Establishment and optimization of in vitro rapid propagation technology of Xiaoguo Tianshi as compatible rootstock for persimmon

Abstract：【Objective】Xiaoguo Tianshi (Diospyros kaki Thunb.) is a kind of persimmon rootstock collected from Dabieshan Mountain area of Hubei Province. Xiaoguo Tianshi has been confirmed to have superior grafting compatibility with persimmon varieties as Taishu, Soshu and Fuyu PCNA (pollinationconstant non-astringent). In recent years, Xiaoguo Tianshi has been widely used in commercial persimmon propagation industry in China. However, large- scale clonal vegetative propagation system for Xiaoguo Tianshi rootstock has not been established. The efficient rooting conditions in vitro for Xiaoguo Tianshi need to further investigation. The aim of this study was to establish the tissue cultureand rapid propagation system of‘Xiaoguo Tianshi’and optimize its rooting conditions.【Methods】 The branches of Xiaoguo Tianshi with robust dormant buds were collected in winter, then cut into stem segments and put into sterilized conical flask with Tween-20 for 1-2 min, lately washed with sterile water for 2-3 times. The buds were soaked in 75% alcohol for 30 s, then disinfected with sodium hypochlorite of different concentrations (0.5%, 1%, 2%, 2.5%) for 6 minutes, washed with sterile water for several times, and the scales outside the buds were removed with scalpel. The tips were placed on MS medium (1/2 NH4NO3 and 1/2 KNO3) plus 1 mg · L^-1 ZT, 0.1 mg · L^-1 IAA, 30 mg · L^-1 sucrose, 7 g · L^-1 agar and 0.6 g · L^-1 PVP-40, cultured at 25 ℃, 2000 lx light intensity and 16 h/d. The in vitro plantlets derived from the primary culture were cut into stem segments of 1-1.5 cm length and trasfered on 6 kinds of proliferation media containing DKW+1 mg·L^-1 ZT+0.1 mg·L^-1 IAA+30 mg·^L-1 sucrose +7 g·L^-1 agar + 0.6 g·L^-1 PVP-40 with betaine at different concentration (0, 10, 50, 100, 1000, 2000 μmol·L^-1 ). The culture conditions were the same as that of the primary culture. Two methods were used for rooting culture of Xiaoguo Tianshi. (1) Dipping method: the base of shoots was soaked with different kinds of plant growth regulators at different concentrations and then cultured on 1/2 MS basic medium+1 mg·L^-1 activated carbon +30 mg·L^-1 sucrose +7 g·L^-1 agar +0.6 g·L^-1 PVP-40. The shoots were dark treated for 10 days and then transferred to the basic medium without plant growth regulator. (2) Two- step rooting method: different kinds and concentrations of plant growth regulator were added into the basic medium, and the 2-3 cm rootless plantlets obtained from proliferation culture were excised and trasfered to the rooting media. After dark treatment of 5, 10 or 15 days, they were transferred into the basic medium without plant growth regulator to induce rooting.【Results】(1) After 30 days culture, embryogenic callus appeared and differentiated into axillary buds and leaves from 62.56% of the primary culture, but there was still a small amount of endophytic bacteria contamination, which needed to further eliminated by subsequent subculture. (2) After treatment with betaine at different concentrations for 30 days, the leaves of the plantlets turned dark green, and the in vitro culture medium supplemented with 1000 µmol · L^{- 1} betaine was most beneficial to the proliferation and growth of the plantlets. The average number of the proliferation rate was 1.59, the average plant height was 3.29 cm, the average number of leaves per plant was 9.49, and the average number of stem nodes per plant was 7.40. During subculture, each 0.5 cm stem with buds of the plantlets was excised for proliferation, and the effective number of subculture stem segment reached 10.46 per generation. (3) After rooting treatment by dipping with 100 mg · L^{- 1} NAA for 10 min, the rooting percentage reached 90.00%, the average total root length was 23.17 cm, the average root surface area was 6.73 cm2 , and the average root volume was 0.19 cm3 . (4) The stem segments were darkly treated with 1/2 MS+0.5 mg·L^-1 NAA+0.2 mg·L^-1 IBA+5 mg·L^-1 MT (melatonin) + 30 mg ·L^{- 1} sucrose +7 g ·L^{- 1} agar +0.6 g ·L^{- 1} PVP-40 medium for 10 days, and then transfered to hormone free 1/2 MS medium. The rooting percentage achieved 71.50% by the two-step rooting method. The average root length, root surface area and root volume were 24.14 cm, 9.74 cm² and 0.27 cm³ , respectively. (5) The survival rate of Xiaoguo Tianshi plantlets was 52.5% after transplanting. The average plant height and stem diameter was 49.17 cm and 6.0 mm at 8 months after acclimatization and transplanting to the field.【Conclusion】An effective protocol was established for propagating Xiaoguo Tianshi as rootstock for persimmon. The better rooting effect was obtained when 0.5 mg · L^-1 NAA+ 0.2 mg ·L^-1 IBA+5 mg ·L^-1 MT was added to the basic medium in dark treatment for 10 days. Dipping method for induction of rooting had the advantages of low cost and simple operation, and has the potential to be to used in the nursery industry.