| Daylily(Hemerocallis fulva) is one of perennial monocotyledonous plants belonging to the family Lilium of Hemerocalluss. Daylily plants are very populous with its beautiful flower colors, shapes and its cutting flowers purpose. While the dayliy plant has several shortcomings such as low reproduction rates, short blossom flowering periods and unscented flowers to influence its widely application in gardens.Producing new varieties which have the features of high reproduction coefficient, special colors, long florescence and perceptible flavour become the aim of breeding. Developing the new daylily varieties with good ornamental features is a major task for breeders.Many new daylily varieties had been achieved by the conventional cross-breeding method. However several features can not be develope by this cross-breeding method in the short run. Fortunately, the genetic transformation technique provides a new way to improve flower characters and quality. So to optimize the regeneration system and establish the agrobacterium-mediated transformation system of daylily plants is the key factor for gene engineering. Daylily is not sensitive to Agrobacterium tumefaciens because it is a kind of monoeotyledons, so we should explore an efficient way to transform extrinsic gene into Daylily.This experiment took stem-tip tissures of Daylily Hemerocallis fulva as material.We established the regeneration system and established the agrobacterium-mediated transformation system of daylily plants. The results are as follows:The regeneration system from stem tips was established by optimizing the regeneration conditons. The main factors which influence the callus induction, differentiation and rooting were studied. The stem tips explants grown on medium MS(2.0mg/L6-BA+0.2mg/L NAA) for30days. The optimal callus induction medium was:MS+4.0mg/L6-BA+0.1mg/L NAA, the induction rate was100.00%; The optimal differentiation medium was:MS+1.0mg/L6-BA+0.2mg/L NAA, while the differentiation rate was more than82.22%; The best rooting medium was:1/2MS+2.0mg/L NAA, while the rooting rate reached93.48%.Based on the efficient regeneration system, an agrobacterium tumefaciens-mediated transformation system was established for Daylily Hemerocallis fulva. A Series of factors influencing the transformation such as pre-culture time, the density of agrobacterium, infection time, co-culture time and it’s concentrations of AS were discussed using calli as inoculation material, then these factors were optimized. Agrobacterium-mediated daylily Hemerocallis fulva transformation was performed according to the callus infection method described as follows. The infected callus which had been subcultured for one or two times grown on differentiation medium (MS+1.0mg/L6-BA+0.2mg/L NAA) for2days in light. Centrifuged the bacterial culture at4000rpm for5min and discard the supernatant. Suspended the cells in LB/MS with200μmol/L Acetosyringone and incubated the culture with shaking (150-200r/min) at28℃before infection(Adjust the OD600=0.1). Infected the callus for20min, then dried them and transferred to Co-cultivation Medium (MS+1mg/L6-BA+0.2mg/L NAA+200μmol/L AS) in dark at25℃for3days; Transferrd the callus to Selection Medium(MS+lmg/L6-BA+0.2mg/L NAA+125mg/L Kan+400mg/L Cef) which have been washed by sterile DW with200mg/L Ceftoxime. After cut the resistant calli and buds down, transferred them to fresh Selection Media with antibiotics at low concentration.110resistant plants regenerated from the resistant callus; Results of rooting selection, Fluorescence detection and PCR assays showed that11transgenic plants were obtainted.It suggested that the exogenous reporter gene (GFP)had been transferred into genome of Daylily Hemerocallis fulva. |
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