In Vitro Plantlet Regeneration And Gene Transfer Method Of Male Plants Of ’Hongyang’ Kiwifruit

Kiwifruit（Actinidia chinensis Planch）is one of important fruits in the world,which is rich in nutrients and has very high levels of vitamin C.‘Hongyang’is an elite red-fleshed kiwifruit cultivar of Actinidia chinensis var.chinensis.It has been deeply loved by consumers because it is not only delicious,but contains lots of red anthocyanins having significant function of antioxidation.Kiwifruit is a dioecious plant,and female plants can fruit only after being pollinated.Lots of studies have proved that pollens of male plants have dramatic effects on both yields and quality of kiwifruit.‘Hongyang’has been cultivated in many regions in China,but it has no uniform varieties of male plants.Tissue culture of female plants of kiwifruit has been extensively studied,but there are few reports on in vitro plantlet regeneration of male plants of kiwifruit and no reports on their genetic transformation techniques.In this study,difficulties in surface sterilization and bud formation of both nodal and leaf explants collected in different orchards in Sichuan Province and Shanxi Province were investigated first;then,effects of plant hormones on multiplication and rooting of buds of male plant MX1 were studied,and a rapid clonal propagation protocol of MX1 was established;finally,some factors affecting Agrobacterium-mediated transgenic transformation effciencies of MX1 were investigated and transgenic plants were regenerated.The main results obtained were list as followings.1.It was very difficult to surface sterilize nodal and leaf explants of the 4 male plants of‘Hongyang’kiwifruit collected in 4 different orchards,but the abilities of nodal section and leaf pieces to form buds were not significantly different among the four male plants.When sodium hypochlorite solutions（1%active chlorine）was used as disinfectant,contamination rates of nodal sections of the four male plants ranged from 75%to 95%after they were sterilized for 20min,and contamination rates of leaf pieces of the four male plants were over 90%after they were sterilized for 15 min.Use of broad spectrum antimicrobial agent Plant Preservative Mixture（PPM）in medium at 0.2%（v/v）did not effectively decrease contamination rates of nodal and leaf explants.Within 5 weeks,most clean and survived nodal sections of the four male plants formed new buds on MS medium having 1.0 mg/L BA and 0.1 mg/L,and all clean and survived leaf explants of the four male plants formed callus first and then adventitious buds on medium containing 0.5 mg/L TDZ plus 0.5 mg/L NAA.2.The buds formed by nodal sections or leaf explants of the four male plants multiplied and grew well when they were subcultured on medium containing 0.3 mg/L BA、0.3 mg/L GA₃and 0.1%（v/v）PPM.3.But multiplication of male plant MX1 was affected significantly affected by different types and concentrations of cytokinins.Among the four tested cytokinins（BA,TDZ,ZT and KT）,BA was the most effective one,and the optimum level for was 0.5 mg/L;on the medium with 0.5 mg/L BA and 0.3 mg/L GA₃,95%of the nodal sections of MX1 produced multiple shoots and average multiplication efficiency was 5 within one month of incubation.4.Among the three tested auxins（IBA,IAA and NAA）,IBA was the most effective one to induce MX1 buds to root,and the optimum level for was 0.5 mg/L.On the medium with0.5 mg/L IBA,95.5%of the buds rooted within one month of induction and averagely a regenerated plant had 8 roots.The regenerated plants grew healthily after being transplanted in pots and the survival rate were 100%after 45 days of transplantion.5.All the leaf pieces excised from the regenerated plants of MX1 formed callus and later adventitious buds within one month of culture on MS medium having 3.0 mg/L BA and 0.2mg/L NAA.Addition of kanamycin（Kan）at 50 mg/L completely inhibited the leaf explants of MX1 to form callus and bud。6.Effects of agrobacterium cell concentrations,infection time,acetosyringone levels in co-cultivation medium and co-cultivation time on genetic transformation efficiencies were investigated using leaves of regenerated plants of MX1 as explants.The results showed that the agrobacterium solution with an O.D.₆₀₀ value of 0.5,immersing leaf pieces in agrobacterium solution for 15 min,addition of AS in co-cultivation medium at 100μM and co-cultivation of infected leaf explants for 3 days led the best transformation efficiencies.Under the optimized conditions,the regeneration efficiency of transgenic plants of MX1 was up to 10%.The transgenic plants were confirmed by both GUS staining and PCR.