Study On The Mechanism Of In Vivo Haploid Induction By Stock6 Derived Line

Haploid breeding shortens the breeding process,reduces costs and improves the breeding efficiency.In vivo haploid induction by Stock6 is widely used by most breeders for its high effectiveness,simple operation and inexpensiveness in maize(Zea mays L.). In this study we try to illustrate the mechanism of haploid induction by Stock6 using Stock6 derived line HZI1,inbred lines Hua24,HZ124b,HZ141 and HZ85,as well as the crosses HZ124b×HZI1,HZ141×HZI1,HZ85×HZI1 and Hua24×HZI1 as materials, applying cytological assay,molecular markers analysis and morphological assay.Main results were described as follows:1.About 0.2%kernels in the F₁ kernels from the cross Hua24×HZI1 had mosaic endosperm of purple aleurone normal half and yellow shrunken half without purple aleurone,and the mosaic kernels had different sizes of sweet shrunken endosperm in the top centre of the kernels.The occurrence of the kernels with mosaic endosperm strongly supported the hypothesis of 'chromosome selective elimination' in maize haploid induction.2.In ovule cells after pollinated 36h from the cross Hua24×HZI1,individual chromosomes were arranged out of equatorial plate in metaphase cell and micronuclei were formed,which were the main evidence of the chromosome elimination.We deduced that chromosome elimination exists in the maize haploid induction by HZI1.3.Chromosome counting in the radicles of inducer HZI1 and Hua24,HZ85 as checks,found that 15%cells were not diploid cells in HZI1 and only 3%cells were not diploid cells in Hua24 and HZ85.Diploid cells were dominated in inducer HZI1,and also 10%were haploid cells,and in 10%radicles of inducer HZI1 were most frequent of haploid cells(＞97%),and were haploid plants in the field.4.In all four crosses,55-86.84%were mixoploid in the radicles of the putative haploid kernels with purple aleurone and colorless embryo,and the cells 2n=10 were more frequent,the frequency was 67.47%,76.74%,45.22%and 27.93%,respectively. 62.5-87.5%were mixoploid in the radicles of kernels with purple aleurone and purple embryo,and the cells with 2n=20 were more frequent,and the frequency was 54.27%, 65.43%,56.05%and 58%,respectively.According to the results of chromosome counting,typesâ… ,â…¡,â…¢of kernels with purple aleurone and colorless embryo were haploids in the field,and other types were pseudo haploids by incorrect selection. Kernels with purple aleurone and purple embryo were diploids and mainly in typesâ…¤,â…¥andâ…¦.5.The results of the changes of cells frequency of the three times radicle collection in cross HZ85×HZI1,showed that the frequency of cells with 2n=10 was increased from 13.29%,to 53.88%,to 70%,and the frequency of cells with 2n=20 was decreased from 65.19%,to 20.87%,to 10%in the radicles of kernels with purple aleurone and colorless embryo.The frequency of cells with 2n=10 and 2n=20 were varied narrowly in the radicles of kernels with purple aleurone and purple embryo,in which cells 2n=20 were dominated and the frequency was about 50%in three EXPs,cells with 2n=10 were below 5%in three EXPs.6.Morphological traits:Plant height,Ear height,Ear leaf length,Ear leaf width, Tassel length,Tassel branches numbers were measured after flower in crosses Hua24×HZI1 and HZ124b×HZI1,and found that there are all significant difference at 0.001 level between Hua24 haploid and Hua24 inbred except for tassel branches numbers, while there are all significant difference at 0.001 level between HZ124b haploid and HZ124b inbred.7.SSR analysis was made in the haploid of crosses Hua24×HZI1 and HZ124b×HZI1 and their parents.All haploids from the cross Hua24×HZI1 shared the same genomic compositions as Hua24 except for plants Nos.862 and 857 with some polymorphic DNA bands,and all haploids from HZ124b×HZI1 have the same genetic compositions with maternal parent.The results of morphology,chromosome counting, and SSR analysis were identical in No.857 haploid,its phenotype were different from other haploids,and have significant difference from others in Ear leaf length,Ear leaf width,Tassel branches,we deduced that No.857 were mixoploid haploid.The results revealed that chromosome elimination after fertilization caused the haploid production in maize.Aneuploid cells,haploid cells and the diploid cells from the progress of chromosome elimination formed the mixoploid tissues,and finally caused haploid plant.The results may be important for the selection of inducers with high frequency and application in maize haploid breeding and lay a foundation for the further cytology research of mechanism of haploid induction.