| Object:(1)Screening the varieties with somatic embryogenesis capacity from some cultivarsof Xinjiang cottons and establishing a high-frequency regeneration system;(2) Establishmentof the efficient genetic transformation system by Agrobacterium-mediated and obtainingtransgenic plants with glyphosate-resistant characteristics.Method:(1)We Compared the effection of the hormone combinations on calli differentiation,embryogenic calli induction, and somatic embryogenesis to establish of regeneration system.(2)By improving culture conditions on seedling stage, determining the co-cultivation durationand the amount of Cef in selective medium, the transformation system was optimized. Andthen, by optimizing somatic embryogenesis capacity of the resistant embryogenic calli andcotyledonary embryos culture methods, the cycle of regeneration was shorten. Through thesemethods, we established an efficient transformation system.Results:(1)Among these tested varieties, only Xlnluzao33and Xincai7have the ability ofsomatic embryogenesis and plant regeneration.(2)For all tested varieties, calli wereeffectively produced on the medium with2,4-D+KT hormone regime, and the calli ratesreached100%. The induction rate of embryogenic calli of Xinluzao33and Xincai7weresignificantly enhanced through reducing the concentration of2,4-D, and could reach60.0%and7.5%, respectively. Embryogenic calli effectively induced on the medium with thehormone combinations of IBA+KT hormone regime; the differentiation rates of Xinluzao33and Xincai7could reach45%and55%, respectively. Further investigation suggested Xincai7showed strong embryogenic capacity than Xinluzao33. Regenerated plants were obtainedfrom the two cultivars within6months and regeneration systems were established.(3)Xinluzao33as experimental objects was used for studying the optimization of genetictransformation system. The dark culture for3d adding normal light training for3d couldmake seedlings strong appropriately, that improved the conversion efficiency. Co-cultivationfor48h and maintaining the concentration of Cef at600mg/L in the select medium were best for the transformation. During the stage of embryogenic calli proliferation,3.8g/L KNO3and subculture cycle for11days were suitable for fast proliferation and keeping better state.Supplemented with4.0g/L Phytagel in MSB medium obviously promoted embryoiddifferentiation, and1.0g/L activated carbon facilitated root development., reduced browningand improved the ratio of normal regenerated plant.(4) PCR and whole plant spraying ofglyphosate detection of the transformed plants, preliminary confirmed target gene fragmenthas been transferred to the acceptor material genome.Conclusion:(1)We obtained two varieties, Xinluzao33and Xincaimian7, which could plantregeneration; and optimized regeneration system.(2)By Agrobacterium-mediated method,were got the transgenic plants of these two varieties, and transformation system wasoptimized. |
