| The haploid breeding of maize is a technology that using anther culture or gynogenesis to produce haploid,and then obtain stable new varieties through natural or artificial doubling. Haploid breeding can shorten the time period of breeding,and if the haploid culture system is used as the target of gene transformation,the foreign gene can be inherited stably in the genome of the offspring,and no separation could appear. So,haploid breeding of maize has great significance either in practice,or in basic research.For a long time,there are some unresolved problems in the anther culture of maize,such as the low induction frequency,the great difference of induction frequency between genotypes,and low survival rate of transplant of regenerated plantlets. According to theses problems,the present article emphasized on the effect of various factors on the anther culture of maize. The results show that:effects of genotypes are very different,and the induction frequency of the hybrids was higher than that of the pure lines,a hybrid "Zhong 0198" with high induction frequency was screened;the induction frequency was optimal when the pollen were at the mid-unincleate stage;the induction frequency of the embryoids and calli in liquid culture medium could increase to more than twofold of that in semi-solid;when the medium was supplemented with 0.5% active carbon,the induction frequency of embryoid or callus increased from 5.25% to 9.35%;15% sucrose was optimal for anther culture of maize,and adjust sucrose concentration from 15% to 10% after 2weeks of culture,can obviously increase the induction frequency;the combination of high level kinetin and low level BA can stimulate the regeneration of somatic embryos;in contrast,the low level of kinetin and high level of BA are beneficial to the regeneration of buds;low temperature (4C) preculture could increase the induction frequency from 3.31% to 11.71%;when the medium was supplemented with 2 mg/1 MET,the root growth of plantlets was promoted obviously;the winter of Hainan Island is optimal for transplant of regenerated haploid maize.In the experiment of gynogenesis,unfertilized female ears were inoculated on medium. The culture medium:N6 + 2,4-D Img/L + NAA Img/L + BA Img/L + CH 200mg/L + colchicine 2 mg/L + sucrose 5% + agar 0.7%. 3 materials were used,26 ears (about 3900 ovaries) were inoculated,and each kind of material can get seeds more or less. The induction frequency was 3.06%,2.29%,and 1.90% respectively. Totally 5 regenerated plantlets were obtained. 3 of them were haploid plants (n=10),and the other two were diploid. The ploidy of plants has been determined by examination of chromosome. When they were transferred into soil,4 of them survived,and only one diploid plant flowered and got seeds normally. When the seeds were sowed in the experimental field,they grow regularly and consistently,appeared the characteristics of self-line. And 2 albinos were appeared in the offspring,indicating they were doubled haploid. Through paraffin section,we can see that the embryogenesis was originated inside the embryo sac. We repeated the experiment by inoculating ovaries of 10 more genotypes of maize,and the results were verified.Gene transformation is an effective approach of maize breeding using biotechnology. The present article tried the transformation of gene into maize ovaries by microinjection. First,a plant expression vector pFBR,which contains the gene encoding flower promoting factor 1 (FPF1) and plant selection marker pat gene,was constructed. The method of in vitro injection was used in which the maize ears were removed from the plant after they were pollinated for 24 h,then,the ear coats were stripped off to expose the ovaries in which several u L of transformed gene solution were injected in clean bench area using glass microneedle. After all the ovaries were injected,the ear was cut into several segments,which then were put into a glass bottlecontaining culture medium and incubated in a growth chamber with fluorescent lamps. Seeds or plantlets could be di...
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