| The species of Medicago genus are widely cultivated around the world and are the most important source of high quality forage, which also helping improve the agro-ecological issues. With the rapid development of grass industry in China, High-yield, high-quality, high resistance forage varieties are extremely demanded. Ploidy research is the basis of alfalfa breeding program, and the theoretical guidance of genetic characteristics study on genus Medicago. This paper take M. polymorpha, M. lupulina, M. truncatula Gaertn, M. sativa cv. Huaiyin, M. saliva cv. Siriver and M. sativa cv. Multifoliar as materials. With different concentrations of colchicine treating on the plant seeds, we establish the colchicine mutagenesis polyploid technology system of Medicago spp. to obtain the new germplasms at different ploidy level.The results are as follows:(1) After the induction of colchicine on the germinating seeds, apparent distortion appeared on the germinated plumules:hypocotyls distorted, and became rodlike, and the radicle growth points had been severely suppressed, the radicle tip grew spherical, the majority fails to grow to plants and eventual dead;(2) Haploid alfalfa was significantly lower than the tetraploid plants on height, the branches number, the internode number and the leaves number on each stem (p<0.05), the leaf area was about half of the tetraploid alfalfa. The suspected octaploid alfalfa which was found in the field was significantly higher than the tetraploid alfalfa on leaf area, height, the branches number, and the leaves number on each stem (p<0.05), but the difference between the internode numbers were non-significant;(3) The number of stomatal guard cell chloroplasts of tetraploid alfalfa was56.02%more than diploid alfalfa, which showed highly significant difference (p<0.05), the suspected octaploid alfalfa was compared with the tetraploid alfalfa, the number of stomatal guard cell chloroplasts was21.62%more than the tetraploid plants;(4) The improved squash method to count the chromosomes of root tip, to establish the chromosome section technical regulations for Medicago spp. As the analysis of chromosome counts showed, the chromosome numbers of the studied6species of genus Medicago were as follows:M. sativa cv. Siriver (2n=32), M. truncatula (2n=16), M. lupulina (2n=16), and M. polymorpha was a diploid species with14chromosomes. In addition, the diploid alfalfa appeared naturally in field with the chromosomes of2n=16, the number of chromosomes of suspected octaploid alfalfa was2n=64, and mixoploidy(2n=46,52,60) were also observed.(5) Ploidy level of each material were measured by flow cytometry,and obtained the DNA content distribution curve, the analysis results were consistent with the chromosome count. After the colchicine treatment, the chromosome ploidy of the variation plants doubled. The DNA content of diploid alfalfa, M. lupulina, M. polymorpha were similar with M.truncatula, but the DNA content of M. polymorpha was slightly lower than M.truncatula(6) The flow cytometer analysis showed that, the extract buffer â…¢, â…£, and â…¤ were not applicable for the nucleus suspension preparation of genus Medicago plants, extraction buffer â… was the right buffer suit for the nucleus suspension preparation. The two kinds of mixed enzyme solutions prepared the nucleus suspension which can form the obvious ploidy peak. Besides, the enzymatic hydrolysis could get more complete protoplast. So, the recommendation was to use of enzymatic hydrolysis to prepare the nucleus suspension of genus Medicago plants, and after purified, we can obtain high quality single nucleus suspension suit for the FCM analysis. |
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