关键词: 长白落叶松, 体胚诱导, 胚性愈伤组织, 体细胞胚胎, 植株再生

Abstract: [Objective] Immature zygotic embryos of Larix olgensis were used as explants to study induction, proliferation and somatic embryogenesis of embryogenic callus in order to reveal the key factors affecting Larix olgensis embryogenic callus induction,and at the same time, to explore the effects of growth regulators of different types and concentrations on proliferation and somatic embryogenesis of embryogenic callus, in subculture process. [Method] Embryogenic callus was induced using immature seeds from superior individuals of three families of Larix olgensis, and the effects of seed collection time, family, concentration of 2,4-D and basic medium on the induction of embryogenic callus were studied. Subsequently, the embryogenic callus subculture on the proliferation medium containing different kinds and concentrations of growth regulators, and the somatic embryos were obtained by the process of somatic embryogenesis. Finally, morphologically normal somatic embryos were selected and germinated, and transplanted after rooting of the somatic embryos. [Result] There was significant differences in induction rate of embryonic callus of immature embryos with different collection times. The induction rate was 5.61% 63 days after pollination, the induction rate was 22.35% 70 days after pollination, and the induction rate was zero 80 days after pollination. In this experiment, the average induction rate of embryogenic callus from the families of‘77-22’,‘77-37’and‘73-50’was 6.69%, 11.17% and 3.11%, respectively. The concentration of 2,4-D had a certain effect on the induction of embryogenic callus, the induction rate was increased with the proliferated of 2,4-D concentration in a certain range. It was up to 11.11% when the concentration of 2,4-D reached 1.5 mg·L^-1. The induction rate began to decrease when the concentration of 2,4-D exceeded 1.5 mg·L^-1. The medium of BM, MS and S were able to induce embryogenic callus, the induction rate in BM medium was the highest, followed by S medium, and the induction rate in MS medium was the lowest. Embryogenic callus culturing for 15 days on BM medium which containing 2,4-D 0.3 mg·L^-1, 6-BA 0.1 mg·L^-1 and KT 0.1 mg·L^-1 can obtain more embryogenic callus, the proliferation rate was up to 345.93%. Culturing for 14 days on BM medium which containing 2,4-D 1.5 mg·L^-1, 6-BA 0.5 mg·L^-1 and KT 0.5 mg·L^-1, the number of somatic embryos per gram embryogenic callus was up to 179.87 on average. The germination rate of somatic embryo and regeneration rate of plantlets was up to 75% and 66.67%, respectively. The survival rate of transplanting was 27.08%.[Conclusion] The embryos of L. olgensis seeds collected 70 days after open-pollination was suitable to induce embryogenic callus, the basic medium BM contains 2,4-D 1.5 mg·L^-1. Embryogenic callus was prone to lose the ability of somatic embryogenesis in the medium with high concentration of exogenous hormones, and the proliferation speed was slow, but the number and the germination rate of somatic embryo were relatively high. An appropriate decrease of the concentration of the growth regulator was beneficial to keep the ability of somatic embryogenesis and proliferation of callus, but the quantity and the germination rate of somatic embryogenesis were somewhat decreased. It was not conducive to the proliferation of callus when the concentration of the growth regulator in the medium was low, but the ability of somatic embryogenesis was maintained for a long time. The proliferation of embryogenic callus can be improved by using NAA 0.15 mg·L^-1 instead of 2,4-D 0.5 mg·L^-1, and the germination rate of somatic embryos can be improved to a certain level. Therefore, according to different purposes at different stages, it was necessary to select the medium containing different types and concentrations of exogenous hormones for subculture.

Key words: Larix olgensis, induction of somatic embryo, embryogenic callus, somatic embryogenesis, plantlet regeneration