Isolated Microspore Culture And Analysis Of Gene Loci For Microspore Embryogenic Ability In Chinese Cabbage(Brassica Rapa L.ssp.Pekinensis)

Since successful production of haploids via an embryo from isolated microspore culture of Brassica.rapa in China in 1990s.Microspore culture producing haploids and/or double haploids is a very useful technique for breeding programs,genetics and functional genomics research in Chinese Cabbage?B.rapa?.The routine protocol of isolated microspore culture of Brassica has been established.However,it is necessary to improve this culture method for easy to operate.And the genetypic variations remain as a nondissolved factor which limits the application of isolated microspore culture technology.In order to improve the culture techniques and gain more insights into the mechanism of microspore embryogenesis,we performed study in relation to development the culture protocol and finding the influence factors in genetypic variation to elucidating the mechanism of microspore embryogenesis.And we also attempts to use microspores as a target for particle bombardment for transformation in Chinese cabbage.The main progresses of study are as follows:1.We developed the microspore culture method for dealing with multiple samples:This is the first report of use machine method isolation microspores in China.Compare with conventional method,use machine method isolation microspores can eliminate human influencing factors.The improved method in this study is a timesaving method for microspore culture of Chinese cabbage and can be applied to multiple samples efficiently.Machine method replace artificial can deal with multiple samples in one experiment and easy adjust the density of microspores culture at short time.Compare microspore isolation using the Beads Cell Disrupter System with the conventional method,the isolated microspores quantities and viability was not significant differences and can objectively reflect the test material's ability of microspore embryogenesis.2.We used two lines of Chinese cabbage as parents for producing F1 on the basis of their different microspore embryogenic ability?MEA?,and used the high MEA parent backcross with BC₂ for production of the BC₂DH population to analyse of the candidate gene on MEA.Based on Construction of Genetic linkage maps with BC₂DH population,which have introgression lines characteristics according to the previoue study work,We attempt to use a new method controlling microspore embryogenic ability gene positioned in the chromosome region by directly compare with DNA marker segreation analysis and test plants phenotype.By linkage analysis,the candidate chromosomal sections were located at the A03 chromosome and indentified 3 markers linked to the candidate gene loci which associated with microspore's MEA.We have screening 11 candidate genes by transcriptome analysis and positioned in A03 chromosome.These candidate genes showed highly expression at the induction stage for microspores of responsive genotypes but not work in the recalcitrant genotypes,which these genes regulated the cell Stress response and closely related to the skeleton rearrangement of cell wall and embryo development.3.By RNA-seq and analysis of microspores of B.rapa in the beginning induction period of 24 hours.We found 5020 genes expressed themselves differentially under heat-stress treatment between different samples.Pathway enrichment analyses revealed that large amount of genes associated with regulated the cell wall cellulosic and pectin esterification metabolism,include the exine and intine.When compared differentially expressed genes between genotypes and pretreatment,the responsive genotypes and recalcitrant genotypes showed more significant than temperature treatment.4.There was a new discovery that histone deacetylase inhibitior Trichostatin A?TSA?could trigger microspore embryogenesis in Brassica.rapa like traditional heat stress did and not need 33?temperature pretreatment.The results of TSA inducing microspore embryogenesis indicate that TSA can alter the epigenetic modifications then regulation of gene expression.By Q-PCR examined the embryo genes of BABY BOOM?FUS3?NAPIN,the study results showed that they were detected at embryo stage with high expression but low expressionin in the beginning of microspore induction period.5.Use microspores as a target for particle bombardment for transformation in Chinese cabbage.The results of this study suggest that particle bombardment of microspores could be an alternative method for genetic transformation of Brassica.rapa,and may overcome the bottleneck of Low shoot regeneration for conventional method in B.rapa and to improve the genetic transformation ratio for microspores highly embryogenic ability.The results of this paper optimized the isolated microspore culture of Chinese cabbage and broaden the application fields of microspores.It has a breakthrough to analyse of the candidate gene on microspore embryogenic ability on Chinese chabbage.