| Perennial ryegrass(Lolium perenne L.), belonging to tribe Poeae, subfamily Pooideae, family Gramineae, was an important cool-season forage and turfgrass. The utilization of the grass was limited due to the poor appearance of leaf texture and the low ability of resistance to overwinter, drought and salinization. In order to seek for new germplasm resources and improve the quality of the perennial ryegrass, a high-efficient tissue culture system was established. The polymorphism of somaclonal variation, including morphology, chromosome and DNA were also studied. Additionally, the 60Coγ-ray irradiation-induced effects on the perennial ryegrass and mutagenic mechanisms of the two type induced chlorophll-deficient mutants were discussed. The main results were summarized as follows: 1. Establishment of a high-efficient system of tissue culture for the perennial ryegrass. Callus could be induced from mature embryo in N6 medium with 10mg·L-12, 4-D, 500mg·L-1CH, 0.5mg·L-16-BA and 0.5mg·L-1NAA. The induction rate of the medium reached 85%. Then the callus was transferred into the differentiation medium that contained N6 medium and 1 mg·L-16-BA, 0.5 mg·L-1 KT, the induction rate was 77.73%. The S1medium with 0.2mg·L-1NAA and 1/2 N6 medium in mass, was greatly suitable for rooting. The survival rate reached 100%. 2. Polymorphism of somaclonal variation. (1)There existed different morphological characteristics between regenerated plants and CK, such as leaf index, leaf color and flower type. Some special physiological types were also obtained. (2)The result of Karyotype analysis showed that the perennial ryegrass was diploid and composed of metacentric chromosomes and submetacentric chromosomes that belonged to "1A"type. Its karyotypical formula was 2n=2x=14=2sm+12m (2SAT). It was also found that chromosome number changes occurred in 40% of cells, and there were an obvious chromosome structural variation (such as fragment, dicentrics, condensation and transposed ring etc.), mitotic aberrations (such as lagging chromosome, bridges etc ) as well as numerical alterations (including haploid, aneuploid and polyploid etc.) in regenerated plants. (3)Somaclonal variation in regenerated plants was tested by RAPD profiles. Eleven arbitrary 10-mer primers were used to amplify DNA. Four of the eleven primers detected polymorphism in the regenerated plants, and the frequency of variation was between 5.8% and 15.4%. Morphological, chromosomal and RAPD analyses of regenerated plants indicated a relatively rich somaclonal variation of the perennial ryegrass. Therefore, the population of the regenerated plants was a source of variation for new germplasm selection. However, the genetic stability was reduced by somaclonal variation. It was suggested that the induction of somaclonal variation should be avoided when tissue culture technology was used as a tool of gene transfer. 3. The 60 Coγ-ray irradiation effects and mutagenic mechanism of two type induced chlorophll-deficient mutants (1) Seed germination and seedling growth were stimulated at the dose rate of 50 Gy of radiation. When the dose rate was between 100-250 Gy, there was a inhibiting effects on the germination and seedling growth. When the rate was 250 Gy, the effects was significant. And the LD50 of the perennial ryegrass was 342 Gy according to the linear regression equation of LD50. Therefore, the optimum radiation dose for perennial ryegrass was 250-350 Gy. (2)Two type induced chlorophll-deficient mutants were obtained by 60Co γ-ray irradiation treatment. One had yellow spaced green leaves (FA and FC), the other had white spaced green leaves (FD). The contents of chlorophyll a, chlorophyll b in the two type mutants were lower than that of the leaves in control (17.5%-48.6%). There was an obvious difference in the ratio of chlorophll a/b between the two type mutants. The ratio of chlorophll a/b in the FA and FC were higher than that of the leaves in control (between 17.5% and 22.5%), whereas the ratio of FD was similar to that of the control. Therefore, both of two type mutants were chlorophyll deficient mutants, and FA and FD were the types with low chlorophyll b content. Compared with the control, FA and FC had lower net photosynthesis rate (Pn), decreased by 73.7% and 29.34%, respectively, but FD had no decrease in net photosynthesis rate. These results indicated that the ability of photosynthesis was different in two type mutants, and the lower chlorophyll content had no effect on the net photosynthesis in FD. The polymorphism was detected in both of the two type mutants by POD, therefore, it could be deduced that the main reason for the mutant occurrence might be gene mutation or gene differentiational expression. AFLP marker did not detect polymorphism loci, which might be due to the limitation of AFLP or largest volume of the total DNA in the species, orthe mutation did not occur in nuclear DNA but in cytoplasm DNA. The results will provide not only a theoretical basis for induced breeding, but also a technique support for selective breeding of new varieties.
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