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Optimization of Flax Efficient Regeneration System and Bar Genetic Transformation

Author: JiaWanZuo
Tutor: WangYuFu
School: Chinese Academy of Agricultural Sciences
Course: Crop Genetics and Breeding
Keywords: Flax Regeneration System Agrobacterium bar gene Genetic transformation
CLC: S563.2
Type: Master's thesis
Year: 2011
Downloads: 29
Quote: 1
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Abstract


Flax (Linum usitatissimum L.) is an important fiber crops and oil crops, flax textile its unique quality much consumers favor. Flax production development with high-yield, high quality, and is closely linked to the cultivation of new varieties and promotion of multi-resistant. With the development of biotechnology, the use of genetic transformation means innovation of flax germplasm resources and breeding new varieties is a very effective way. In this study, fiber flax varieties \as the basis for herbicide resistance bar gene into linen. The main findings are as follows: (1) flax hypocotyl tissue culture regeneration system optimization study of genotype, explant of select parts of the body, the basic medium, plant growth regulating agents affect the flax plant regeneration factors were optimized, flax hypocotyl efficient tissue culture regeneration system. The results show that flax seed disinfection using 75% ethanol for 3min, with 0.1% mercuric chloride sterilization 3min best results; flax hypocotyl lower explant; Y4 medium (MS1 NAA 0.02mg / l 6-BA 1mg / l), the highest flax callus induction and differentiation rate reached 98.89% and 95.56%, respectively; optimum rooting medium is 1/2MS NAA 0.001mg / l; Pikes Huaxing 009 compared to the regenerative capacity of the strongest varieties are No. 2 in Central Asia. (2) Agrobacterium-mediated genetic transformation system optimized plant expression by the freeze-thaw method the vector pCAMBIA3301 successfully introduced into Agrobacterium tumefaciens strain LBA4404 efficient regeneration system, combined with the already established flax transient gus gene expression of Agrobacterium-mediated genetic transformation The main influential factors in the process of optimization, including screening agents and antibiotics off bacteria concentration, pretreatment of explants, Agrobacterium dip conditions, co-culture time. And herbicide PPT as a screening agent, for the first time established a flax varieties \Optimized parameters, the bar gene screening agent the PPT concentration of 1.5mg / l the npt II genetic screening agent kanamycin concentration of 50 mg / l; off bacteria antibiotic Ampicillin Sodium 300mg / l, ceftriaxone sodium 400mg / l; under the slight peeling treatment after hypocotyl preculture 3 days GUS transient expression rate can reach 96%; to MS1 liquid medium as re-suspension of Agrobacterium, regulating bacterial concentration OD600 of = 0.8, disseminated 30min; optimum total culture time 4 days and so on. (3) of transgenic plants detected GUS histochemical chemical detection of total training after the transient expression of gus gene within the organization a lot of expression; transformed seedlings GUS staining and PCR identification, preliminary determine the bar gene has been integrated into the linen gene group.

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CLC: > Agricultural Sciences > Crop > Economic crops > Fiber crops > Fiber crops > Flax
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