Abstract: 【Objective】L938 is a wide grafting compatible and cold resistant rootstock selected from date plum. The cultivation practice of fruit trees in Shandong has shown that it has good grafting compatibility with non-PCNA (pollination constant non-astringent) persimmons such as ‘Fuyu’ and ‘Taishu’, and suitable to be applied as a cold resistant non-PCNA persimmon rootstock in Northern China. However, the superior line L938 of date plum is a male plant and cannot produce seeds (which is prone to trait segregation and has poor affinity for offspring), making its large-scale reproductive application a challenge. Therefore, using tissue culture rapid propagation technology to establish a regeneration system for date plum superior line L938 and efficiently reproduce its asexual rootstock can provide technical support and reference basis for variety promotion and tissue culture seedling large-scale breeding. 【Methods】This study used persimmon rootstock L938 as the test materials and focused on exploring the effects of different explant materials, disinfection time, and plant growth regulator concentration combinations on its regeneration process. (1) Using L938 new shoots (May), dormant buds, and new shoots (April) as explants, which were rinsed with laundry detergent and placed under flowing water for 30 minutes before disinfected with 0.1% HgCl2 for 40 minutes. Then the effects of different explants on the initiation of cultivation of date plum superior line L938 were studied. (2) Using the stem segments during the growth period of L938 as the test material, cut the middle and upper parts of the new shoots into branch segments before soaking them in 5% laundry detergent water for 5 minutes, and then rinse them under running water for 30 minutes. After disinfected the segments with 0.1 g/L HgCl2 for 5 different time points (10, 20, 30, 40, 50 minutes) in the ultra-clean workbench, they were inoculated on 1/2MS medium and the effect of different disinfection times on the implantation of external implants were studied. (3) Date plum tissue culture seedlings growing on 1/2MS+1.0 mg/L 6-BA+0.1mg/L NAA medium with a plant height of about 1.5 cm were selected for subculture treatment. In the ultra-clean workbench, axillary buds of date plum tissue culture seedlings were cut off and inoculated on 1/2MS, (1/2N) MS, and 1/4MS basic medium containing 0.1 mg/L IAA+2.0 mg/L ZT, respectively. Then the effects of different basic media (1/2MS, (1/2N) MS, and 1/4MS) on the subculture of date plum superior line L938 were studied. (4) The robust date plum superior line L938 single bud tissue culture seedlings with a plant height of 2.0-3.0 cm were treated with six plant growth regulator combinations, namely NAA (0.5, 1.0 mg/L) +IBA (1.0, 2.0, 4.0 mg/L), as induction rooting treatments. Then the effects of different concentrations of NAA+IBA combinations on their rooting rate, root length, root surface area, and root volume were studied. 【Results】(1) After 30 days, the pollution rate, browning rate and survival rate of new shoots (May), dormant buds and new shoots (April) were counted. The results showed that compared to dormant buds and new shoots (May), the new shoots (April) were the most suitable explants for the initiation and cultivation of the superior line L938 of date plum. After 40 minutes of disinfection with 0.1 g/L HgCl2, the survival rate of the new shoots (April) explants could up to 71.33%. (2) The explants were disinfected with 0.1 g/L HgCl2 for 5 periods of time (10, 20, 30, 40, 50 minutes), and 30 days later, the contamination rate, browning rate, and survival rate of the explants were calculated, respectively. The results showed that prolonging the disinfection time of HgCl2 can reduce the contamination rate of explants. The best disinfection effect was achieved by treating with 0.1 g/L HgCl2 for 40 minutes, with a survival rate of 35.3%. (3) The axillary buds of date plum tissue culture seedlings were cut off and inoculated into 1/2MS, (1/2N) MS, and 1/4MS basic media containing 0.1 mg/L IAA+2.0 mg/L ZT, respectively. After 30 days, the proliferation coefficient of new shoots and the grading of new shoots of the tissue culture seedlings were counted. The results indicated that 1/2MS medium is more suitable for subculture of date plum L938 tissue culture seedlings, with a new shoot proliferation coefficient of 4.4. (4) The single bud of tissue culture seedling of date plum superior line L938 was inoculated into six combinations of NAA (0.5, 1.0 mg/L) and IBA (1.0, 2.0, 4.0 mg/L) to induce rooting. After 90 days, the root length, root surface area and root volume were measured. The results showed that in the 1/2MS basic medium treatment, 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 superior line L938. Results showed that using new shoots (May) as explants the survival rate could reached 71.33% after 40 minutes of disinfection with 0.1 g/L HgCl2. Then 1/2MS medium is more suitable for subculture of date plum L938 tissue culture seedlings, with a shoot proliferation coefficient of 4.4. Finally, 1.0 mg/L NAA+1.0 mg/L IBA is the most suitable combination for inducing rooting, and a complete efficient in vitro regeneration and rapid propagation technology system of persimmon rootstock superior line L938 has been successfully established, which can provide scientific support and reference basis for the large-scale breeding of tissue culture seedlings.
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