Isolation And Culture Of Protoplast From Potato Leaf And Suspension Cell

Original diploid potato cultivar contains rich resistance genes such asdisease-resistant genes, anti-insect genes and resistance to adverse environment genes, andpurple potatoes possess characteristic genes related with nutrition and health care value.Therefore both of them could be the potential sources of potato breeding and somatichybridization. However, the protoplasts isolation and culture were not very successful forOriginal diploid potato cultivar which had become a limiting factor of somatic fusion toobtain hybrid plants. In this study, original diploid potatoes lines of47-33NDK15-33,5-19NDK33-38and purple potatoes variety GH were used as materials to study theeffect factors in protoplast isolation and culture. The results were showed as follows：1. AgNO3had an obvious promoting effect on the growth of potato in vitro plants. Whenthe concentration of AgNO3was1.5mg/L the fresh weight and root weight of potato lines5-19" and "47-33" in vitro plants were the highest, and the fresh weight reached to0.2731gand0.2115g, and the highest root weight were0.0407g and0.0454g, respectively. Theapplication of1.0mg/L AgNO3significantly increased the root length, the root length of5-19" and "47-33" plants in vitro were increased by40.56%and216.91%compare with thecontrols, respectively, however, the height of in vitro plant and the number of the leaves weredeceased. Additional, the leaf area of plant in vitro of5-19" was the largest on theconcentration of2.0mg/L AgNO3, which was95.03cm2, and the leaf area of47-33reachedthe largest value of102.46cm2, after treated with1.5mg/L AgNO3.2. The highest callus inductive frequency from stem of5-19,47-33and GH in vitroplant were obtained in MS medium supplemented with2.0mg/L NAA and2.0mg/L BAP,they were100%,100%, and86.67%, respectively. Callus secondary cultured for3times canbe used for the preparation of suspension cells. GH suspension cells cultured in MS liquidmedium supplemented with1.0mg/L NAA,2.0mg/L2,4-D and250mg/L CH for4d neededto secondary culture. The suspension cells could be used a favourable material to dissolveprotoplast after it were successive transfer cultured for3~4times.3. The leaves of potato in vitro plants were cultured in MS medium with silver nitratetreatment for21days were used as the material to separate protoplasts. When the top leavesof potato in vitro plant incubated in enzyme solution of0.35M osmotic pressure including 1.5%~1.5%cellulase and2.0%pectinase for14hours at28℃in the dark gently shaking orstanding one hours and then shaking13hours, the effect of protoplasts dissociation was thebest. After purifying the protoplasts with0.45M sucrose, the high yield and quality ofprotoplasts were obtained. Under cultured in CM-1liquid medium of0.35M osmoticpressure supplemented with1.0mg/L NAA,0.5mg/L2,4-D, and0.4mg/L BAP, the firstdivision frequency and the secondary division frequency of protoplasts reached13.85%and1.36%, respectively.4. The method of floating purification was better than interface floating purification methodfor leaf protoplast. Protoplasts should be purified by filtration through sieves directly and thenrinsed2~3times.5. When GH suspension cells placed in enzyme solution containing a mixture of enzymes(2.0%~2.5%cellulase,0.25%macerozyme,0.5%pectinase),0.30~0.35M osmoticpressure, then incubated at27～28℃in the dark, with standing one hour and then shaking(50r/min)13h, the yield of protoplasts reached the highest of8.37×105protoplasts per gramFW, the vigour of the protoplasts were the highest (>95%). When protoplast were plated at ainitial density of2.5~5.0×105/mL, they first divided in2days, second divided in3～4days, and formated small cell groups in6～10days, the rate of little callus was14.25%~34.21%. The first division frequency and the secondary division frequency of protoplastscultured on the KM8P medium supplemented with1.5mg/L2,4-D could reach66.59%and35.24%, respectively, which was more suitable for suspension cell protoplast culture.In this study, a stable dissociation system of protoplast for two original diploid potatoand a purple potato varieties has been established, better culture conditions of leaf orsuspension cell protoplast have been obtained, which laid a foundation for potato breeding bycell hybridization technology making use of the good genes from three potato varieties.