Study On Tissue Culture And Karyotype Of Several Orchid Filial Generation In Cymbidium

The research was based on Cymbidium hybridum imported from Korea by Taishan Academy of Forestry in Taian and Cymbidium orchid varieties cultivated in Taishan Yi Xiang garden.The study mainly included the following three aspects. The first was to research the non-symbiotic germination on crossing embryos (immature seeds). From the fruits of different developmental stages, the basic medium germination aspects, the system of asymbiotic propagation was established. The second was to research tissue culture technique of the PLBs germinated from hybrid embryos. From the basic medium, a large number of elements, sucrose concentration, additives and other aspects, the optimum medium was selected for the propagation of the offspring PLBs to provide a systematic and rational frame of reference for research and production practice. The third was to identify the filial generations early and clear the genetic background, including the ploidy,chromosome morphology, karyotype parameters, to provide reference and basis for breeding of hybrid offspring when cross breeding.The results are indicated as follows:1. Hybrid embryos between Cymbidium hybridum and Cymbidium as the explants are first cultured to select non-symbiotic germination optimum harvest time and the optimal germination basic medium. Embryo age of 150 days or so is the best harvest time, and the germination rate was the highest. The optimal germination medium was B11 + sucrose 20 g / L + agar 8.0 g / L + AC3.0g / L.2. Hybrid PLBs between Cymbidium hybridum and Cymbidium as the explants for subculture , the best medium was selected for PLBs proliferation and differentiation. Different combinations had different propagation multiple in different medium, however, the optimal subculture basic medium were KC + banana 150g / L. Exogenous hormones on the proliferation and differentiation are promoted, in which NAA as the auxin class to the best cytokinin to 6-BA is the best. NAA concentration of 0.2mg / L was the best to the hybrid PLBs proliferation. 6-BA and NAA concentration ratio was 4 to 5:1, which was most profitable to differentiate; GA1.0mg / L on the subculture was also promoted. In summary, the best subculture medium was KC + NAA0.2mg / L +6- BA0.8mg / L + GA1.0mg / L + banana 150g / L + sucrose 20 g / L + agar 8.0 g / L.3. In the hybrid PLBs root culture, macroelement and sucrose concentration on rooting is extremely significant. The optimal concentration of mineral salts was 1/4MS.The rooting rate was up to100% after 60 days, while average number of root up to 2.4, the average root length of up to 1.79cm. Root growth status was the best when the sucrose concentration was 30 g / L, average number of root up to 3.43, the average root length up to 1.34 cm.. Therefore, the hybrid PLBs rooting medium was 1/4MS + NAA0.2mg/L +sucrose30 g / L + banana 150g / L+ sucrose 20 g / L + agar 8.0 g / L.4. Hybrid plantlets were to quit dealing with the roots when transplanted to sphagnum substrate , which survival rate was 81% after 100 days and the average height was 11.89cm. From this we can deduce that root pruning treatment can stimulate the formation of new roots and improve survival rate of seedlings. Seedling vigor was significantly brought the matter to be dealt with than without.5. Through karyotype analysis of Cymbidium hybridum male parent and filial generations, early identification of hybrids was made. The experiment results showed that Cym. Longibracteatum and Cym. KLR565 are tetraploid. They are respectively crossed with C.Longibracteatum and C. faberi ,and somatic chromosome number of their filial generation are 2n = 3x = 60.Therefore, filial generations can be identified as true hybrids. Somatic chromosome number of C. Summer Queen and C. Saint Raphin are2n = 2x = 40, diploid. They are crossed with C.Longibracteatum and their filial generations had common with their parents in chromosome morphology. Their relative chromosome length, average arm ratio, asymmetry index and other indicators tend to the intermediate value of parents, which can identify them to be true.6. Filial generations practical and theoretical karyotype had differences, which showed that artificial hybridization broke the formation of long-term evolution system suitable for parental chromosomes. All parents and filial generations chromosome structure were mainly composed with metacentric and sub-metacentric chromosome. The ratio of the longest and shortest chromosomes was between 2.01 to 4.2.Chromosomes percentage of arm ratio >2:1 was not more than 35. By the karyotypic classification Stebbins (1971), all parents and filial generations are 2B except C. Summer Queen which was 2C. Thus, the number of chromosomes of Cymbidium was relatively conservative, the variation was small and the karyotype symmetry was also high.