Research On Tissue Culture And Genetic Transformation System In Lotus(Nelumbo Nucifera)

Lotus is the largest aquatic herb cultivated in China,which has significant values in food,ornament and medicine.This important and versatile plant has been cultivated more than 2,500 year in China,and is currently grown in a large scale.With the rapid development of molecular biology technology,more and more researchers started to work on molecular breeding and gene function study in lotus.At present,lotus tissue culture and genetic transformation system have not yet been constructed,which impeded the genetic function study on lotus one space.Therefore,in this study,a)we tested different varieties,and selected different lotus organs as explants for callus induction.The culture medium,hormone combinations,as well as culture conditions were also optimized.In addition,b)an optimized aseptic seedling cultivation system was also developed.Meanwhile,we also tried to use the aseptic lotus seedling to induce hairy roots for lotus.The results are summarized as follows:1.Using embryos collected from fresh seeds at 18 days after pollination as explants,a rapid lotus propagation system was established through primary culture,subculture,plantlets hardening,and transplantation.Results showed that MS medium supplemented with 0.5 mg/L 6-BA+0.5 mg/L NAA+30 g/L sucrose+0.5 g/L activated carbon+8 g/L agar was the optimum for the explant growth.The average amount of stem nodes was up to 3.9 per explant and the explant induction rate was up to 85%.In the subculture,the optimal concentration of sucrose in the medium was 80 g/L.When dividing aseptic seedling for subculture,cuttings with two stem nodes showed the highest multiplication efficiency,up to 6.8.Aseptic seedlings were suggested to be sub-cultured every 50 days.High multiplication rates were maintained up to 6 round of subcultures.Rooted in vitro plantlets were transplanted to pots and grown outside under natural conditionsduring May to July.The survival rate of transplanted plantlets was as high as 83%.With this in vitro propagation system,1465 seedlings could be developed from a single lotus seed within one year.Thus,in this study,we established a rapid in vitro propagation system for lotus.2.Callus induction was performed among 21 lotus genotypes.Great variation in callus induction efficiency was observed among those genotypes.’Shilihe 1’exhibited the highest induction rate of class I callus,up to 37.29%.Compared to the stored lotus seeds and the rhizome tips,mature embryos collected from fresh lotus seeds had much less endophytes,and can be easily sterilized,thus are excellent explants for lotus aseptic seedlings cultivation.The highest efficient callus production was induced on immature cotyledon and embryo explants cultivated on MS medium containing the optimized combination of 3 mg/L 2,4-dichlorophenoxyacetic acid（2,4-D）and 0.5mg/L 6-benzylaminopurine（6-BA）.In addition,lotus callus induction was influenced by several other factors,including lotus genotypes,light conditions,the developmental stages of explants,and the seasons of explant sampling.For better callus induction,it was suggested to collect cotyledons from lotus seeds at 9 DAP（Day After Pollination）during early Julyand to cultivat the explants under darkness.Histological observation showed that callus cells were much bigger in size and were arranged irregularly compared with those cells before callus induction.Furthermore,combing of transcriptomics and metabonomics analysis,the reasons of lotus callus browning were explored.In this study,a high efficient callus induction system was developed for lotus,and possible reasons causing lotus callus browning were studied.3.We also tried to develop a hair root culture system for lotus.Agrobacterium rhizogenes were to infect sterile lotus seedling explants and maintain the concentration of bacteria solution at OD₆₀₀=0.8,the infection time was 30 min.After10 days,red fluorescence signal can be observed in wound area of sterile seedlings under primary culture.By using 18 DAP lotus seed germs as explants for agrobacterium infection,and a small amount of hairy roots with red fluorescence signals were successfully induced.This indicates that the induction system of using agrobacterium rhizogenes to infect lotus explants to induce hairy roots is feasible.However,the hairy roots grow slowly and the induction success rate is extremely low.In-depth research is needed for optimization.In summary,we established an efficient lotus aseptic seedling culture and in vitro propagation system,optimized the lotus callus induction system,carried out preliminary exploration work on the production of lotus hairy roots by Agrobacterium rhizogenes.The results of this study provide useful technical supports on lotus functional genomics studies.