| Huaiqing chrysanthemum (Dendranthema morifolium), one of the Four Famous Huai Medicine, they are major in Wen County, Wu Zhi, Bo Ai of Henan province (old Huaiqing government had jurisdiction over these area, so they were named), there are three functions in ornamental, medicinal and tonic value. Chrysanthemum belongs to cross-pollinated plant, while its germplasm is preserved by field planting at present, this preservation method is so single that can not ensure its purity, long period preservation of using field planting preservation had resulted in germplasm mixture and valuable germplasm resources hand been lost seriously. ultra-low temperature preservation technology was developed at the late 20th century, and this technology is the only practical good way of long-term and stability preservation plant germplasm resources. On the one hand, it can save space, avoid germplasm loss because of subculture, pests and diseases, and climatic factors, on the other hand, the needed equipment is simple, and doing is easy. Based on this, we had carried out the technical studies of vitrification cryopreservation chrysanthemums germplasm, and gotten the following results:1. The technology system of tissue culture and rapid propagation for Huaiqing chrysanthemum had been established. There were two ways of propagation Huaiqing chrysanthemum, that is, based-axillary branch and multi-bud type. The best medium of the former was MS +6- BA 0.2 mg·L-1 + NAA 0.2 mg·L-1, and that of the latter was MS +6- BA 1 mg·L-1 + NAA 2 mg·L-1. The best rooting medium was MS + PP333 0.5 mg·L-1.2. The technology system of vitrification cryopreservation Huaiqing chrysanthemum germplasm had been established. It was as belows: Firstly, the Huaiqing chrysanthemum plantlets were cultured in vitro on the rapid propagation medium for 40 d. Secondly, they were cold-hardened for one week at 4℃. Thirdly, stems with one axillary bud (about 0.5 cm) were excised from cold-hardened plantlets and precultured for 3 d on MS medium containing a mixture of Glu 0.24 mol·L-1 + Gly 0.4 mol·L-1 at 4℃. Fourthly, the stems were loaded with 60% PVS2 for 20 min at room temperature. Sixthly, following loading, the stems were dehydrated with modified PVS2-2 (Gly 30% + EG 15% + DMSO 10%) for 60 min at 0℃prior to a plunge into liquid nitrogen directly. Seventhly, after immersed in liquid nitrogen for at least 1 d,the stems were rapidly thawed in a water bath at 40℃for 1～3 min. Subsequently, the stems were washed gradiently with MS medium supplemented with 1.0 mol·L-1, and 1.2 mol·L-1 respectively, each time 10 min, then transferred them to the medium of rapid propagation as described previously. Finally, put the materials in the dark for 15 d in the light, callus began formation, after cultured for 30 d, transferred them to light. Callus began change green when cultured for 7 d under the light, the buds were looked when cutured for 25 d～30 d, and the buds developed normal plants when cutured for 60 d under the light.3. Stability analysis of the regeneration plantlets after vitrification cryopreservation had been done. There were no difference between the plantlets after cryopreservation by vitrification (named as RPC) and the plantlets preserved at room temperature (named as CK) on the average increased value of internodal length, plant height, leaf number, and the contents of soluble sugar, soluble protein and chlorophyll, while relative conductivity and contents of malonaldehyde (MDA) and superoxide anion (O2·-) reduced, the activities of peroxidase (POD) and superoxide dismutase (SOD) increased in the leaves of RPC. Isozymes analysis of SOD and POD showed that there was a high unity of between electrophoresis zymography of RPC and that of CK, but there were difference in the width and light degree of some bands. So the established cryopreservation vitrification technology system could not only maintained morphological, some physiological stability and Isozymes, but also enhance the capacity of anti-membrane lipid peroxidation of Huaiqing chrysanthemum. |
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