| Lycium is a genus of the family Solanaceae.The common types in Northwest China include Lycium ruthenicum Murr.,Lycium barbarum vr and Lycium chinense Mill.vr.Among them,L.ruthenicumis riched in anthocyanin content and L.barbarum owned high polysaccharide content are deeply loved by people.However,with the wild resources of L.ruthenicum decreased sharply and the people's requirements for the health value of L.barbarum increased,carrying out cell engineering research of Lycium became an important thing.Therefore,L.ruthenicum growing naturally in the Qinghai-Tibet Plateau and L.barbarum cultivated artificially were chosen as experiment materials in this study.Based on the study of their regeneration system,the isolation and culture of their protoplasts were studied.These would provide technical support for the subsequent somatic cell hybridization between L.ruthenicum and L.barbarum.The results obtained were as follows:1.Two regeneration systems were established by using sterile leaves of wild L.ruthenicum.The direct organogenesis is differentiated by leaves,and the indirect organogenesis is differentiated by calli.The best medium for callus induction was MS+1.5 mg·L-1 2,4-D,with induction rate of 100%;the optimum medium for callus differentiation was MS+1.5 mg·L-1 6-BA+0.1 mg·L-1 IBA,with 39.4 shoots per gram of calli;the suitable medium for direct shoot induction was MS+0.5 mg·L-1 6-BA+0.3 mg·L-1NAA,with induction rate of 92.9%,and 18.1 shoots per explants.Using the leaves of sterile seedlings of L.barbarum as explants,the direct organogenesis was established.The optimal medium for direct induction of adventitious buds with leaves was MS+1.0 mg·L-1 6-BA + 0.5 mg·L-1 NAA.The adventitious bud induction rate was 53.3%.After adventitious buds were transferred to the MS medium without hormones,the roots could form within two weeks.2.Using the leaves of L.ruthenicum and L.barbarum as materials,the protoplasts were isolated by enzymatic hydrolysis.The effects of enzymolysis time,concentration of cellulase,mannitol pretreatment and sucrose concentration on protoplast isolation and purification were studied.The optimal enzymolysis time and cellulase concentration were 5 hours and 1.5% in L.ruthenicum,while those were 6 hours and 2% in L.barbarum,respectively.The yield and viability of protoplasts were increased after their leaves were pre-treated by mannitols for 30 minutes.The sucrose of 18% was the optimal concentration for purifying leaf protoplasts of L.ruthenicum and L.barbarum.Using calli from L.ruthenicum and L.barbarum as materials,the effect of enzymolysis time,concentration of cellulase,callus amount,growth state of calli and sucrose concentration on protoplast isolation and purification were studied.The optimal enzymolysis time and cellulase concentration were 8 hours and 2% in L.ruthenicum,while those were 12 hours and 1% in L.barbarum,respectively.Three grams of calli grown for 12 days in 10 m L of enzyme solution obtained the best yield in L.ruthenicum,while those were 1 gram of calli grown for 9 days in L.barbarum.The optimal concentrations for purifying callus protoplasts of L.ruthenicum and L.barbarum were 17% and 18%,respectively.3.The solid-liquid double-layer culture with modified MS liquid medium was better than other combinations in the protoplast culture of L.ruthenicum.The first division was observed after 2 days,the division frequency after 10 days of culture was 45.9%,the colony formation rate after 20 days of culture was 22.9%,the calli were formed,and the regenerated plants were obtained by differentiation.The best culturing method of L.barbarum protoplasts was liquid shallow culture with modified MS liquid medium.The first cell division was observed after 3 days,the division frequency after 10 days of culture was 42.4%,the colony formation ratio after 20 days of culture was 32.5%,and the calli were formed.4.The genetic stability of the regenerated calli and plants were analyzed by flow cytometry?FCM?,molecular marker and esterase isozyme.FCM results showed that parental materials and regenerated materials were all diploid.ISSR molecular markers showed that the average genetic similarity coefficient between the regenerated plants and the mother plant was 0.875 in L.ruthenicum,whereas that was 0.899 between the regenerated calli and mother calli in L.barbarum.Except a few of minor bands,the major bands of peroxidase and polyphenol oxidase had not obvious differences between the regenerated materials and the mother-materials.These results indicated that the calli and plants regenerated from protoplasts of L.ruthenicum and L.barbarum maintained high genetic fidelity,but there were still some somaclonal variation. |
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