Establishment of seedling formation from embryo culture in vitro of Diospyros lotus L.

【Objective】This study addressed the immature embryo culture system of Diospyros lotus L. and established an efficient seedling formation system.【Methods】Using embryos at different developmental stages as materials, comparative experiments were conducted to investigate the effects of embryo age, medium type, sucrose concentration, and combinations of IBA and 6-BA on embryo culture. Rooting conditions were optimized via dark culture, activated carbon/ink treatments, and root cutting, followed by detailed acclimation and transplantation protocols. The basic media were MS, 1/2MS, and (1/2N) MS; the sucrose concentrations were 30, 50, and 70 g · L- 1 ; the IBA concentrations were 0.04, 0.08, and 0.12 mg·L-1 ; and the 6-BA concentrations were 0.2, 0.4, and 0.6 mg·L-1 . The pH value of the medium was 5.7-5.8, and 600 mg · L- 1 of polyvinylpyrrolidone (PVP) was added to prevent browning. The medium was sterilized at 121 ℃ for 20 minutes in a high-pressure sterilizer. There were 10 bottles for each treatment, with 3 young embryos inoculated in each bottle, and the experiment was repeated 3 times. The different embryo development stages were 45, 55, 65, and 75 days after flowering, and the number of effective embryos that could be taken out within 10 minutes and 20 minutes at each development stage was counted, with 3 repetitions. After the young embryos were peeled off, they were inoculated on the medium of (1/2N) MS + 0.04 mg·L-1 IBA + 0.2 mg·L-1 6-BA + 50 g·L-1 sucrose + 7 g ·L-1 agar + 600 mg·L-1 PVP. Root cutting treatment: The embryos were inoculated on the medium of (1/2N) MS + 0.5 mg·L-1 IBA + 0.4 mg·L-1 6-BA + 30 g·L-1 sucrose + 7 g·L-1 agar + 600 mg·L-1 PVP. Induc-tion of rooting of the seedlings: (1) Dark treatment for 3 days after inoculation; (2) Adding 3 g·L-1 of activated carbon to the medium; (3) Adding 1% of ink to the medium; (4) Control. The medium for the treatments was (1/2N) MS + 0.08 mg ·L-1 IBA + 0.2 mg ·L-1 6-BA + 50 g ·L-1 sucrose + 7 g ·L-1 agar + 600 mg ·L- 1 PVP. When the activated carbon was added, the agar content was 9 g ·L- 1 , and the proportions of other substances remained unchanged.【Results】The best time for embryo extraction of Diospyros lotus L. was 55 days to 65 days after pollination; the most suitable medium was (1/2N) MS medium. After the young embryos were inoculated on the medium, the germination rate of the young embryos was high, the seedlings grew quickly, the leaves were large and dark green, the average height of the seedlings was 2.9 cm, the average weight of the seedlings was 0.4 g, the average length of the lateral roots was 4.32 cm, and the average number of lateral roots was 6.32; when the sucrose concentration in the medium was optimized to 50 g · L- 1 , the average height of the seedlings was 3.35 cm, the average weight was 0.42 g, the average number of lateral roots was 6.80, and the transplantation survival rate was 51.5%, which was the most suitable sucrose concentration for the growth and development of the young seedlings; when the young embryos of Diospyros lotus L. were inoculated on the medium containing (1/2N)MS + 0.04 mg·L-1 IBA + 0.6 mg·L-1 6-BA+ 30 g·L-1 sucrose + 7 g·L-1 agar, the germination rate of the embryos of Diospyros lotus L. was high, the seedling weight was 0.42 g, the main root length was 8.81 cm, the lateral root length was 6.45 cm. All inoculated embryos developed lateral roots, with no incidence of lateral- root- deficient seedlings, and the transplantation survival rate reached 62.7%; the root cutting treatment could transform the seedlings without lateral roots among the seedlings into normal plants, and the effective conversion rate exceeded 85%. After the conversion, the average number of lateral roots reached 6.47, and the maximum number of lateral roots could reach 25; dark culture for 3 days after the inoculation had the best effect on inducing rooting. After the seedlings were formed, the leaves of the seedlings were dark green, the growth was quick, and the average number of lateral roots reached 4.75.【Conclusion】This study established a standardized seedling formation system via embryo culture for D. lotus, which would provide a reference for genetic improvement and large-scale seedling production for the species.