| Maize (Zea mays L.) is one of the most important cereal crops, being used both as human food and animal feed. Usually it may take 4-6 years to breed a pure line by using traditional breeding method. But, haploids production via in vitro androgenesis or in vitro gygogenesis can accelerate the process and enhance breeding efficiency. In maize, in vitro-produced haploids can save 2-3 years. The investigations were aimed at establishing an effective protocol for in vitro production of maize haploids, and understanding the mechanism of in vitro gygogenesis. The effect of various pollination treatment for maize ovaries on haploid production was investigated, including duration of pollination treatment, mode of pollination, season of pollination, et al. Two different chromosome doubling techniques was compared. The plants derived from maize ovaries were subjected to genetic analysis using SSR molecular marker. The results were as follows:1. Embryo induction medium in maize ovary culture was N6 basal medium, supplemented with 3.0mg/L 2.4-D, 500mg/L casein hydrolysate, 690mg/L L-proline, 30g/L-120g/L sucrose , 10mg/L silver nitrate. Embryo differentiation medium was MS basal medium, contented with 0-1mg/L KT, 500mg/L, 690mg/L L-proline, 30g/L-60g/L sucrose. Medium for rooting was 1:1 dilute MS salts, MS organic compounds, enriched with 0.5mg/L NAA, 15-20g/L sucrose.2. Cold pre-treatment led to increased embryogenesis in maize. Ovaries without cold pre-treatment failed to produce embryo. On the contrary embryogenesis occurred. The embryo induction frequency was 3.33% in ovaries with 15 hpp after cold pre-treatment for 60 hours at 5C. The embryo production ratio was 1.55% in ovaries with 17 hpp after cold pre-treatment for 24 hours at 5C.3. Hpp was an important factor affecting induction result. Haploid plants can be obtained from pollinated ovaries with hpp ranged from 15 hours to 22 hours . The highest induction frequency was 02.%, occurred in ovaries with 19 hpp. However, for ovaries growing in the greenhouse, the hpp should be prolonged to 26-30 hours for haploid induction. The highest was 0.18%, occurred in ovaries with 29 hpp.4. Better result achieved in self- pollination than cross-pollination. The hpp for cross-pollination was found to be shorter than the one for self-pollination for in vitro gynogenesis.5. Gygogenesis efficiency related to pollination season. The ovaries collected in spring (February, March) and autumn (September, October) were more responsive to in vitro gygogenesis than ones in summer (July). The best result occurred in spring (0.109%). The second in autumn (0.105%). The poor result obtained in summer (0.058%).6. A total of 281 plantlets was obtained in two years. Among of them, 24 were determined to be haploids after chromosome counting. Two haploids were diploidized successfully after applying colchicines. One haploid shed pollen spontaneously and set seed.7. Twenty different SSR primers showing polymorphism between 178 and Huang C were used to genetic analysis for .diploid plants from maize ovaries. A R1 generation plant , whose progenyrevealed a high level of homogeneity for many agromorphological traits such as : plant height, leaf shape, the location of ear, etc, was homozygous at 20 loci ( 2 loci per chromosome), showing paternal ( Huang C ) bandings at 11 loci and maternal (178 ) bandings at the other 9 loci. It was concluded that the R1 diploid plant originated from an unfertilized haploid cell.8. The haploid shed pollen spontaneously was also subject to be genetic analysis using SSR molecular markers. SSR analysis revealed that it was homozygous at 20 loci, showing paternal (Huang C ) bandings at 15 loci and maternal (178) bandings at the other 5 loci.The above results demonstrated the it is feasible to induce maize gygogenesis by in vitro culture of pollinated ovaries.
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