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Studyonoptimizationofagrobacterium-mediated Transformation System and Aphid-resistant Gene Transformation in Maize

Author: LiHaiXia
Tutor: HuYanMin
School: Henan Agricultural University
Course: Crop Genetics and Breeding
Keywords: Corn Callus Aphid resistance genes Antibiotic Genetic transformation
CLC: S513
Type: Master's thesis
Year: 2010
Downloads: 78
Quote: 0
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Abstract


Corn is an important food, feed and industrial raw material crops occupy a very important position in China's national economy. In recent years, corn aphids showed worsening trends, local area from secondary pests rise as a major pest, caused serious damage to corn production. The test immature embryo culture regeneration system using Agrobacterium-mediated genetic transformation aphid resistance genes research aimed through the transformation system optimization from mint extract the aphid resistance gene transferred to the recipient cell regeneration plants, creating the foundation aphid resistance of new maize germplasm, breeding for resistance to the aphids corn varieties. The the test corn callus induction, regeneration, antibiotic concentration, Agrobacterium infection conditions, a more detailed discussion, the following results: Agrobacterium-mediated genetic transformation of maize receptor genotype is an important factor affecting the rate of regeneration, conversion efficiency there is a big difference between the different genotypes. The many genotypes more difficult induced callus quality, especially inbred line plant regeneration difficult. The trial of four inbred lines and hybrids immature embryo induction characteristics were screened, the results show that the ,87-1 inbred lines and Qi 319 × Zheng 22 and Chang 7-2 x Hi Ⅱ A in seven hybrids good callus is an ideal material for corn transformation system, especially in Chang 7-2 × Hi Ⅱ A best results. These genotypes in the differentiation before its callus different concentrations of 2,4-D pretreatment, the results show that the use of 2,4-D-free medium pretreatment effect Preferably, the regeneration rate than control increased significantly, and can significantly accelerate the regeneration process. Transformed to antibiotic selection marker gene, determine appropriate screening pressure is important. Of this study cefotaxime (Cef) and G418, kanamycin (Kan), glufosinate (PPT) of maize embryogenic callus growth and differentiation. The results showed that: in the Agrobacterium-mediated genetic transformation of maize, the bacteriostatic agent CEF concentration of 100mg / L is more appropriate; The Kan, G418 and PPT suitable concentrations of 6-10mg / L ,15-25mg / L and 9mg / L. In this study the expression of the GUS gene, respectively, affect the conversion efficiency of some of the conditions were optimized, such as the infection time, addition of acetosyringone (AS), off bacteria manner OK. The results showed that after addition of 100μM AS GUS positive rate significantly increased; dip time of 10 minutes after treatment was dried for 4 hours, removing bacteria, Agrobacterium on callus damage lighter, obtained resistant calli; use different The dip solution had no significant effect on the conversion. Zheng 22 × 87-1 and Chang 7-2 x Hi Ⅱ A in resistant plants staining revealed GUS positive expression rates were 37.5% and 28.12% of the plant expression system the the carrier pBJ40-EβF and with automatically deleted , pNCX-EβF were transformed into Agrobacterium LBA4404 strain of corn callus dip transformation, PCR detection will be resistant callus, preliminary evidence of anti-aphid gene was integrated into the maize genome.

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CLC: > Agricultural Sciences > Crop > Cereal crops > Corn ( maize )
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