| Stylosanthes guianensis is a perennial herbaceous plant in the genus Stylosanthes of the family Leguminosae and it's an excellent tropical pasture. Induced polyploid breeding is one way of germplasm innovation. Studies of polyploid induction of Stylosanthes guianensis cv. Reyan No.5 by chromosome doubling technique of colchicine treatment were conducted and homozygous autotetraploid plants were obtained. In addition, rapid identification of the polyploid of Stylosanthes guianensis and differences of some biological characteristics of Stylosanthes guianensis of different ploidies were performed. The results of this test are as follows:Influence of different concentrations and durations of treatment of colchicine on the polyploid induction of callus, seedling and bourgeoning seeds of Stylosanthes guianensis cv. Reyan No.5 have been investigated in detail. The results showed that:①Callus dipping method. The frequency of adventitious buds formed from calli was very high after the calli were treated with low colchicine concentrations for short time, with variation frequency being relative low. The incidence rate of tetraploid was 10.42% with the treatment of colchicine of 0.75% in concentration and for 12 h.②mixing culture method. The variation frequency gradually increased with the enhancement of colchicine concentrations and the extension of the treating time, however, but fell when the concentration reached 0.006%. The incidence rate of tetraploid was 8.54% when the colchicine concentration attained 0.004% and the mixed culture for 10 d.③seedling injection method. The variation frequency of seedings was very high, even up to 96%, but most of them were mosaic and returned to diploid under fast cytology identification. The variation frequency of seeding basically went up with the increase of colchicine concentrations and the lengthening of the treating time, but fell with the long-term treatment of colchicine at high concentration, paralleled with high mortality of seedlings. The incidence rate of tetraploid was 10% when the colchicine concentration was 0.20% and the injecting time was 48 h, and this was the best result.④Bourgeoning seeds dipping method. The variation frequency was very high, even up to 100%, but the incidence rate of tetraploid was not high, and the incidence rate of tetraploid with this method combined with rooting culture was 6.67%. The incidence rate of tetraploid was 8.47% after the swollen hypocotyls were dissected to in vitro culture.The rapid polidy-level identification has been established for the plants showed morphologic and Stomatal variation: the material are collected from test-tube plantlets at 9:00 am or potted plants at 10:00am, treated with 0.002mol/L 8-hydroxyquinoline at 4℃for 2h, cleaned with distilled water, fixed with Carnoy's fixative at 4℃for 7h, cleaned with distilled water, then softened with 1mol/L HC1 for 4 min, cleaned with distilled water, dyed with carbolfuchsin for 10 min, and pressured onto glass slides for observation. The cells which have uniform size and obviously much bigger than the known diploid nucleus are identified as polyploid nucleus.The plants identified as polyploid plants were further used for chromosome counting and the plants in which the chromosome numbers of somatic cells were 40 (2n=4x=40) can be confirmed as tetraploid plants.Comparison of the tetradploid plants induced through colchicine treatment with the normal diploid plants showed that the tetraplont plants have giant organs, such as stoma, leaf, stem, flower, microspore and seed significantly bigger than those of the diploid plants. Both the density of stoma and the activity of microspore in the tetraploid plants are significantly smaller than those of the diploid plants. The crude protein content of dry matter of the tetradploid plants is significantly higher than that of the diploid plant ,the crude fat content being significantly lower than that of the diploid plants and the crude fiber content significantly higher than that of the diploid plants. |
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