Physiological And Molecular Basis Of Seed Germination In Calanthe Tsoongiana

Calanthe tsoongiana T.Tang et F.T.Wang,a rare and endangered orchid species in china,is a kind of perennial herb of Orchidaceae Calanthe.A single capsule derived from pollination contains numerous tiny seeds,A plenty of uniform seedlings can be obtained through collecting unripened capsules for aseptic germination,we devote our efforts to utilize seeds to propagate for the purpose of protecting rare orchid species.In addition,seeds of terrestrial orchids with special structure characteristic are difficult to germinate in vitro and its germination process is distinct from general plants.Protocorm is an important go-between structure during seed germination.In order to unravel the essential physiological and molecular mechanisms underlying seed germination and seedling establishment,morphological observation,paraffin section,endogenous hormone detection and RNA-Seq was applied to investigate the developmental process from seed to seedling in C.tsoongiana.(1)According to morphological observation and paraffion section,we have found that larger cells and nucleus existed in embryo at the time of seeds absorb water resulting in swelled embryo which had no differentiated epidermis tissue.The white embroy turned into green protocorm gradually after embryo breaked through seed coat,one end of protocorm with shoot apical meristem had strong cell division and similar morphological characteristics.With the cells divided constantly,leaf primordium formed at one end of protocorm accompanied with digitation.In the meantime,transparent ciliums apperaed in another end of protocorm which functioned as imbibing water and nutrient.Root primordium formed in basal part of protocorm and ultimately developed into young root during the growth and differentiation of shoot apcial.The seedling was established as soon as the the leaf primordium and root primordium differentiated into leaf and root,respectively.On the basis of morphological observation and paraffion section,developmental process from seed to seedling was divided into six stages:(S0)unripened seed stage;(SA)swelled and activated seed stage;(SB)globular protocorm stage;(SC)finger protocorm stage;(SD)the differentiation of root and leaf stage;(SE)seedling stage.(3)The experimental materials at six stages divided as above was used to detect seven endogenous hormone content including ABA,IAA,GA3,BR,JA-ME,IPA,ZR.Among them,the conenet of ABA,IAA,JA-ME and IPA fluctuated greatly during this whole process,ABA content declined sharply during seeds absorb water and then kept a lower level.While IAA content displayed an significant up-and-down trend in the whole developmental process.Additionally,JA-ME content increased drastically in globular protocorm stage and continues at this high content in the following stages.IPA content maintained a slight growth and reached the peak at finger protocorm stage.The content of another two hormones,BR and ZR,fluctuated slightly during the differentiation of protocorm.Therefore,ABA,IAA,JA-ME and IPA may act on seed germination,while IAA,IPA,ZR and BR had a relation to the differentiation process from protocorm to seedling.(4)Samples of four stages from swelled embryo stage to leaf and root differentiation stage were used for RNA-Seq.73 528 Unigenes were obtained after carrying on processing to raw reads.When comparing against to public databases,48.10%,34.17%,13.53% and 31.43% of Unigenes were annotated to NR,Swissprot,KEGG and GO database,respectively.There were 6 comparison groups(PB VS SA,PC VS SA,PD VS SA,PD VS PB,PD VS PC,PC VS PB),and the number of DEGs(differentially expressed genes)were 10 029,9 277,8 056,6 784,3 560 and 1 038,respectively.GO enrichment analysis has showed that DEGs mainly involved in cell process and metabolic process,while KEGG pathway enrichement analysis indicated a number of DEGs participated in photosynthesis,phenylpropanoid biosynthesis,starch and sucrose metabolism,etc.(5)DEGs were further analyzed and genes related to endogenous hormones and organ morphogenesis were screened in C.tsoongiana.GA synthetic genes KAO2 and CPS1 and metabolic gene GA2ox1 were significant differentially expressed between PB and SA stages.However,genes related ABA have complex molecular regulatory mechanisms,NCED1 and ZEP were key genes determining ABA biosynthesis,while CYP707A4 controls ABA metabolism.Auxin synthetic and metabolic gene,TSA1 and DAO,showed opposite expression trend and both presented a high expression level.AUX1 was the staple gene responsible for auxin transport in meristem of protocorm.Genes belong to ARR-A and ARR-B transcription factor family playing a crucial role in cytokinin signal transduction.What’s more,CUC2,AS1 and STM interacted with each other to regulate the formation of meristem and leaf.