| Melastoma sanguineum belongs to Melastoma, Melastomataceae. It has not been developed, which with short flowering and poor adaptability even though it has high values in ornament and the potential of beautification of urban environment. Polyploid breeding is an important approach to improve ornamental quality and resistance for M. sanguineum. M. sanguineum was used as research materials in this paper, conducting the following researchs: on discovering the seed germination, the induceing polyploid for germinative seeds by colchicines was investigated, as well as its growing point. Establishment of regeneration system gave the platform to carry out the research of induceing polyploid for stems and callus treated by colchicines. Polyploids were identified by morphology, stomatal character, chromosome counting and flow cytometry. This study aimed to explore a rapid and efficient method to induce polyploid, breed new M. sanguineum cultivates with strange leaf, bigger flower and higher resistant character. The main results showed as follows:(1) The study of regeneration systemThe optimal medium of callus was MS+0.5 mg·L-1 6-BA+2.0 mg·L-1 2,4-D+0.1 mg·L-1 NAA, in which the induction rate was 96%, under 12 h/d light conditions. The proliferation rate increased with concentration of NAA. The best mutipulication medium was MS+0.5 mg·L-1 NAA+0.5 mg·L-1 6-BA, mutipulication rate was 1.9. The differentiation rate of callus was low, which induced by leaf. It increased with concentration of 6-BA. The best differentiation medium was MS+0.1 mg·L-1 NAA+2.0 mg·L-1 6-BA, whose differentiation rate was 10%. The best rooting medium was 1/2 MS+0.1 mg·L-1 NAA, whose rooting rate was 88.89%.(2) The study of polyploidy inducementIn experiment on seed germination, the germination rate of the control was 31%, while the highest germination rate for treatments was 58%. Low level of 6-BA in combination with GA3 may promote seed germination. The best treatment combination for seeds germination was obtained according to range analysis, which was to soak the seeds for 24 hours in GA3 50 mg·L-1, IAA 100 mg·L-1,6-BA 20 mg·L-1, respectively, under 23℃.The study of field induction showed that variant plants obtained easily with the method of growing point treatment, the highest mutagenic rate was 45% with 0.2% colchicines for 4 days, compared to the treatment of germinative seeds whose mutagenic rate just 6.67%, obtaining polyploid 11 and 5, respectively, by the identification of morphology, stomata characteristic and flow cytometry. As induced materials, stems were better than callus in the treatment under aseptic condition. The mutagenic rate of stems was 11.67% soaked for 24 hours by 0.1% colchicines, while 26.67% with the treatment that medium added by 0.1% colchicines for 10 days, obtaining polyploidy 10 and 6 by flow cytometry. The optimum compage of concentration and time of colchicines for callus was medium added by 0.1% colchicines for 10-20 days, whose doubling effect was 6.67% with 3 polyploidy by identification of stomata characteristic, chromosome counting and flow cytometry.The ployploid had thick leaves, widening stems in morphological shape. The stomatal density was shorter than diploidy, the size of stomatal guard cells was larger, while the chloroplast number was increased. The chromosome number of polyploidys was 40, that of diplois was 24. Flow cytometry is a fast and accurate method to identify the ploidy, which showed that the DNA content in tetraploid was two times that of the diploid with chimerals. |
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