| Doubled Haploid population with homozygosity of all loci derived from spontaneous Diploidization or induced by treating haploid plants with an ant mitotic agent, which constructed permanent segregation population. It can be directly applied to either actual breeding improvement or to modern genetics analysis such as molecular mark and soon on. The techniques of establishment DH population include two key processes that are obtaining doubled haploids and identification the chromosome level of them in the shortest time possible. At present, the techniques of anther culture have been developed well, but the frequency of spontaneous chromosome doubling in tobacco is very low and the efficient protocols for production of dihaploid plants have not been developed. This study compared the efficiency of different methods of diploidization in tobacco and optimise the chromosome-doubling treatment by applying DMSO (dimethylsufoxide) to colchicine, and we successfully obtained two DH populations which included 144 and 42 doubled haploid lines respectively for the first time. Meanwhile, we went ahead of others to evaluate 6 genetic parameters of agronomic traits of tobacco by using the established DH population. The main results are listed as follows: 1. Study on doubling method of haploids in tobacco. For the first time, we compared four methods of doubling in tobacco, the result indicated: application 4g/L colchicine for four methods of doubling was the most efficient among other concentration. The efficiency of colchicinesin7applied directly to anther excised from the flower buds of tobacco just before anther culture was the highest at 75% among four methods, followed by that of leaves pre-treated by colchicines before regeneration was (36.67%) and haploid plantlets was soaked in colchicine was (32.35%); and the efficiency of colchicine soaked the auxiliary bud was the lowest, only reached to 17.78%. Although, the former two methods brought about higher score in doubling, the more abnormal plantlets and intensive labour were companied. So, the method of soaking young plantlets in colchicine was chose to construction DH population for economical aim.DMSO, it was added respectively to colchicine solution at concentrations of 10g/L~30g/L to optimise the technique of doubling haploids. The result proved that it obviously improved the efficiency of colchicine for doubling haploids in tobacco and this effect increased with soaking duration. Application of 4g/L colchicine solution mixed with 20g/L DMSO to soak haploid plantlets for 48h gained the highest efficiency of colchicine doubling. With soaking plantlets in colchicine more than 12 h, the frequency of doubling in two tobacco groups was raised to 2.30~2.93 (plantlets treated with 4g/L colchicine for 48 h) times respectively according to controlling methods. Fathomer, it's more economical that application of DMSO to low concentration colchicine could lead to the higher doubling efficiency in high concentration colchicine.2. Rapid determination of ploidy level of chromosome in tobacco.The study revealed that measurement of the number of stoma chloroplast in guard cell could be considered as a simple and accurate method to determine haploid, diploid or polyploid of tobacco. The result of identification by this method was above 93%, which is in consistent with those observations of fruiting of the plants. There were about 90% plants that owned below 14 chloroplasts in stoma were picked out before transplanting by counting the number of stoma chloroplast in guard cell. We selected undoubled haploid at early time when young plantlets just grew the forth leaf to treated it with otherIVdoubling method to save time and accelerated establishment DH population.3. Evaluation on genetics parameters of agronomic traits of DH population in tobaccoThe heritability, the number of segregating genes, the skewness and the kourtosis of 6 quantitative traits were analysed with two DH populations. The result showed: The heritability of major... |
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