| Licorice (Glycyrrhiza uralsensis Fisch.), an perennial herbal plant of Glycyrrhiza Leguminosae, is one of the most important Chinese medicinal plants and one of the most important ecological protection plants to prevent the wind and bind the sand in desert and semi-desert districts. Moreover, it is commonly used in food and beverage, cigarrate and cosmetic industries. As the wild populations of licorice had been decreased rapidly and the genetic diversity had been threatened seriously for the unplanned destructive gathering in recent years, studies on the genetic diversity and plant tissue culture techniques have the most important significance both in theory and practice. In the paper the genetic diversity level of 18 wild populations of licorice was evaluated measured by AFLPs, and the factors affecting the callus inducement, character regulation, regeneration in vitro, micropropagation and the accumulation of the secondary metabolites glycyrrhizinic acid and total flavornoids in calli and plantlets were studied. The results were as follows:1. Fifteen pairs of selective AFLP primer combinations yielded a total of 759 fragments from 360 single plants 18 populations of wild licorice, of which 527 fragments were polymorphic (69.43%) and the average number of allels variation per primer was 35.1.2. The wild licorice possessed the medium genetic diversity. Nei's gene diversity index ranged from 0.13 to 0.19 with the average of 0.25, the Shannon genetic diversity index ranged from 0.19 to 0.28 with the mean of 0.39. The populations from Ningxia district had the highest genetic diversity, then the Innermogolia, Xinjiang, Heilongjiang and Shanxi province, the populations from Gansu jiuquan had the lowest genetic diversity. Among 18 populations, thepopulation from Lingwu Ningxia autonomous region had the highest genetic diversity. Genetic similar coefficient among the 18 populations was from 0.8559 to 0.9679. The genetic distance between the populations ranged from 0.0326 to 0.1556. 18 populations were clustered into 4 groups by UPGMA, which significantly correlated with the geographic distance of the materials.3. The optimized processs for determinating the total flavones was as follows: the flavonoids was supersonic extracted for 5 minutes twice at 32°C after soaking for 6 hours with the solvent of 75% methnol, then checking the absorbency after preparing the chromogenic solution by adding 10% alkali with the standard sample of liquidritin. The method was stable, simple and replicable and the recovery percentage was between 95.0% and 101.4%.4. The suitable culture media for induceing the callus from the explants hypotocal, radical and cotyledon were MS+2,4-D0.5-(-6BA1.0~2.0, MS + 2,4-D0.5 +6BA2.0 or MS + 2,4-D1.0+6BA2.0 separately. To regulate the characters of four types calli, continuously subculture had no significant effects, and changing the kind of plant growth regulators was more effective than changing the concentration.5. There were two ways of morphogenesis for licorice. For direct way, when cultured in MS+TDZO.lmg/L+NAAO.lmg/L, hypotocal and cotyledonary nodes may initiate the bud directively. For indirective way, calli from the hypotocal may initiate the calli first then differentiate into adventious buds and roots with the rate as high as 52%. When regulated in different medium, the light yellow or green hard calli from cotyledon may differentiate with the regeneration rate only 3~12%. No differentiation was examined from radical calli.6. The optimized micropropagation system was established as follows: the cotyledonary nodes from 7-day-old germ-free plants were cultured on MS+TDZ0.1mg/L+NAA0.1mg/L+sugar20~30g/L to induce more adventious buds;the segment with one axillary bud was subcultured on the improvement MS+6BA1.0~2.0mg/L+NAA0.1mg/L and the proliferation efficient being from 4 to 32;rooting medium: l/2MS+VB2l.0~2.0mg/L with the rooting percentage 86%;when the adventious roots was 1~2 cm the plantlets could be transplanted to the steriled vermiculite directly without any exercising, then sprayed 1/800 carbendazim CIPAC. The survival rate was about 96%.7. The content of total flavornes in hard initial calli from cotyledon explants was the highest, then loose calli from cotyledon, hypotocal and radical. For the loosecalli from cotyledon, IAA and 6BA were the most effective regulators on increasing the accumulateion of total flavornes,the highest content and yield was 0.0803 % and 41.876mg separately with IAA2mg/L+6BA0.5mg/L. The total flavornes was the highest when plantlets were cultured for 30 days. IAA was the most effective plant harmony on increasing the accumulation of the total flavornes. Two lines F5 A and F6A were screened , of which the prolifer efficient and the total flavornes contents was 28 > 1.3031% and 27^ 1.2872% separately. After replanted for 2 months, the flavonoid content of the leaves and stems and the roots were 2.0623%, 4.5992% and 0.1164%, 0.5041 %0As the materials used in the studies were from the main districts where the wild licorice grow and AFLP technique was stable and replicable, the results were reliable for the resources protection, identification, evaluation, cultivation and breeding of the licorice. The system of regeneration and micropropagation could be the platform for the gene transformation, resource storage, polyploidy breeding and the variety increasing. |
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