Study On The Protoplast Culture And Gene Transformation Of Actinidia Arguta

A.arguta is an important species in Actinidialts fruits are hairless, juicy and flavorful. A.arguta contains very high amount of Vitamin C. It is a very hardy species,since the plant can safely survive in severe winter (-38℃).The other valuable characters of the species are insect and disease-resistant.To date, few biotechnological studies on the species have been reported, less than suspension culture, protoplast culture and gene transformation. In this study, we have established the systems of tissue culture and suspension culture and gene transformation, obtained cell colonies from leaves, leaf calli and suspension calli protoplast, and for the first time gotten calli from leaf calli protoplast. We have also successfully transferred GUS gene into A.arguta protoplasts by PEG method.Cultured on twenty MS media added with different auxins, stem segments and leaves of in vitro seedlings of A.arguta produced calli and bud differention. For stem explants, l/2MSo, MSo and MS media supplemented with 0.8mg/L IAA and 1.0 mg/L ZT gave the best results in term of bud differention and the growth of the regeneration shoots. While the optimal media for leaf explants were MS media supplemented with 0.5mg/L 2,4-D, 2.0 mg/L ZT and 0.3 mg/L NAA or 2.0 mg/L ZT and 0.3 mg/L NAA.The white or light green calli from MS'media supplemented with 2.0 mg/L ZT and 0.3 mg/L NAA were cultured in ten MS liquid media added with different auxins. The best liquid medium for suspension culture of A.arguta was MS supplemented with 1.0 mg/L 2,4-D and 0.3 mg/L NAA.Leaves from tissue culture, leaf calli and suspension calli of A.arguta were used for protoplast isolation. Culture condition for donor materials affected not only protoplast49isolation but also protoplast culture later on. Newly expanding materials which cultured in darkness about 2-7days at low temperature(4℃) for A.arguta were found to be suitable for protoplast isolation and culture. Protoplasts from different materials were cultured in MS (devoid of NHt*), B5, KM8p, NT and CPW media added with different auxins, the ideal medium for A.arguta protoplast culture was MS medium. ZT and 2,4-D or KT were essential for A.arguta protoplasts to divide. MS supplemented with 1.0 mg/L 2,4-D +0.5 mg/L ZT and 0.5 mg/L ZT + 0.5mg/L 2,4-D were suitable media for calli and leaves respectively. The first division of the A.arguta protoplasts took place about 5-7 days of culture, followed by 2nd at 14 days of culture. Regenerated cell colonies were seen on 30th day of culture, and finally the colonies developed into calli visible to the naked eyes on 55th day.GUS gene was successfully transfered into A.arguta protoplasts by PEG method. Transformation was affected by material condition, different PEG solution and the time of heat stimulus.40% PEG (MW6000, final concentration was 8.2%) and mixture solution of CPW and 0.2M CaCl2 (v/v) were used in the study.These studies offered new opportunities to facilitate and expedite further research of the somatic hybridization and gene tranfermation of A.arguta.