Studies On Rapid Propagation And Morphogenesis In Tissue Culture About Curculigo Orchiodes Gaertn

As a traditional Chinese herb, Curculigo orchiodes Gaertn. has important medical value, and the need for it is growing day by day. But the nature resources are decreasing steadily for over exploitation and its slow growth and reproduction rate. In this article,rapid propagation of C. orchiodes in tissue culture was reported for the first time; the processes of callus induction and morphogenesis have been traced with cytohistological methods; and the genetic stability of regenerate individuals was studied through the comparison of karyotypes and curculigoside content between the wild and the cultured. The optimal culture conditions were established by single factor tests. The leaf blade segments were cultured as explants on MS medium supplemented with different phytohormones. The results showed that the calli were able to induced on the medium supplemented with low level hormones; the higher level of 6-BA or Kin, the more severity of browning; the ratio of embryogenic calli were higher in dark condition than in light condition; the leaf blade was more fit for inducing embryogenic callus than the leaf sheath; Exogenous auxins,NAA or 2, 4-D, inhibited either formation or development of buds. Cultured on medium â… (MS+NAA 0.1+6-BA 1.0) or on medium â…¡(MS+2,4-D 0.05 + Kin 1.5 ) in dark condition, the calli could reach an induction rate of 100%. When the calli were transferred onto hormone-free medium and cultured in light condition for several days, buds were emerged with roots, and plantlets were formed. Respectively, the breeding coefficients were 11 and 9, and the surviving rates were 35% and 78% one month after transplant. Cytohistological observations were carried out on calli and buds formation. The studies revealed that regenerate individuals had been obtained through different ways: the organogenesis type on medium â… and the embryoid type on medium â…¡. There were significant differences between these two ways. The structures such as dividing centers, trachea cells and vascular nodules were observed and other two evident characters, the bud primordium and vascular system, were formed in the former. Organogenic nodules were the basic regenerative structures producing large amounts of adventitious shoots. In the latter, similar to the embryogenesis of monocotyledon, different stages of embryogenic cells, from single cell, two-four-to-multiple-cells,then spherality-to-shield-embryoids, and then the mature embryoid consisted of scutellum,coleoptile, and coleorhiza, could be observed. Besides, we observed some abnormalities in torpedo embryoids, which were an embryo development stage in dicotyledon and not in monocotyledon, derived from tissue culture. Polarity was established in the embryoids, and distinct borderline appeared around the embryoid; suspensor disappeared early during the embryoid formation. The organogenetic plantlets usually connected together by callus. This went against to rooting and separating, and that would bring on damage to the plantlets easily when being severed from each other and would results in low surviving rate after transplant. Reversely, the somatic embryogenic plantlets usually kept independent to each other which would help to increase surviving rate after transplant. The facts that different morphogenesis ways the plantlets have taken on medium with different hormones may have resulted in the different surviving rates after transplant. And different auxins and cytokinits in different strengthes was the key factor which resulted in different morphogenesis ways. The karyotypes of C. orchiodes were studied for the first time. The results showed that the number of the somatic chromosomes of C. orchiodes was 2n=4x=36, belonging to tetraplont, and the karyotype formula was 2n=4x=36= 12st+20sm+4m. The karyotype belonged to "3C", according to Stebbins (1971) classification of karyotypic asymmetry. The coefficient of karyotypic asymmetry was 73.80, according to Arano(1963). In order to investigate the genetic stability of regenerated plants in tissue culture, comparison of karyotypes had been made between the wild and the cultured C. orchiodes. The results showed that the karyotypes of cultured plants were normal and the variation of chromosomes hadn't been founded. Measured by ultraviolet (UV) spectrophotometer, It was founded that curculigoside contents were very close between the wild and the cultured (including plants from medium â… and â…¡), which were 1.67%,1.54% and 1.51% of dry rhizome weight respectively. These illuminated that the plants reproduced by tissue culture were able to keep genetic stability and the rapid propagation method of C. orchiodes were credible and feasible. The curculigoside synthesis was observed in callus. Dark condition was favorable to the accumulation of secondary metabolism. The total amount of curculigoside was 1.21% of the cell dry weight. So the calli cultured in dark condition also have a high applicable value.