Study On Regeneration System From Leaf In Vitro And Protoplast Isolation And Culture Of Black Chokeberry

The black chokeberry ( Aronia melanocarpa ) is a new small fruit. It has been cultivated in many countries around the world. However, it is difficult to improve cultivars by normal breeding means like cross breeding because of the limited valuable genes of dominant characters. Biotechnology such as genetic engineering and somatic hybridization is a new effective means in germplasm enhancement by which misogamy can be overcame.In this experiment callus inducement from black chokeberry leaf in vitro, establishment of regeneration system, establishment of suspensions, protoplast isolation, cell culture and protoplast culture were studied. The experiment intened to establish regeneration system of leaf and cell suspensions and get cell and protoplast regeneration plant. The experiment result provided scientific information to further study on gene transfer and somatic hybridization.1. Callus induce of black chokeberry leaf in vitroCallus induce from black chokeberry leaf in vitro was studied with different treatments of plant-growth regulator, light intensity, pH and sucrose. The results showed that: The callus type and growth were affected by the category and concentration of plant-growth regulator. Callus did not form in treatments of cytokinin BA and CPPU, and did in all treatments of auxin alone or combination of cytokinin and auxin. 95% rate of callus induce was obtained in MS medium supplemented 2,4-D 1.0 mg/L. The optimum conditions for callus culture were MS + 2,4-D 1.0 mg/L + sucrose 3%, pH 6.0 and 3 000 1x light indensity, 25℃ in the day for 14h, 22℃ in the dark for 8h, by which the best results of callus fresh weight and dry weight were obtained. Only by 2,4-D was the friable-looking type of callus induced. Others auxin, cytokinin and their combinations induced the hard type of callus.2. Establishment of regeneration system of black chokeberry leaf in vitro Establishment of regeneration system of leaf was affected mainly by plant-growth regulator. The experiment results showed that combinations and concentrations of cytokinin and auxin affected the re-differentiation. NAAinduced rooting; NAA0.5mg/L treatment appeared the optimal rooting result of 90%. There appeared bud re-differentiation when appropriate concentration combinations of cytokinin and auxin were applied. The highest re-differentiation rate reached 50%, only 2 shoots per leaf, was obtained with treatment of MS medium supplemented BA3.0mg/L and NAA0.5mg/L in one step induce method. In two steps induce method, WPS medium supplemented CPPU2.Omg/L and 2, 4-DO.lmg/L was used for callus induce first. Then the callus induced was transferred to WPS medium supplemented CPPU 2.0 mg/L. The bud re-differentiation rate was 93.3% with 15.2 shoots per leaf on average. Effective regeneration system was established by two steps induce method.3. Establishment of suspensions of black chokeberry.Suspension was easy to be establisded with the white friable-looking callus induced by MS+2, 4-D,but was not established with the green hard callus induced by MS + BA 2.0 mg/L + NAA 0.5 mg/L. The result of suspension culture in MS medium was better than that of B5,WPS,AA and N6.The optimum conditions for suspension culture and cell growth were MS medium supplemented 2,4-D 0.5mg/L, sucrose 3%, pH6.0, inoculation of 2.0g/30ml, and lid culture. The density of cell in medium was 4. 97 X 107ml.4. Protoplast isolation of black chokeberryUsing cell as suspension culture materials. The factors of influence protoplast isolation such as concentration combination of Onozuka R-10 and Pectolyase Y-23, protoplast isolation time, protoplast isolation methods and mannitol concentration and suspension culture time were studied. The optimum conditions were the concentration of Onozuka R-10 20g/L and Pectolyase Y-23 0.5g/L with mannitol 0.6mol/L, enzymolysis 12 hours. The yield of protoplast was 17. 7X 105/g FW. The static isolation methods was better than isolation method on shaker at 30r/min.5. Cell culture and protoplast culture of black chokeberryCells were inoculated in shallow liquid MS medium with BA 1.Omg/L,2,4-D O.lmg/L and 3% sucrose. Cell colonies were produced at plating density of l.OXlOVml, but did not 0. 5X105/ml. The cell division was observed in plating method with MS medium supplemented BA 1. Omg/L, 2, 4-D 0. Img/L, 3% sucrose and 0.6%agarose LMP. Protoplast of black chokeberry inoculated in solid MS medium with BA l.Omg/L, 2,4-D 0.1mg/L,0.6mol/L mannitol,l% sucrose and 0.6% agarose LMP developed cell wall at culture density of l.OXlOVml.