Embryo abortion observation and in vitro embryo rescue technique of ‘Manaohong’ cherry

Abstract: 【Objective】Immature embryo abortion is the key factor limiting the efficiency of fruit tree breeding. With the conventional propagation, the abortive cherry seeds cannot germinate into seedlings.However, before embryo abortion occurs, the immature embryos can be rescued by tissue culture techniques, which may possibly obtain seedlings. Using in vitro culture, the present work attempted to solve the problem of embryo abortion in cherry‘Manaohong'. The time course embryo development after full-blossom was investigated. Based on the observation, an in vitro embryo rescue system was established.【Methods】Young embryos of‘Manaohong'cherry were sampled every 5 days from the 15th day after the full-blossom. The collected embryos were used for anatomical observation to obtain changes in the abortion rate, according to which the embryo abortion period was determined. In addition, germination of embryos at different developmental stages was investigated. The germination rate and embryo development index (PF) were used as the indicators to determine the optimal embryo rescue time. In the embryo rescue system, immature embryos at different periods after sterilization were transferred into a medium containing differential concentrations of thidiazuron (TDZ) , indolebutyric acid (IBA) and 6-benzyladenine (6-BA) for embryo germination. The optimal hormone concentration ratio was determined based on a L9 (34) orthogonal experiment. Using MS as the basic medium, the growth statue of immature embryos was observed after 30 days of cultivation; the germination rate was recorded; and the optimum growth regulator ratio for seedling germination was screened out. In order to determine the hormone ratio for multiplication, the seedlings were transferred into a MS medium supplied with several growth regulators, i.g. TDZ, IBA, 6-BA and gibberellic acid (GA3) at differential concentrations. The growth regulator concentration ratio was selected using a L9 (44) orthogonal experiment. After 30 days, the growth status of embryos under each treatment was observed; multiplication coefficient was recorded; and medium for multiple bud induction and proliferation were selected. The obtained cherry seedlings with good growth status were rooted in the medium of MS + 0.2 mg · L-1 IAA (Indole-3-acetic acid) and then transplanted into nutrient soil, and the survival rate was calculated.【Results】The results showed that the abortion rate of cherry embryos increased slowly from day 15 to day 25 but sharply from day 25 to day 30, and no significant change was observed from day 30 to day 35. Therefore, the large-scale abortion of immature embryos started around 30 days after full blossom. By morphological observation, the normal embryos were full, smooth and round, while the abortive ones were shriveled.Germination of the embryos at different developmental stages after the full-blossom, from day 15 to day25 was tested. The normal embryo development index was lower than 0.30, and the embryo germination rate was 0. Thus, it was difficult to rescue the embryo at this period. The PF value increased with development of the embryos, but the germination rate of embryos varied at different developmental stages. At day 25, the germination rate was the highest, reaching 34%, which was significantly higher than the germination rates in other developmental stages. Therefore, the best embryo rescue time was 25 days after the full-blossom. In the embryo rescue system, the immature embryos at 25 days after the fullblossom were transferred into media containing different proportions of growth regulators for embryo germination. The best medium for embryo germination was MS + 0.5 mg · L-1 TDZ + 0.5 mg · L-1 6-BA +1.0 mg · L-1 IBA, which gave a germination rate of 70%. After germination and growth of young embryos, cherry seedlings were obtained. The seedlings were transferred into medium with MS as the basic medium supplemented with differential concentrations of growth regulators to screen out the suitable medium for bud induction and proliferation. The best medium for shoot multiplication was found to be MS + 0.5 mg · L-1 TDZ + 0.1 mg · L-16-BA + 1.5 mg · L-1 IBA + 0.5 mg · L-1 GA3, which generated a growth coefficient of 5.45. Stem sections with buds of 2 to 4 cm in length were transferred into the rooting medium and cultured for 30 days. Acclimated seedlings were then selected and transplanted into sterilized nutrient soil with a survival rate reached up to 90%.【Conclusion】In this study, the stage of massive embryo abortion was determined in‘Manaohong'cherry. A stable embryo rescue system for‘Manaohong'cherry was established and regenerated cherry plants were successfully obtained.