Studies On Polyploid Induction Of Cassava In Vitro

The cassava is a very important food and energy plants in Euphorbiaceae family, and the number of chromosome is2n=36. Generally polyploidy have enhanced resistance, enlarged plant organs and other characteristics, so researching cassava polyploid breeding has great significance for improving the yield of cassava and innovating cassava germplasm resources. However polyploid breeding of cassava is still in the initial stage. Studying cassava polyploid induction with the material of SC8and ARG7has not been reported. In this paper, colchicine immersion and mixed culture methods were used to treat stem with buds, cotyledon segments and multiple shoot clumps of cassava SC8and ARG7for polyploid inducing in tissue culture conditions. A variety of identification methods were used to identify the mutants. The main results were as following:(1) Regeneration system was established from the study of in vitro culture of the five species. The two high effective regeneration systems were obtained. The result showed that somatic embryos induced rate of SC6is highest among5recipient materials. The somatic embryos induced rate of all tested cultivars achieved more than90%. Adding AgNO3to the regeneration medium significantly reduced callus formation and improved the development of shoots. AgNO3at5mg/L has been considered the most suitable concentration to be used in this study. In addition, it was found that enlargement culturing stem with buds for3-5d was conducive to the proliferation of multiple shoots through the study of the proliferation of multiple shoot clumps. This method was an innovation of this experimental. The best medium for clump shoot regeneration was MS+1.0mg/L6-BA+0.1mg/LNAA+0.1mg/L GA3.(2) The stem with buds, cotyledon segments and clustered buds were treated with colchicines by soaking, or adding into the medium to induce polypoids. The results showed that different species were in different level of sensitivity to colchicine, the optimal treatment (the concentration of colchicine and the processing time) was different relatively. For SC8cassava, using colchicines on stem with buds with soaking method could obtain better mutagenic effects, and the percentage of induced variation was31.3%; For ARG7cassava, using colchicines on multiple shoot clumps with soaking method could obtain better mutagenic effects, and the percentage of induced variation was36.7%. It was found that enlargement culturing stem with buds was more conducive to improving polyploidy induction rate.(3) Ploidy tests of induced plants in the initial stage can be carried out through observing appearance of plants. Compared diploid cassava with tetraploid cassava, the induced tetraploid plants showed that the leave became shorter, thicker and the leave index became smaller. At the same time the mutate plantlets were deformed and color of the leaves became deeper. The internode lengths were shorter than the average of normal cassava. Those characters of rose could be taken as the advance index in testing tetraploid cassava. By comparing the polyploidy plants with normal diploid ones in cytology, the guard cells and stomas showed bigger with lower density, the number of chlorophyll showed more than diploid plants. As well as through the chlorophyll content and the leaf water content's determination, polyploidy compared with diploid had obvious difference.(4)Established and optimized the optimization system of chromosome sectioning of cassava in vitro. Taking stem tips of cassava in vitro as materials, microscopic examination technology of cassava chromosome was studied. The results showed that the optimal sampling time of stem tips was about8:30～10:00A.m. Pretreating stem tips with mixturing solution of0.1%colchicine solution and8-hydroxyquinoline at4℃for3hours got the best result. Then stem tips were fixed with the fixative solution (alcohol:chloroform:glacial acetic acid=6:3:1) at4℃, hydrolysed with1mol/L HC1for10min at60℃and stained with modified Carbol fuchsin solution. With this method a good effect of chromosome squashing had be obtained.(5) Through the chromosome counting method, morphological variation of the identification of polyploidy plants, the results showed:SC8(2n=4x=72), ARG7(2n=4x=72), whether to be tetraploid need further evidences.