Study On Somatic Hybridization Of Lycium

Lycium is genus of the family Solanaceae.The common types include Lycium ruthenicum,Lycium barbarum and Lycium chinense in Northwest China.Most of the wild Lycium growing in this area has a strong to genetic resistances,especially L.ruthenicum,which is a potential natural genetic resource pool.In this study,L.ruthenicum growning naturally in the Qinghai-Tibet Plateau and L.barbarum cultivated artificially in Ningxia were chosen as experiment materials.Based on the development of their high frequency protoplast division and plantlet regeneration,somatic hybridization between wild and cultivated Lycium were performed.These would provide scientific references for the transferring of stress-related genetic characterization of wild plants and provide useful materials for the further researches on the variety breeding of Lycium.The results obtained were as follows:1.The optimal explants for calli induction were Lycium leaves.In 0.5 mg·m L^-12,4-D+MS medium,the calli induction rates were 100%of both L.ruthenicum and L.barbarum.2.The best material for isolating protoplasts was the leaf calli of Lycium that have been subcultured once.In 0.5 mg·m L^-1 mannitol enzyme solution,the protoplast yield of L.ruthenicum was 7.77×10⁶ pieces·g^-1,and the vitality was 91.98%.The protoplast yield of L.barbarum was 7.01×10⁶ pieces·g^-1,and the vitality was 84.09%.3.The best way to culture Lycium protoplasts was liquid shallow culture.The frequency of protoplast division of L.ruthenicum was 60.9%after 7 days of culture,and the frequency of cell mass formation was 72.2%after 14 days of culture.The frequency of protoplast division of L.barbarum was 34.5%after 7 days of culture,and the frequency of cell mass formation was 72%after 14 days of culture.In 1.5mg·m L^-1 6-BA+0.1 mg·m L^-1 IBA+MS solid medium,the protoplasts of L.ruthenicum were differentiated into regenerated plants.In 0.5 mg·m L^-1 2,4-D+MS solid medium,the protoplasts of L.barbarum were differentiated into regenerated calli.4.In order to conduct asymmetric hybridization research,the protoplasts of L.ruthenicum were treated with ultraviolet light,and the protoplasts of L.barbarum were treated with IOA.The frequency of protoplasts vitality and division of L.ruthenicum decreased with the increase of UV irradiation time,and the optimal passivation time was 20 minutes.The frequency of protoplasts viability and division of L.barbarum decreased with the increase of IOA concentration,and the optimal passivation concentration was 0.2 mmol·L^-1.5.After passivation treatment,the protoplasts of L.ruthenicum and L.barbarum were fused.The concentration of PEG fusion agent was 40%,the fusion time was 15minutes,the ratio of protoplast mixture to PEG fusion solution was 1:3,and the fusion rate can reach 13.42%.Use liquid shallow to culture fusion products,in 1.0 mg·m L^-12,4-D+MS medium,the first cell division started after 3 days,the second cell division appeared after 1 week,cell mass was formed after 2 weeks,and micro-calli were formed after 4 weeks.Transfer the micro-calli to MS solid medium with 0.5mg·m L^-1 2,4-D,the regeneration calli was formed in 2 months.6.The calli regenerated from fused protoplasts were analyzed by ISSR,ITS,chloroplast genome differences and isozymes.The regenerated calli were highly similar to L.ruthenicum in chloroplast genome,and they were similar to both parents in nuclear genome,but prefered L.ruthenicum.