A study on the factors influencing rescue success of the embryo in stenopermocarpic grape

Abstract:【Objective】The objective of this study was to explore the factors affecting embryo development and embryo germination in the process of seedless grape embryo rescue so that effective regulatory measures will be taken improve embryo rescue in vitro, which will help improve efficiency of breed- ing seedless grape.【Methods】The effects of exogenous hormones (IAA and 6-BA) and natural nutrients (grape juice, coconut powder, banana puree, and walnut puree) on embryo germination were stud- ied with embryos taken from Zaoheibao fruitlet 100 days after anthesis (DAA) in order to screen the best medium for embryo germination. In addition, ovules from two seedless grape varieties, Wuhecui- bao and Lihongbao were harvested at 28-38 DAA and 28-32 DAA, respectively. These two seedless grapes are varieties bred by Shanxi Fruit Research Institute from Guibao × Centennial seedless, and both belong to Vitis vinifera L. The effects of different sampling periods and different in vitro culture time on embryo development rate was studied, and the polyembryony development was recorded. The rate of polyembryony development from in vitro ovules in seedless grapes at different sampling stages was compared. Then, the embryos were isolated and cultured in vitro. Embryo germination medium was solid WPM medium, which was selected through seed embryo germination test. In this experiment, the embryo germination rate, seedling formation rate, normal seedling rate and abnormal seedling rate of different embryo types were analyzed statistically.【Results】Adding exogenous hormones and natu- ral nutrients during embryo germination had a significant effect on embryo germination and seedling formation. The treatment with addition of 0.1 mg·L-1 IAA+0.2 mg·L-1 6-BA+0.5 g·L-1 grape juice generated the highest the embryo germination rate and seedling formation rate, which were 86.67%±6.67% and 70.00%±0.00%, respectively. During in vitro culture of ovule, the ovule development rate was dif- ferent at different sampling stages. Before the initial stage of embryo abortion, ovule development rate increased with the delay of sampling date. Sampling at the beginning of embryo abortion, the ovule development rate reached a maximum level, and after this stage, there was a significant drop. The embryos obtained from the ovules of Wuhecuibao and Lihongbao after 8 and 9 weeks of in vitro culture didnot germinate, but the embryos obtained after 10 weeks of culture had the highest germination rate (≥50.00%). Analysis of phenotypic differences between Wuhecuibao ovules cultured for 8 and 10 weeks showed that embryo cultured sampled after 10 weeks of cultured were mature (bright white embryos), while those after 8 weeks were immature. Polyembryony occurred in all the three seedless grape embry- os. The polyembryony development rate of the in vitro ovules of Wuhecuibao increased first and then decreased with the delay of sampling. Maximum value was observed at the beginning of embryo abor- tion. The variation trend was consistent with the development rate of ovule in vitro. There were signifi- cant differences in germination rate and seedling formation rate among different types of embryos. The embryo germination and plantlets development in vitro differed among stages of embryo development. Through comprehensive evaluation and analysis with entropy weight TOPSIS, it was concluded that cotyledon embryo had the best germination performance, which in other types of embryos followed an or-der of torpedo embryo>golbular embryo>heart embryo>malformed embryo.【Conclusion】Addition of 0.1 mg·L-1 IAA+0.2 mg·L-1 6-BA+0.5 g·L-1 grape juice into the medium produced the highest embryo germination rate and seedling formation rate. The highest rate of in vitro embryo development and the highest rate of polyembryony were obtained when ovules were taken prior to the initiation of embryo abortion. None of the embryos obtained from ovules in vitro cultured for 8 and 9 weeks germinated, while the embryos obtained after 10 weeks of in vitro culture could germinate, and the germination rate was ≥50.00%. There were significant differences in germination performance among embryos with dif- ferent shapes. The germination performance the rescued embryos followed an descending order of coty- ledon embryos> torpedo embryo > golbular embryo > heart embryo> malformed embryo.