| Potato is one of the largest crops planting in the world, with high yield, strong adaptability, nutrient- rich, wide range of uses and other characteristics. The potato original cultivation has a variety of quality traits and resistance genes, purple potatoes are rich in anthocyanins and have high nutritional value and medicinal properties, both of them were important gene pool and high quality material of potato variety improvement and somatic hybridization breeding. The potato original cultivation of NDK 47-33, NDK 5-19 and purple potato of GSAP-H were used as materials in this experiment to investigate and obtained the stable system of separation, purification, cultivation and fusion of protoplast from suspension cells. Meanwhile the changes of anthocyanin content from the callus organ growing in vitro and protoplast culturing also were studied in this experiment. The main results are as follows:1. Young leaves and stems of potato plant in vitro were good materials for cullus induction, and the best of callus induction medium was MS + 2.0 mg/L NAA + 2.0 mg/L 6-BA + 0.5 mg/L 2,4-D, the callus induction frequency was up to 100% in this medium. The best subculture medium of calli was MS + 1.0 mg/L NAA + 2 mg/L 2,4-D + 250 mg/L CH. The callus had a brightly colour, a fast growth rate, a loose structure after subcultured for 3 times, and these calli were the optimal materials to establish potato suspension cell in this experiment.2. Three suspension cell lines of potato NDK 47-33, NDK 5-19 and GSAP-H there were the best physiological status and the strongest divition rate as they were cultured in liquid MS medium with 1.0 mg/L NAA, 2 mg/L 2,4-D, 250 mg/L CH under the temperature conditions of 24 ℃, 120r/min shaker speed. Suspesion cells should be subcultured after they grew in medium for 4 days. Do not continuous culture more than 6 generations for every potato line of suspesion cell.3. The optimum enzymolysis ingredient for isolation protoplast of suspension cell was WPS solution adding with 2% cellulose, 0.5% pectinase and 0.25% macerozyme., the osmotic pressure of WPS solution was adjusted to 0.3M by sucrose. the suspension cells subculturing for 4 times were treated by the solution above mentioned and were isolated at the speed of 80 r/min, at the temperature of 26 ℃ for 14 h, the highest production and vitality of protoplast was obtained that was up to 8.17×106 individual/g and 95%, respectively.4. The shallow liquid culture is one of the methods of protoplasts cultivation. It was the best methods to improve the frequency of protoplast divition of potato suspension cells in this experiment. As the density of purified protoplast was sdjusted to 2.5×105/ml, in this medium of VKM adding with 0.5 mg/L BAP and 2.0 mg/L 2,4-D, the first and second division of protoplastsfrom suspension cells were observed by microscope after they were cultured for 2 d and 10 d. Further culture this protoplasts about 30 to 40 d, some of cell mass were founded by our eyes, and the frequency of protoplast division and cell mass were up to 76.72% and 31.46% for potato variety of “GSAP-H”. Furthermore, 23 regeneration plants were obtained in this experiment after the cell mass from protoplast were cultured in MS medium with 1.0 mg/L ZT, 1.0 mg/L BAP, 0.1 mg/L NAA, 2 g/L AC and 1% mannital for 50 to 60 days. Testing the anthocyanin content from 36 kinds of cell mass which were random selected from all of protoplast cell mass, the change range of anthocyanin content was from 8.464 to 4477.44 nmol/g·FW, there was the highest anthocyanin content of 4477.44 nmol/g·FW in cell mass of G-12 by detection.5. The protoplast from potato lines of “NDK47-33” and “GSAP-H”were used to proceed somatic hybridization by PEG chemical fusion or electrofution. The results showed that using the concentration of 25% PEG 6000 treated two kinds of protoplast(proportion for 1:1) for 15 min, the total PEG fusion frequency was up to 52.24%, and the 2 to 3 cells fusion rate was up to 42.84%. Meanwhile, the results from electrofusin showed that the best condition of cell fusion was alternating electric field strength for 112 V/cm, Alternating current action time for 10 s, DC pulse field strength for 2240 V/cm, the pulse wide for 50 us, DC pulse times for 2 times, in which the total cell fusion frequency and 2 to 3 cells fusion frequency were up to 62.15%,49.34%, respectively.6. Purple callus of suspension cultures regenerated materials, study on exogenous hormones, sugar sources, types and proportions of n, medium p H conditions, illumination and temperature on anthocyanin content of callus. Experimental results showed that 4 mg/L 6-BA+1.5 mg/L is 2,4-D+1.0 mg/L NAA hormone combinations accumulation of anthocyanin; 45 g/L sucrose as a carbon source or osmotic regulation and the media NO3-and NH4+ ratio 3:1 of the highest anthocyanin content. In addition, all light and medium p H 4.8, the temperature is 25 degrees could significantly promote the accumulation of anthocyanins, anthocyanin 32.19% higher than dark. Can also greatly promote callus of anthocyanin accumulation. Experimental results gained from callus of potato for the future laid the foundations for industrial production of anthocyanins. |
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