A study of abnormal seedling occurrence from rescued embryos of seedless grapes and their transformation into normal seedlings

Abstract: 【Objective】The objective of this study was to explore the causes of abnormal plantlets in the process of embryo rescue of seedless grapes and to find effective measures to prevent the occur- rence abnormal seedlings. With embryo rescue, efficiency of seedless grape breeding can be improved.【Methods】Abnormal seedlings were used as the test materials. These abnormal seedlings developed from embryos rescued in vitro from self-pollination of four seedless grape (Vitis vinifera L.) cultivars‘( Thompson seedless’‘Flame seedless’‘Heshi seedless’and‘Ruby seedless’) and embryos from dif- ferent crosses of seedless grapes. The effects of different parent genotypes and different embryo germi- nation media on the emergence of abnormal seedlings rescued from seedless grape embryos were studied. The 4 different parent genotypes were‘Thompson seedless’‘Flame seedless’‘Heshi seedless’and‘Ruby seedless’. The 5 different media were MS, MM3, MM4, ER, and modified MM3. Different methods for transforming abnormal seedlings into normal seedlings were studied in vitro. The methods included 4 different culture media and different subculture durations. The media included WPM (W1), WPM + 6-BA 2.0mg·L^-1 (W2), WPM + 6-BA 2.0 mg·L^-1 + IAA 2.0 mg·L^-1 (W3) and WPM + IAA 2.0 mg·L^-1 (W4). These media were all added with agar 6 g·L^-1 + sucrose 20 g·L^-1 + activated carbon 2.0 g·L^-1. Normal seedlings were observed and their number was counted after 45 days. The different subculture  durations were 7 d, 14 d, 21 d and 28 d, and normal seedlings were counted after 60 days.【Results】It was difficult to rescue‘Thompson seedless’embryos. The proportion of abnormal seedlings from res- cued embryos of‘Thompson seedless’and‘Heshi seedless’was higher that from the other cultivars. The 5 embryos development media were used to culture in vitro embryos from self-pollination of the four seedless grape cultivars. The percentage of abnormal seedlings was always highest on the MS me- dium. The percentage of abnormal seedlings of‘Thompson seedless’was 33.3%,‘Flame seedless’37.5%,‘Heshi seedless’33.3%, and‘Ruby seedless’23.1%. The abnormal germination embryo rate of the 5 media followed a pattern of modified MM3< MM3< ER< MM4 < MS. Therefore, MS medium was not suitable to be used as the rescuing medium for seedless grape embryos. The abnormal grape seedlings were classified into 5 types: (1) albino seedlings, (2) vitrified seedlings, (3) seedlings with ex- cessive growth, (4) plantlets with contorted cotyledon and no roots, and (5) plantlet without shoot tip. All these different types of abnormal seedlings could be transformed into normal seedlings using appro- priate media. The percent of seedlings transformed to normal ones was highest (24.6%) when cultured on WPM+6-BA 2.0 mg·L-1 +IAA 2.0 mg·L-1, and lowest (8.1%) when cultured on WPM. After leggy seedlings were cut for subculture every 14 days, the percentage of normal seedlings was higher (33.3%) than other intervals. Albino and vitrified seedlings were not able to turn into normal seedlings. Finally, these normal seedlings were hardened and transplanted in the field, and the operation steps for seedling hardening and transplantation into fields were summarized.【Conclusion】Abnormal plantlet occurrence from rescued embryos of‘Thompson seedless’,‘Heshi seedless’was high, especially cultured in MS medium.WPM+6-BA2.0mg·L-1+IAA2.0mg·L-1 +agar6g·L-1 +sucrose20g·L-1 +activatedcarbon 2.0 g·L-1 was suitable to normalize the abnormal seedlings. For leggy plantlets, the transformation rate was high with a subculture duration of 14 days, reaching 33.3%. The abnormal plantlets produced by embryo rescue of seedless grapes can be transferred to normal plantlets on proper media and subcultured in a proper way. The abnormal plantlets except albino plantlets and vitrified plantlets could be transformed into normal plantlets.