| The diploid species Chrysanthemum nankingense with many excellent genes is one of the wild Chrysanthemum species, native to China. According to the difference in ploidy level, it was hard to obtain hybrids in the crosses between Ch. nankingense and cultived chrysanthemum (mainly in hexaploid and aneuploid). The induction polyploidy could overcome the distant hybridization barrier between species. The study used nodal segments of Ch. nankingense as explants for establishing sterility system. The tetraploid of Ch. nankingense was induced by the colchicine treatment of nodal segments. Ploidy level was determined by an analysis of stomata size, flow cytometry and chromosome counting. The morphological characteristics and the responses of the tetraploid to the imposition of heat, cold, drought and salinity tolerance were compared with those of the original diploid. In addition, artificial hybridizations between the diploid and tetraploid of Ch. nankingense ((?)) and cultivated chrysanthemum (Ch. morifolium'Zhongshanzixing') ((?)), and the genetic performance of their F1 hybrids were studied. The detailed results were listed as follows:(1) The best medium for lateral bud sprouting was MS+BA 2.0 mg·L-1+ NAA 0.2 mg·L-1. Nodal segments treated in 500 mg·L-1 colchicine solution for 48h was suitable for induction tetraploids with the highest induction rate 15.38%. Tetrploid plants identified by stomata size were verified by flow cytometry and chromosome counting. The accuracy for identification of tetraploid plants by stomata was 90.69%. So it was considered as a credible index for determineing tetraploid plants of Ch. nankingense in the early. Compared to the diploid plant, tetraploid plants got shorter, their stems got thicker, leaves were larger and thicker, flowers and pollen grains were larger.(2) In response to heat stress, the diploid LT50 was 1.18℃greater than that of the tetraploid, while in response to cold stress, the tetraploid LT50 was 2.32℃less than that of the diploid. Semi-lethal temperatures suggest that cold tolerance is improved by polyploidization, but the heat tolerance is reduced. Under drought and salt stress, the leaves of the diploid suffered more damage than those of the tetraploid. The activity of peroxidase (POD) and relative water content (RWC) in the tetraploid were higher than those in the diploid, while its malondialdehyde (MDA) content maintained a lower level. The content of chlorophyll (a+b) in the tetraploid was higher, and decrease of its content was postponed in tetraploid compared with the diploid under salt stress. It suggested that polyploidization could alleviate oxidative stress, maintain good water balance and higher chlorophyll (a+b) content, thereby enhanced the drought and salt tolerance in colchicine induced tetraploid.(3) Pollen viability of the diploid and tetraploid were higher and the pollen grains could germinate on the sigma of Ch. morifolium 'Zhongshanzixing' well. But no seed was obtained in the combination between the diploid of Ch. nankingense and Ch. morifolium 'Zhongshanzixing' while a number of interspecific hybrids were obtained by crossing tetraploid of Ch. nankingense with Ch. morifolium 'Zhongshanzixing'.The chromosome number of Ch. morifolium'Zhongshanzixing', tetraploid of Ch. nankingense and their hybrids were 54,36 and 45, respectively. The chromosome commonest configurations at metaphaseⅠper pollen mother cell in Ch. morifolium'Zhongshanzixing', tetraploid of Ch. nankingense and their hybrids were 27Ⅱ,1.70Ⅰ+13.23Ⅱ+0.03Ⅲ+1.90Ⅳand 5.32Ⅰ+13.84Ⅱ+2.79Ⅳ+0.16Ⅵ, mainly with bivalent. Plant height, leaf width, and petiole length of hybrids were significantly positive in mid-parent heterosis of hybrids, while their internode length, leaf index, inflorescence diameter, floral disk diameter, and floret number were significantly negative in mid-parent heterosis. Doubling the chromosome of Ch. nankingense could overcome the distant hybridization barrier with the cultivated chrysanthemum. |
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