Study Of Fertilization Condition And Development Process In Vivo Haploid Induction In Maize

As the large-scale application of technology for inducing maize haploids in vivo by Stock6 derivative in maize breeding, the selection of inducers with high frequency and chromosome doubling technology have made great progress. However, the mechanism of inducing haploid is still in speculation or hypothesis state. In this study, the super-sweet maize inbred, Hua24 as the seed parent crossed with the high frequency inducer line, HZI6 which was independently cultivated by our research group as pollen parent. The fertilization condition of central cells and egg cells and development process of embryoes and endosperms at 25-65h,8d,12d,15d and at maturity were studied after Hua24 x HZI6 pollination. The goal was to reveal the mechanism of inducing haploid. The result would direct the selection of high frequency inducer and the application of technology for inducing maize haploids in vivo, and provide the example for the study of the other plants haploid. In addition, it would facilitate understanding the double fertilization more comprehensively. The results were as follow:1. The line HZI6, Hua24 and normal Corn inbred lines were selected to hybrid each other or self-fertilization. The setting rate was examined. The high setting rate of Hua24 self-fertilized or Hua24×normal corn showed that the egg cells and sperms of Hua24 were normal. The high setting rate of HZI6 x Hua24 showed that the egg cells of HZI6 were normal. However, the setting rate of self-fertilization HZI6 and Hua24×HZI6 was very low and some kernels were abnormal, which showed that the kernels of abnormal development were caused by the pollens of inducer.2. Based on the ovule whole stain-clearing 25-65h after Hua24 x HZI6 pollination, ovules of an average 54.19% that embryoes and endosperms both divided, ovules of an average 45.80% that embryoes or endosperms alone developed, ovules of embryoes and endosperms both divided should include haploids and diploids, which the rate of haploid induction was 4.77% according to the data of 12th and 15th day after pollination. So the ovules of normal diploid embryo and triploid endosperm were about 50%, and the other half ovules developed only embryo or endosperm or developed into haploids. About 9% of ovules had one sperm in synergid in embryo and endosperm ovules of division, in ovules of the endosperms division and egg cells no division, the 70% of ovules had sperms in synergid cells, and the egg cell is clearing, is similar as the unpollination.3. Based on the ovule whole stain-clearing 25-65h after Hua24 x HZI6 pollination, the ovule of 37.74%-43.57%, an average of 40.51% of the egg cells that didn't divide while the endosperm divided normally.44.68% of seeds developed without embryos at 8th day after pollination, seeds of 42.46% were abortive at 12th day and 15th day after pollination, seeds of 44.31% were wizened at the seed mature stage. These values were very similar. So speculated that the ovule which the egg cells had no division after pollination, no embryo at 8 th day, abortion at 12th and 15th day, wizened at mature.4. Hua24 x HZI6 embryoes and root tips of 10 or 10 and 20 coexistence, SSR markers dections suggested that all the bands were materal Hua24, and endomitosis phenomenon at cytology detection. These suggested that the haploids were maternal origin. The cells of 20 chromosomes were doubled naturally during development by endomitosis.5. Young embryoes of about 0.5-1.0% by cytological or SSR markers detection were aneuploidy, of which chromosomes of every cell were 19 or 21 in aneuploidy embryoes, Presumably, the sperms of inducer HZI6 were 11 or 9 chromosomes. That is to say, and the embryoes of 19 chromosomes lacks the the eighth chromosome, so Speculated that fraction of aneuploidy sperms in inducer HZI6 could also live, even accomplished fertilization. The endosperms of about 1.85% had 40 chromosomes and the embryoes had no development by cytological detection the 6th day after Hua24 x HZI6 pollination It was speculated that two sperms fertilized the central cells and egg cell had no fertilization.