| Glycyrrhiza inflate Batal. is not only a typical Chinese medicine in our province, but also an important fixing-sand plant in western desert, semi-desert region. Over-harvesting and disease lead to wild licorice was almost extinct. Despite of large-area artificial cultivation, the effective component was lower than wild licorice. So selection the licorice of high-efficacy component by breeding becomes extremely important. However, there are still many unsolved problems in licorice conventional cultivation:abortion in microspore development process, long flowering period (3 to 4 years). In our experiment, Glycyrrhiza inflate Batal. was used to optimize the licorice callus induction, protoplast and culture for accessing to the fine licorice lines by Cell fusion in the future. The result was shown as follows:1. Optimum induction system for licorice callus(1):The frequency of callus from hypocotyl and cotyledon were significant different under the concentration of NAA and 2,4-D, and the highest frequency of hypocotyl callus was obtained at 1.5 mg.L-1,67.83% and 67.70%, respectively. However, the frequency of cotyledon reach maximum in the concentration of 0.5 mg.L-1 NAA and 1.0 mg.L-1 2,4-D, were 73.33% and 66.29%, respectively.(2):The frequency of callus and browning from hypocotyls and cotyledon were found at a significant level in different concentration of 6-BA. In the combination with 2,4-D, the callus frequency achieved the highest level at the 2.0 mg.L-1 6-BA,were 87.12% and 70.77%, respectively; but when it combined with NAA, the highest frequency of callus and browning from hypocotyls and cotyledon were existed at the 0.5 mg.L-1 and 2.0 mg.L-1 6-BA respectively. The inducement rate were 74.49% and 74.69%.(3):The different concentration of NAA,2,4-D and 6-BA have significant effect on callus browning frequency. The average lowest browning frequency were presented at the 1.0 mg.L-1 2,4-D and 1.5 mg.L-1 NAA, were 38.53% and 25.42%.Moreover, the browning of hypocotyl and cotyledon can be effectively curbed under the 1.5 mg.L-1 and 1.0 mg.L-1 6-BA, and the browning frequency rate were only 40.71% and 44.80%.(4):According to interaction of exogenous hormones on the rate of callus induction and browning,we got the optimal combined hormones of hypocotyl and cotyledon callus induction were:MS+1.5mg/L NAA+0.5mg/L+6-BA, MS+2.5mg/L NAA +0.5mg/L 6-BA, respectively.2. The establishment of licorice hypocotyl, leaf and callus protoplast isolation system(1):The impact of cellulase in callus induction was greater than Pectolase, and the domestic cellulase was better than import in the respect of solubility and effect on isolation. The highest yield and viability of protoplast on hypocotyl, leaf and callus were obtained from the enzyme combination of 2.0% cellulose and 0.5% Pectolase. were 1.44×105 month·g-1, 72.8%,1.25×105month·g-1,70.5% and 1.1×105 month·g-1,69.4%, respectively.(2):Tender hypocotyls, seeding age of 10-15d leaves and 5 times subculture of callus can obtain a number of protoplasts. The hypocotyls growth 5d in sugar-free MS medium and leaves seedling age of 11d were the best material for protoplast isolation, and yield and viability were 1.19×105 month·g-1,65.2%; 1.02×105 month·g-1,61.16%, respectively. We get the optimal consequence of protoplast isolation after 5 times subculture, however, yield and viability were only 8.64×104month·g-1,58.84%, which is sharply lower than the hypocotyls and leaves.(3):Pretreatment have significant effect on protoplasts isolation of callus, and the heat treatment was better than cold, the highest yield of protoplast can reached 1.08×105onth·g-1 under the deal with 8h. Besides, Shaking after standing can obtain higher yield protoplast.3.Protoplast cultureThe different composition of medium, culture density and culture method have significant effect on protoplast division. In KM8P medium,0.3mol.L-1 sucrose as carbon source, using nurse culture, 1×105month·g-1 as culture density, both time of the first division of protoplast and cell clusters formation were earlier.
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