广亲和砧木小果甜柿离体快繁技术建立及优化

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

【目的】近年来广亲和砧木类型小果甜柿已经在我国柿产业中广泛应用,但尚未建立起规模化无性系营养繁殖体系,尤其是离体培养的生根条件亟待优化,旨在建立小果甜柿组培快繁体系,并优化其生根条件。【方法】以小果甜柿为试材,重点探讨无菌离体培养过程中的生长调节剂种类、处理时间,以及暗处理等因素对其试管苗生根的影响。【结果】(1)休眠芽经75%乙醇消毒30 s,再使用1%次氯酸钠消毒6 min,消毒效果最佳,消毒后的休眠芽接种到含MS(Murashige&Skoog)(含1/2 NH4NO3和1/2 KNO3)+1 mg·L^(-1)玉米素(zeatin,ZT)+0.1 mg·L^(-1)吲哚乙酸(indole-3-acetic acid,IAA)+30 mg·L^(-1)蔗糖+7 g·L^(-1)琼脂+0.6 g·L^(-1)聚乙烯吡咯烷酮-40(polyvinylpyrrolidone-40,PVP-40)的初代培养基上,可获得62.56%的无根试管苗;(2)取初代培养试管苗侧芽接种于DKW+1 mg·L^(-1)ZT+0.1 mg·L^(-1)IAA+1000μmol·L^(-1)甜菜碱+30 mg·L^(-1)蔗糖+7 g·L^(-1)琼脂+0.6 g·L^(-1)PVP-40的培养基上,平均分蘖数1.59个,株高度3.30 cm,叶片数9.49片·株-1,茎节数7.40节·株-1,继代培养时可按照0.5 cm切取带芽茎段继续增殖培养,有效继代数目可达10.46个·代-1;(3)用100 mg·L^(-1)萘乙酸(1-naphthaleneacetic acid,NAA)浸蘸生根处理茎段基部10 min,转入1/2 MS培养基,生根率90.00%,平均总根长度23.17 cm,平均根系表面积6.73 cm^(2),平均根系体积0.19 cm3;(4)茎段在1/2 MS+0.5 mg·L^(-1)NAA+0.2 mg·L^(-1)IBA+5 mg·L^(-1)褪黑素(melatonin,MT)+30 mg·L^(-1)蔗糖+7 g·L^(-1)琼脂+0.6 g·L^(-1)PVP-40的培养基中暗处理10 d后,再接种至无激素1/2 MS培养基上,生根率71.50%,平均总根长度24.14 cm,平均根系表面积9.74 cm^(2),平均根系体积0.27 cm3;(5)生根后的组培苗经闭瓶和开盖炼苗后移栽,成苗率52.5%,移栽至大田8个月后平均株高度49.17 cm,茎粗6.0 mm,可用于接穗的嫁接繁殖。【结论】构建的小果甜柿高效离体快速繁殖体系,为优质甜柿苗木的规模化营养繁殖提供了科学依据。

【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 culture and 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(0,10,50,100,1000,2000μmol·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 1000μmol·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 cm^(2),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 cm^(2)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.