Micropropagation Characteristics Of Cymbidium With Different Ways Of Life And Molecular Mechanism Of Light-induced Shoot Regeneration In Vitro

Cymbidium orchids have important ornamental,economical,and cultural values.According to their ways of life,they are divided into three major types:terrestrial Cymbidium,epiphytic Cymbidium and saprophytic Cymbidium.In this study,micropropagation characteristics of Cymbidium with different ways of life were studied from four aspects,including the induction,proliferation,differentiation of intermediate propagule and seedling strengthening.Based on this,molecular mechanism of light-induced shoot regeneration and the differences between terrestrial and epiphytic Cymbidium were also studied.These studies provide a reference for the establishment of efficient micropropagation technological system and further study on molecular mechanism of micropropagation characteristics in Cymbidium.The main results are as follows:（1）There were obviously different in micropropagation characteristics of Cymbidium with different ways of life.Seeds of terrestial Cymbidium including C.sinense were germinated slowly to form rhizomes,while those of epiphytic Cymbidium such as C.erythrostylum germinated quickly to form protocorms.Seeds of C.macrorhizon,a saprophytic Cymbidium,germinated slowly to form protocorms.After cultivation on MS medium without exogenous phytohormones in darkness for 40 days,the proliferation coefficient of intermediate propagule of C.sinense‘Qi Jian Bai Mo’,C.ensifolium‘Xiao Tao Hong’,C.aloifolium as well as C.‘Guo Feng’was 1.84,2.42,3.74 and 2.01,respectively.When cultured on MS medium without exogenous phytohormones under light,the rhizomes of terreatrial Cymbidium such as C.sinense‘Qi Jian Bai Mo’did not differentiate,while the protocorms of epiphytic Cymbidium such as C.erythrostylum and the rhizomes of C.‘Guo Feng’quickly differentiated to form a complete plantlet.（2）When cultured on MS medium under dark conditions,no addition or addition of IAA and 6-BA exogenous hormones separately or simultaneously,the intermediate propagule of C.‘Guo Feng’and.C.erythrostylum weie not differentiated.The shoots regenerated from the rhizomes of C.‘Guo Feng’stopped to develop when cultured in darkness.Light quality had a significant effect on shoot regeneration of C.‘Guo Feng’,when cultured under white light,blue light and red light,the shoot differentiation rates were99.33%,95.83%and 35.83%,respectively.According to the morphological changes,the process of shoot regeneration of C.‘Guo Feng’was divided into three stages,which were de-etiolation stage（0-5 d）,bud differentiation stage（5-10 d）,and bud growth stage（10-40d）.（3）The average contents of auxin and cytokinin during light-induced shoot regeneration of C.‘Guo Feng’rhizome were 29.7 ng.g^-1FW and 13.7 ng.g^-1FW,respectively,which were significantly higher than that cultured in darkness(24.9 ng.g^-1FW and 12.9ng.g^-1FW,respectively).Meanwhile,the expression levels of CRY3,IPT3,CRR2,YUC8,ARF11,IAA3,AUX1 and PIN3 genes were significantly up-regulated,on the controry that of CKX9 and GH3.6 genes were significantly down-regulated.（4）The endogenous auxin content in rhizomes of C.sinense‘Qi Jian Bai Mo’cultured on medium without exogenous phytohormones under light was significantly lower than that of C.‘Guo Feng’,while there was no significant difference in cytokinin content between them.Compared with C.‘Guo Feng’,the expression levels of CRY1α,CRY3,COP1,HY5,IPT3,AHK4,ARR5 and ARF11 genes was significantly up-regulated,on the contrary,the expression levels of LOG8,YUC8,IAA3 and PIN3 genes was significantly down-regulated in the rhizomes of C.sinense‘Qi Jian Bai Mo’.（5）The bud differentiation rate of rhizomes in C.sinense‘Qi Jian Bai Mo’was154.7%when cultured on MS medium with 0.01 mg·L^-1 IAA and 3.0 mg·L^-1 6-BA under light,while that of the rhizomes cultured on MS medium without IAA and 6-BA was 0.0%.The contents of endogenous auxin and cytokinin in the rhizomes of C.sinense‘Qi Jian Bai Mo’were significantly increased when cultured on medium supplemented with exogenous phytohormones,and the expression levels of LOG8,CKX9,AHK4,YUC8,IAA3,ARF11,PIN1C,PIN3 and PINlike were significantly increased,however the expression levels of CRY3,COP1,IPT3 and GH3.6 were significantly reduced.Through the above research,micropropagation characteristics of Cymbidium with different ways of life were clarified.The changes of endogenous phytohormones and gene expression during light-induced shoot regeneration of Cymbidium were demonstrated,and the reasons underlying the differences in shoot regeneration ability in vitro between terrestrial and epiphytic Cymbidium was found.The results have important reference value for further study on molecular mechanism of cell totipotency and improving the efficiency of industrial seedling production in Cymbidium.