Establishment of in vitro rapid propagation technology for an elite persimmon rootstock line, L938

Abstract: 【Objective】L938 is a widely compatible and cold resistant rootstock selected from Diospyros lotus L.. The cultivation practice in Shandong has proved its good grafting compatibility with nonPCNA (pollination constant non-astringent) persimmons such as Fuyu and Taishu, and can be applied as a cold resistant rootstock for non-PCNA persimmons in northern China. However, the elite line L938 of date plum is a male plant and cannot produce seeds, making its large- scale application a challenge. Therefore, in vitro rapid propagation technology of L938 for efficient reproduction of its asexual rootstock is important for large-scale nursery stock production of persimmon.【Methods】This study used persimmon rootstock L938 as the material and explored the effects of different explant materials, disinfection times, and plant growth regulator combinations on its regeneration process. New shoots (May), dormant buds, and new shoots (April) of L938 were used as explants, which were rinsed with laundrydetergent and placed under flowing water for 30 minutes before disinfecting treatment with 0.1% HgCl2 for 40 minutes. Effects of different types of explants on the initiation culture of L938 were studied. Stem segments during the growth period of L938 were used as the test material. The middle and upper parts of the new shoots were cut into segments before soaking in 5% laundry detergent water for 5 minutes, and then rinsed with running water for 30 minutes. After the stem segments were disinfected in 0.1 g · L-1 HgCl2 for 10, 20, 30, 40, or 50 minutes in an ultra-clean workbench, they were inoculated on 1/2MS medium and the effect of disinfection time on the plant regeneration from the explants were studied. In vitro seedlings with a plant height of about 1.5 cm growing on 1/2MS + 1.0 mg ·L-1 6-BA + 0.1 mg ·L-1 NAA medium were selected for subculture treatments. In an ultra-clean workbench, axillary buds of the in vitro seedlings were cut out and inoculated on 1/2MS, (1/2N) MS, or 1/4MS basic medium each containing 0.1 mg ·L-1 IAA+2.0 mg ·L-1 ZT. Then the effects of different basic media (1/2MS, (1/2N) MS, and 1/4MS) on the subculture of line L938 were studied. Robust in vitro shoots with a height of 2.0-3.0 cm were treated with six plant growth regulator combinations, namely, NAA (0.5 or 1.0 mg ·L-1 ) +IBA (1.0, 2.0, or 4.0 mg ·L-1 ), for root induction. Then the effects of different NAA+IBA combinations on rooting rate, root length, root surface area, and root volume were studied.【Results】After 30 days, the pollution rate, browning rate and survival rate of new shoots (May), dormant buds and new shoots (April) were investigated. The results showed that compared to the dormant buds and the new shoots (May), the new shoots (April) were the most suitable explants for in vitro culture of L938 date plum. After 40 minutes of disinfection with 0.1 g ·L- 1 HgCl2, the survival rate of explant from the new shoots (April) was up to 71.33%. 30 days after the explants were disinfected with 0.1 g ·L-1 HgCl2 for 10, 20, 30, 40, or 50 minutes, the contamination rate, browning rate, and survival rate of the explants were calculated. The results showed that prolonging the disinfection time with HgCl2 could reduce the contamination rate of the explants. The best disinfection effect was achieved by treating with 0.1 g·L-1 HgCl2 for 40 minutes, with a survival rate of 35.3%. The axillary buds of were excised from the in vitro seedlings and inoculated onto 1/2MS, (1/2N) MS, or 1/4MS basic media containing 0.1 mg ·L- 1 IAA+2.0 mg ·L-1 ZT. After 30 days, the proliferation coefficient of new shoots and the grading of new shoots from the in vitro seedlings were recorded. The results indicated that 1/2MS medium was more suitable for subculture of date plum L938, with a new shoot proliferation coefficient of 4.4. Single buds of from the tissue culture seedlings were inoculated into medium supplemented six combinations of NAA (0.5, 1.0 mg ·L-1 ) and IBA (1.0, 2.0, 4.0 mg ·L-1 ) to induce rooting. After 90 days, the root length, root surface area and root volume were measured. The results showed that in the treatment of 1/2MS＋ 1.0 mg·L-1 NAA+1.0 mg·L-1 IBA medium, the rooting rate, root length, root surface area and root volume were 93.33% , 29.08 cm, 19.53 cm2 and 1.05 cm3 , respectively.【Conclusion】This study first screened the most suitable explants and disinfection time for the initiation culture of the date plum Line L938. Results showed that when new shoots (May) were used as explants, the survival rate could reach 71.33% after 40 minutes of disinfection with 0.1 g · L- 1 HgCl2. Then 1/2MS medium was found to be most suitable for subculture of date plum L938 tissue cultured seedlings, with a shoot proliferation coefficient of 4.4. Finally, 1.0 mg·L-1 NAA+1.0 mg·L-1 IBA was the most suitable combination for inducing roots. The study established an efficient in vitro regeneration and rapid propagation technology system of persimmon rootstock superior line L938.