| In this paper, compared germinate status of 11 peanut species on MS medium and 1/2MS medium, calculated shoot induce rate of different genotype peanut on four mediums, observed sterilizing effect and effect on seed germinate of different disposal time of 75% Alcohol and 0.1% HgCl2. The result indicated: after seeds were deal with Alcohol for 20~30s and HgCl2 for 10~12min, we gained high germinate rate and good sterilize efficiency: all peanut species' germinate rate on 1/2MS medium higher on MS medium, shoot induce rate of shanyou 523 was 93.1%, the highest on shoot induce medium 1. The worst shoot induce status was shanyou 31, this specie's shoot induce rate only was 9.5% on shoot induce medium 2.This experiments use simplification of a little amount extraction in base method, simple and quick boil method and kit method to extract plasmid pCB302-3, and use a pair of specificity primers proceeds the PCR (polymerase chain reaction) amplification, the difference withdraws to every kind of method of the very PCR amplification outcome and plasmid DNA proceeds the agar sugar gel electrophoresis analyzes, the result expresses a little amount extraction method in base method and kit method is effective, the purity of plasmid DNA extracted by these methods is high with good output, and repetition is good, coming to a the molecular biology experiment the request. A little amount extraction in base method had the stability and economic characteristics, and very in keeping with majority laboratory usage.Genetic transformation of peanut using Agrobactierium-mediaed approach is still technically challenging. On the base of studies on the factors affecting the transformation, such as the selective pressure of PPT, the concentration of the Agrobacteria, the timing of infection and co-cultivation, The optimized procedure is as following. After pre-cultured for 3 days on MS medium containing 3mg/L BA, 0.8mg/L NAA, 2mg/L AgNO3 and 6mg/L Gin, leaf explants were dipped into an Agrobacterium suspension (OD60CN0.3) for 5-7min. The leaflets were then co-cultured with the Agrobacteria on a shoot induction medium in the dark. Two days later, the explants were transferred to above pre-culture media with 500 mg/L Cb, 300mg/L Cef, and 0.25mg/L PPT. This optimized protocol has been successfully used to transfer the CpTI and Bar genes into peanut (Arachis hypogaea L.cv.Shanyou). A total of 24 regeneration plants were produced in 3 months. Out of these, seven plants were proven to contain a characteristic 310bp DNA segment of the CpTI gene by PCR analysis. The integration of foreign gene into the peanutgenome was further confirmed by southern blot analysis, PCR -southern blot analysis of progeny plants (T|) demonstrated that the transgenes are stably integrated in the genome of transgenic peanut plants and inherited by the offspring. The expression of the insect-resistance gene in the transgenic plants was observed through the bioassays.In addition, this experiment studied the influence of two assisting methods on transformation, negative pressure-assisted and Sonication-assisted transformation, estimated the disservice to explants of different intensities of negative pressure and the time of sonication. Experiment results showed:low intensity of negative pressure(30 movements) and short time of sonication(2 minutes) caused little infection to shoot regeneration of explants, which helps to improve the percentage of anti-PPT shoots regeneration, although prolong the time of shoots regeneration. We got 7 transgenic plants from Negative pressure-assisted method, 4 transgenic plants from Sonication-assisted method, 6 transgenic plants and 3 transgenic plants separately of foreign gene's integration into the peanut genome was confirmed by Southern blot analysis.
|
PDF Full Text Request