| Rape is a cruciferous Brassica,and it is one of the most important oil crops in China.With the increasingly fierce international competition,rapeseed production in China is also facing many challenges.Increasing the seed yield,the high oil content of rapeseed planting resources are particularly scarce.As a transcription factor,growth-regulating factor plays an important role in regulating plant growth and development.Studies have shown that rape growth-regulating factor Bn GRF2 can increase the oil content of Arabidopsis thaliana seeds and increase the grain weight,but there haven’t research on Bn GRF2 regulation of rape yet.In addition,weed hazards are one of the factors that limit the development of rape industry.With the increasing shortage of rural labor in our country,the artificial weeding can not meet the demand of rapeseed production and increase the production cost.Therefore,in this study,Bn GRF2 and glyphosate resistance gene EPSPs were applied to rape by transgenic means to study the effect of bivalent gene on rape growth.At the same time,it also laid a foundation for obtaining new oilseed rape germplasm resources with higher seed oil content and herbicide resistance.In this study,the hypocotyls and leaves of Brassica napus L."Qing Za No.5" restorer line "1831R"（QZ-1831R）and Brassica campestris L.H165 were used as the research object.The orthogonal test of 6-BA and 2,4-D,6-BA and GA3 with different concentrations were designed to select the best medium for callus and bud regeneration from hypocotyls and leaves of Brassica napus and Brassica campestris.On the basis of this,eight varieties（lines）were used as experimental materials to study the effects of different genotypes on the regeneration system.In order to select the optimum concentration of NAA/IAA for rooting of Brassica napus and Brassica campestris,four kinds of superior genotypes were studied and five NAA / IAA concentration gradients were designed.In addition,on the basis of Agrobacterium tumefaciens-mediated method,the effects of different time-treated ultrasonic methods and vacuum permeation pressure vacuum permeation method on the transformation were investigated.At the same time,the study used four antibiotics that were most suitable for removing Agrobacterium in the process of transformation.And they are designed for different concentration gradients.The main results are as follows.1.Explore the impact factors of high frequency regeneration system of rape（1）.The best concentration of 6-BA in the callus induction medium of QZ-1831 R hypocotyl and leaf was 2 mg · L-1,that best concentration of 2,4-D was 0.5 mg · L-1;The optimum 6-BA concentration for callus induction in hypocotyls and leaves of H165 was 2 mg·L-1,and the 2,4-D concentration was about 1 mg·L-1.The best concentration of 6-BA in bud regeneration medium was 4 mg · L-1 and the concentration of GA3 was 1.5 mg·L-1.Therefore,2.0 mg·L-16-BA,0.5mg·L-1 2,4-D can be added to the callus induction medium of Brassica napus;2mg·L-16-BA,1.5mg·L-12,4-D can be added to the callus induction medium of Brassica campestris.4 mg·L-1 6-BA,1.5mg·L-1 GA3 can be added to bud regeneration medium.The results showed that different genotypes had different effects on plant regeneration.The callus induction rate of hypocotyls was QZ-1831R> H165> LY-C20> QZ-482R> GZ-3219R> H403-3> H403-2 in turn.The callus induction rate of the leaves was GZ-3219 R> H403-3> QZ-1831 R≥QZ-482R> LY-C20≥H165> H403-2 in turn.In bud regeneration,the regeneration rate of hypocotyls and leaves of Brassica napus was all higher than that of Brassica campestris.（2）.Genotype had a great effect on the regeneration ability of rape,and QZ-1831 R and GZ-3219 R were the best in Brassica napus,and H165 and H403-3 were the best in Brassica campestris.（3）.The optimal rooting medium for QZ-1831 R and GZ-3219 R was 1/2MS + 0.2 mg·L-1NAA(0.4 mg·L-1 IAA).And the optimal rooting medium for H165 and H403-3 was 1/2 MS + 0.5 mg·L-1（NAA/IAA）.This indicated that Brassica napus was easier to take root than Brassica campestris.2.Detection and molecular genetic transformation mediated by Agrobacterium tumefaciens（1）.The optimum ultrasonic treatment time was 15s;When the vacuum pressure was 0.05 Mpa and the vacuum time was 2min,the conversion efficiency of hypocotyls and leaves was better than other treatments;When the concentration of antibiotics was 500-600 mg·L-1,the growth of Agrobacterium was effectively inhibited.When the antibiotic concentration was less than 500 mg·L-1,the inhibitory effect of Carb was the best.But at the stage of bud regeneration,300 mg·L-1 Cav treatment had the strongest ability to regenerate shoots（2）.After the Glyphosate resistance screening and PCR detection,the conversion rate of hypocotyl and leaf of Brassica napus QZ-1831 R was the highest,which were respectively 4.83% and 4.98%,The transformation rate of hypocotyls and leaves of H403-2 in cabbage cultivars was the highest which were 4.4% and 4.2%,（3）.The qRT-PCR analysis of transgenic rapeseed plants of the T1 generation results show that in addition to QZ-1831 R,the expression of Bn GRF2 in the other six cultivars was significantly higher than that in the control plants.And the expression of Bn GRF2 gene in QZ-482 R,LY-C20 and H403-2 was overexpressed.The Bn GRF2 which had effects on the oil content and thousand-grain weight were studied in seven transgenic cultivars.It was found that the oil content and thousand-grain weight of GZ-3219 R,QZ-482 R,LY-C20,H165,H403-2 and H403-3 were significantly higher than those of WT except QZ-1831 R. |
PDF Full Text Request