| Cymbidium sinense is an important ornamental flower originating in Guangdong province with numerous cultivars,long plantation history and extensive market prospect.However,traditional cultivars are difficult to propagate via tissue culture,and could not achieve the need of orchid industry development.Thus,it has extremely important realistic meaning to create Cymbidium sinense resources and new cultivars easy to propagate,and study the molecular mechanism of the difficulties of seedling production in C.sinense in vitro for promoting the development of seedling industry of C.sinense.In this thesis,we studied the methods to create C.sinense resources easy to micropropagation by sexual hybridization,and excavated the candidate genes related to shoot regeneration capacity by the use of the comparison analysis of endogenous phytohormone dynamic changes and RNA-seq during proliferation and regeneration procession of rhizomes of C.sinense ‘Qijianbaimo',a traditional cultivar difficult to micropropagation and C.‘Xiaofeng',a new cultivar easy to micropropagation.The main results include the following aspects:1.Crossability of C.sinense was closely related to the parents' genetic relationship.Crossability of inter-cultivars was the highest with 70.69% capsule harvested rate,followed by inter-species with 30.99% capsule harvested rate,crossability of inter-generic was the lowest with 4.62% capsule harvested rate.Among the same type of crossing,crossability depended on the parents,for example,among inter-species crosses,the capsule harvested rate of combination between C.sinense and terrestrial species or C.sinense and hybrid Cymbidium was higher(61.25% and 48.89%),while the capsule harvested rate of combination between C.sinense and Cymbidium hybridum was low(4.62%),among 40 pairs of inter-generic combinations,the capsule harvested rates of combinations between C.sinense ‘Xiaoxiang' and Epilaeliocattleya Butterfly Kisses ‘Mendenhall',C.sinense ‘Qijianbaimo' and Grammangis ellisii were 50% and 83%,respectively.The major performance of incrossability of C.sinense was column incompatibility.Percentage of seed with embryo were related to the genetic relationship of parents.Combinations of inter-cultivars and inter-species had the high percentage of seed with embryo(87.57% and 89.61%)and combinations of inter-generic had the low percentage of seed with embryo(38.41%).The initiate germination time of hybrid seeds significantly correlated with the developmental stage of fruits.The initiate germination time of hybrid seeds collected 170-190 days after pollination was short,whereas that of hybrid seeds collected over 220 days after pollination was long.Relative seed germination quantity was related to the genetic relationship of parents.Relative seed germination quantity of inter-cultivar combination was the highest,followed by inter-species combination,relative seed germination quantity of intergeneric combination was the lowest.2.Investigations were carried out on micropropagation characteristics of 79 hybrids from the combination of C.Frigdaas Green King gx.SCAU Herald Spring Gp.‘Yunv' × C.sinense ‘Huangyehongmo'.It was found that their proliferation capacity were divided into very strong(28%),strong(25%),medium(25%),weak(19%)and very weak(3%),their shoot regeneration capacity were segmented into very strong(11%),strong(20%),medium(38%),weak(20%)and very weak(9%),and their rooting and seedling strengthening capacity presented as very strong(3%),strong(14%),medium(32%),weak(30%)and very weak(21%).The intermediate propagule included rhizome(47%),rhizome-like(20%),medium type(28%)and PLB-like(5%),and morphology of their seedling had Cymbidium hybridum type(33%),Cymbidium hybridum like type(24%),Chinese Cymbidium like type(31%)and Chinese Cymbidium type(12%).Of the 79 hybrids,the morphology of d26 and d521 was Chinese Cymbidium type,their intermediate propagules were rhizome-type with 155.9% and 126.4% of proliferation rate,618.6% and 430.1% of shoot regeneration rate and 83.7% and 82.0% of seedling production rate,respectively.Their efficiencies of micropropagation were significant higher than that of C.sinense ‘Qijibanbaimo'.3.Rhizome of C.‘Xiaofeng' was similar to that of C.sinense ‘Qijianbaimo' in structure,but the growth rate was faster than that of C.sinense ‘Qijianbaimo'.During proliferation culture,the apical meristem continuously grew to form new nodes,therefore,the rhizomes elongated without new branches,endogenous auxin content in the rhizomes of C.‘Xiaofeng' continually increased,on the contrary,it continually decreased in the rhizomes of C.sinense ‘Qijianbaimo'.There were also different in the contents of cytokinins and methyl-jasmonate between two cultivars at different stages of proliferation.Results of RNA-seq analysis showed that there were 9392 differentially expressed genes(DEGs)between C.‘Xiaofeng' and C.sinense ‘Qijianbaimo',which presented different expression pattern in the process of proliferation.These DEGs were mainly down-regulated in C.sinense ‘Qijianbaimo',and were significantly enriched in the pathways related to cell wall formation,response to endogenous stimulate and carbohydrate metabolism,however,they were mainly up-regulated in C.‘Xiaofeng' and significantly enriched in the pathways related to hormone biosynthesis and photosynthesis.4.On the same culture medium and conditions,the process of shoot regeneration was faster,the mean number of shoot regenerated was more and the shoot sizes was bigger in C.‘Xiaofeng' than that in C.sinense ‘Qijianbaimo'.During shoot regeneration process,endogenous auxin content in rhizomes of C.‘Xiaofeng' rapidly increased at the first stage,then slowly increased at the second stage,in contrast,it slightly decreased at the first stage,then rapidly increased at the second stage in rhizomes of C.sinense ‘Qijianbaimo'.As a result,the ratio of cytokinin-toauxin maintained stability in rhizomes of C.‘Xiaofeng',and it increased first,then decreased in C.sinense ‘Qijianbaimo'.Results of RNA-seq analysis indicated that there were 8625 DEGs between C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' during the process of shoot regeneration,which presented different expression pattern during shoot regeneration.The DEGs with upregulated profile in both cultivars were significantly enriched in the pathways related to meristem formation for C.sinense ‘Qijianbaimo',and enriched in the pathways related to leaf formation and auxin metabolism in C.‘Xiaofeng'.5.Through quantitative real-time PCR(qRT-PCR)analysis,sequence analysis and function analysis,8 candidate genes related to shoot regeneration were screened out.Among them,there were 3 genes from YUCCA(YUC)family,2 genes from GRETCHEN HAGEN 3(GH3)family,1 gene from CYTOKININ OXIDASE/DEHYDROGENASE(CKX)family,1 ARABIDOPSIS HISTIDINE KINASE4(AHK4)-like gene and 1 gene from CYTOCHROMES P450(CYP450)family.During shoot regeneration process,YUC-like candidate genes were up-regulated in C.‘Xiaofeng' and GH3-like candidate genes,encoded proteins both had IAA amido synthetase activity,were up-regulated in C.sinense ‘Qijianbaimo',and supplement of yucasin,an inhibitor of YUC enzyme,obviously inhibited shoot regeneration of C.‘Xiaofeng',but had little influence on shoot regeneration of C.sinense ‘Qijianbaimo',suggesting that these genes might be together affect auxin content at the first stage of shoot regeneration.CsAHK4 had an extra nucleotide sequence compared to CxAHK4,and CxCKX9 had two extra nucleotide sequences compared to CsCKX9,indicating that alternative splicing might existed.CsAHK4 encoded protein had complete AHK4 sensor domain,which could receive cytokinin signal.Both CsAHK4 and CsCKX9 were up-regulated in C.sinense ‘Qijianbaimo' during shoot regeneration.Protein sequences identity between CsCYP450 and CxCYP450 were only 87.30%.Phylogenetic analysis indicated that CxCYP450 had more closely relationship with PeCYP450 from Phalaenopsis equestris compared to CsCYP450.CsCYP450(CxCYP450)contained a provisional fatty acid omega-hydroxylase domain,demonstrating that it might participate in biosynthesis of some kind of important lipid compounds.CsCYP450(CxCYP450)expressed lower in young rhizome and test-tube seedling of C.sinense ‘Qijianbaimo' than that of C.‘Xiaofeng'.Through the above study,the technical system to develop C.sinense resource easy to propagate in vitro was established,and 2 resources of C.sinense easy to micropropagation were obtained;The difference in micropropagation characteristics between C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' and its physiological mechanism were defined,and 8 candidate genes related to shoot regeneration capacity were screened out.These established a solid foundation for further understanding molecular mechanism of rhizome differentiation and developing new cultivars easy to micropropagation in C.sinense. |
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