| Chinese cabbage(Brassica rapa L.ssp.pekinesis)is an important vegetable crop in the genus Brassica in the family Cruciferaceae.Transgenic technology is an important way to study crop gene function and breed new varieties.The existing genetic transformation system of Chinese cabbage has the problems of difficult regeneration of explants and extremely low genetic transformation efficiency of traditional agrobacterium,and it is of great significance to build a new genetic transformation system that is efficient,stable and suitable for Chinese cabbage.In this study,the DH line of Chinese cabbage was used as the experimental materials.The genetic transformation of microspores and pollen by magnetic nanoparticles and genetic transformation with microspores by particle bombardment were investigated.By using the new transformation method the key genes involved in regulation of anthocyanins in purple Chinese cabbage was studied.The results are as follows:1.Genetic transformation of microspores and pollen by magnetic nanoparticle method was investigated.The DNA loading range of magnetic nanoparticles and the magnetic nanoparticles with a diameter of 100 nm were used for 0.5,0.75,1.0 h magnetic conversion time gradient were examed.Only sporadic GUS-transformed microspores and pollens were obtained.Those diameter magnetic nanoparticles can realize the genetic transformation of thin-walled pollens of lily,sweet pepper and cucumber.Further research on nanoparticle size and magnetic field strength are require and this method may become a new way for the genetic transformation of Chinese cabbage microspores.2.The Chinese cabbage DH strains suitable for particle bombardment were screened.Microspores were used as explants to withstand the stress of multiple bombardment and continued induction to obtain embryos.Statistics of microspore viability and embryoid-induced regeneration ability showed that under the combination of bombardment times(3,4,5)× bombardment pressure(900,1100,1350 psi)×bombardment distance(3,6,9 cm),after bombardment and culturing for 72 h,the average viability of microspores in the treatment group ranged from 74.76% to 88.55%,while that in the control group was88.09%.Compared with the control group,there was no significant difference in the viability of microspores.There was no significant effect on the embryos after treated by particle bombardment method,when the number of bombardment time was 4,the number of embryos produced in the treatment group was 16-236/dish,and the control group was 117/dish.In the cell culture category,the number of embryos induced by microspores was not necessarily related to the gene gun bombardment parameters.3.The new genetic transformation system with optimal parameters of Chinese cabbage microspores mediated by particle bombardment was established.The bombardment parameters of the biolistic method were 4 times-1350 psi-3 cm,4 times-1100 psi-3 cm,4 times-900 psi-3 cm and they had the high transient expression efficiency.The average number of transformed microspores was 21.67,11.67,and 11.67 per dish(3.5 m L).When the bombardment times were 4,the bombardment pressure was 900 psi,and the bombardment distance was 6 cm,the microspores obtain the most genetically transformed embryos,and the transformation efficiency reached 10.82%.4.In this study on the regulation of anthocyanin metabolism in purple Chinese cabbage the important regulatory gene Dark＿Pur was cloned.The analysis found that the gene has three exons and two introns,and its structure and sequence are similar to those of purple mustard.By using microspores of Chinese cabbage DH line as explants,the Dark＿Pur gene was transformed by particle bombardment method,and transformed Chinese cabbage seedlings showed purple color.This new Chinese cabbage microspore biolistic-mediated genetic transformation system is useful for in-depth analysis of the genes in Chinese cabbage. |
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