{- 1} IBA+0.10 g · L^{- 1} Ac, and dark-treated for 7 days, and the rooting coefficient could reach 2.30.【Conclusion】The most suitable medium for primary culture of three types of grape rootstocks was MS medium. The optimal concentrations of 6-BA, IBA and GA3 for 5BB and 1103P grape rootstocks were 2.00 mg·L^-1 , 0.25 mg·L^-1 and 0.50 mg·L-1 , respectively. The optimal concentrations of 6-BA, IBA and GA3 for SO4 grape rootstocks were 1.52 mg·L^-1 , 0.25 mg·L^-1 and 0.36 mg·L^-1 . The optimal dark culture time for rooting of the three types of grape rootstocks was 7 d, and the Ac concentra-tion was 0.10 g ·L^{- 1} . The optimal IBA concentration for rooting of 5BB and SO4 grape rootstocks was 1.50 mg · L^{- 1} . The rooting coefficient of 5BB grape rootstocks was 2.60, and that of SO4 grape rootstocks was 1.47. The optimal IBA concentration for rooting culture of 1103P grape rootstock was 1.00 mg·L^-1 , and the rooting coefficient was 2.30. In the study it was found that the effects of different media and plant growth regulators on the stem rapid propagation of three types of grape rootstocks were preliminarily explored, and a reference for the rapid propagation and production application of grape rootstocks was provided.