| A. arguta is an important species in Actinidia. Its fruits are juicy, flavorful and contain very high amount of Vitamin C. It is a very hardy species, since the plant can safely survive in severe winter (-38 ℃). Also, its fruit can be eaten directly.At the present time, few biotechnological studies on the species have been reported, less than suspension culture, protoplast culture and GFP gene transformation. In this study, we have established the systems of tissue culture, suspension culture and gene transformation, obtained cell colonies from the protoplasts of leaves, leaf callus and suspension callus, and for the first time gotten micro-callus from leaf callus protoplast. We have also successfully transferred GFP gene into A arguta protoplasts by PEG method.As far as the medium was concerned, we studied four kinds of medium, i.e. MS+30g/L sucrose, MS+lOg /L sucrose ,l/2MSo+30g /L sucrose ,l/2MSo+10g /L sucrose,and found out MS+30g /L sucrose was the best medium to the growth of seedlings. To the density of 2,4-D, 0.5~1.0 mg/L was much suitable for the callus inducement. We also got the result that the best material to get protoplast was the white friable callus .which was easy to divid in the MS medium with 2,4-D1.0 mg/L +ZT0.025 mg/L +1.0%PVP. The best liquid medium for suspension culture of A. arguta was MS supplemented with 2,4-D 1.0 mg/L + NAA0.3 mg/L.Leaf callus and suspension callus of A. arguta were used for protoplast isolation. Culture condition for donor materials affected not only protoplast isolation but also protoplast culture. The result showed materials especially leaves dealt with in the darkness about 5~7days at low temperature(4℃) were much better for protoplast isolation and culture. Protoplasts from different materials were cultured in MS (removed NH4+), NT, KM8p, and CPW media added with different auxin, the ideal medium for A. arguta protoplast culture was MS medium. MS medium supplemented with 2,4-D1.0 mg/L +ZT0.025 mg/L +1.0%PVP was suitable for both callus and leaf protoplasts. The first division of the A. arguta protoplasts took place after 5~7 days in the MS medium, the 2nd division happened after 14 days. Regenerated cell colonies were seen on the 30th day of culture. Threekinds of methods to culture the protoplast were used, and the result showed that the protoplasts buried in the 0.5% agarose developed and divided much quicker than the double-deck culture and suspend culture .As far as the GFP gene transformation is concerned, the results showed that GFP gene was successfully transferred into A.arguta protoplasts by PEG method. The physiological condition of material, different PEG solution and the time of heat stimulus affected transformation. The protoplasts from white friable callus were suspended in CPW13 solution, then incubated briefly with plasmid DNA for 10 min to allow sufficient contact between the DNA and the plasma membrane. A 40% (w/v) PEG6000 solution was added to bring the final PEG concentration to 20% (w/v). PEG solution must be added slowly and dropwise, because the rapid addition of concentrated PEG can cause a sharp increase in osmotic potential and result in fusion or breakage of protoplasts. After a 20 min incubation, PEG was diluted with CPW13 solution, and after centrifuging the protoplasts were cultured in MS medium. Heat shock for 5 min in 45 癈 water was included in this procedure.These studies offered new opportunities to facilitate and expedite further research of the somatic hybridization and gene tranfermation of A. arguta.
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