In Vitro Culture And Polyploid Induction Of Lychnis Senno

Lychnis senno Siebold et Zucc, belonging to Caryophyllaceae, is native to the Tianmu Mountain area, Lin'an County, Zhejiang province, China. Specific habitat environment is required for the survival and growth of this species. Thus, it is rare and threatened. The purpose of this study was to improve the germination rates of Lychnis senno seeds and develop a protocol for propagation in vitro and utilization for conservation and sustainable utilization of this species, to improve the ornamental qualities of L. senno by means of in vitro polyploidization. The cytological and morphological characteristics of polyploidy were further studied compared with the diploids. The main results are as follows.(1) Because L. senno seed germination took 5⁶ months and was sporadic ex vitro or in vitro culture, various germination rates were obtained by treating seeds with 250 mg/1 GA3 during 1-6 month's period of storage. The germination rate reached the highest of 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4℃.(2) The segment with axillary bud as explants in vitro culture, the proper medium of multiplication was MS basal medium supplemented with 1 mg/1 BA and 1 mg/1 NAA and the multiplication coefficient was 2.8. Rooting rate reached 91.7% on a half-strength MS medium supplemented with 0.5 mg/l NAA. What's more, callus was induced from leaves of L. senno. As a result, the better medium for callus induction was MS medium supplemented with 0.5 mg/l BA, 1.0mg/l NAA and 1.0 mg/1 2,4-D. The differentiation rate of callus was 4.2% on MS basal medium supplemented with 2.0 mg/1 BA and 1.0 mg/1 2,4-D.(3) The chromosome analysis of regenerants showed that L. senno is diploid contained 24 chromosomes. In order to improve its ornamental value, in vitro induction of tetraploid L. senno was conducted via various concentrations of cholchicine treatment in two ways. The induction rate of tetraploid was better by treatment of liquid than that of solid. Survival rate and induction rate were proper in 400 mg/L cholchicine than others, 58.3% and 16.68% respectively.(4) Plant polyploidy was first analyzed by Flow cytometry and then chromosome numbers of the root tips were observed under a microscope to confirm the FCM results. Itshowed that the tetraploid contained 48 chromosomes, twice the normal diploid number of 24.(5) Meanwhile, their cytological and morphological characterizations were also conducted. The results showed that tetraploids were shorter than diploids. The leaves of tetraploids were shorter and wider than those of diploids. Furthermore, the flower diameter of tetraploid was 1.0 cm larger than that of diploid with similar flower structure and contorted staminal filaments. Transmission electronic microscope indicated that chloroplast number in foliar cells is separately 7.2 and 13.0, and starch granule size is 2.2 and 1.5, in diploid and tetraploid. Further more, stomatal density and sizes of diploid and tetraploid plants were observed. Diploid plants had more stomata (9.8/view) than polyploids (tetraploid 4.8) . Compared with the diploids, tetraploids had bigger stomatal areas.In conclusion, L. senno can be multiplicated through in vitro culture. 4000-6500 plantlets were obtained from a segment of L. senno in vitro for 6 months. Thus, the cost of each plant is Y0.12 according to our calculation. Consequently the preservation of germplasm resource was accomplished economically during a short time. Moreover, the morphological characterizations of regenerant were improved after transplanting into the field compared with wild plant. In addition, tetraploid was acquired by cholchicine induction and more compact than diploid. Accordingly, its commercial value is under further investigation.