Histological And Transcriptome Analysis Of De Novo Bulblet Regeneration In Lilium Spp.

Lilium spp.is the second most important bulbous flower worldwide.Vegetative propagation using detached scales is major propogation method for lily.De novo bulblet regeneration from detached scales,is the same biological process as de novo shoot organogenesis from detached leaves in model plants.Considering scales as modified leaves,bulblet regeneration has its own features in bulbous plants.Dissection of molecular network underneath de novo shoot organogenesis has been carried out deeply and extensively.Due to lack of genome information and transgenetic obstacles,although being an important process with both academic and practical meaning,there is no systematic study so far on bulblet regeneration process of lily.Cytological basis,phases defination and transcriptional mechanism remain to be elucidated.This study conducted comparisonal analysis bsetween Lilium brownii and its variant,Lilium brownii var.giganteum,together with a commercial variety Lilium 'Sorbonne' as taxa control.These three taxa differ in bulblet regeneration speed,regeneration rate and propagtion coefficient.Key stages and meristematic cell lineage are first characterized by historical and cytological studies.Transcriptome analysis was then carried out by hybrid sequencing of PacBio and Illumina to allow mining of key pathways and involved candidate genes.The regeneration of bulblet relies on dedifferentiation of pre-embryogenic cells,thus parallel study with exogenous cues for dedifferentiation induced by 2,4-Dichlorophenoxyacetic acid was carried out.Main results are as below:1.Defination of regeneration stages and tissue origin of bulblet in response to wounding and exogenous 2,4-DPre-embryogenic cells for direct organogenesis of lily bulblet is identified to be xylem-pole pericycle-like cells,parenchyma cells and epidermal cells to the adaxial side of the vascular bundles in scale,who are the founder cells for regenerated bulblets.Active time-course of these cells is analyzed.Starch granules is potentially providing energy via active division,but not critical cue for dedifferentiation and division.Exogenous 2,4-D application accelerated dedifferentiation and division of pre-embryogenic cells drastically,and induce dedifferentiation and divisions of relative quiesent parenchyma cells to the adaxial interval side of neighbouring vascular bundles.Histological and Transcriptome analysis convergly point out,pre-embryogenic cells are tissue foundationn for de novo bulblet regeneration with wounding signal as the vital cue,while 2,4-D can greatly enhance dedifferentiation and maintenance of stem cells.2.First-time Establishment high-quality full-length transcriptional dataset for lily de novo bulblet regeneration15Gb Pacbio data was obtained for each of three lily taxa described above,with N50 of 4,199,5,422 and 5,199 bp.Annoation of transcriptional factors and against Plant Resistance Gene database,PRGdb showed similary among the three,but also exhibited specie-specific annotations.10Gb Illumina data was obtained for each of 66 samples involved in four key regeneration stages,with and without 2,4-D treatments,three lily species and two tissues including scales and basal plate,derived from Illumina sequencing.48,279,48,397 and 57,965 genes was found respectively for the three lily.High mapping rates of illumina reads to Pacbio reference sequence were obtained with the average of 71.50%,80.79%and 81.85%for the three species.This dataet would be important and useful reference for Lilium spp.,as well as propagule regeneration in other bulbous plants.At the meantime,Illumina sequencing was conducted on the whole in vitro bulblet of Lilium brownii var.giganteum,whose scales are used as explant in regeneration experiment.60,121 unigenes were obtained,with an average length of 830 bp,N50 1,396 bp,whose functional annotation points out carbohydrate metabolism among top active pathways,which further support the importance of carbohydrate metabolism for in vitro bulblet of lily as a storage organ.3.Interspecific variability elucidated by histology and transcriptome data converglyLilium brownii var.giganteum and Lilium brownii were fast meristem regeneration species while Lilium 'Sorbonne' launched regeneration relatively late.This is confirmed by transcriptome analysis:low pearson correlation between samples of early and late regeneration stages was found,samples of ealry and late stages were clustered rather far away from each other by Principal Component Analysis,large number of differentially expressed genes was found as early as at 8 days post culture(DPC)for fast regeneration species compared to 14 DPC in slow specie.Dedifferentiation and divisions of cells were found to be accelarated drastically by 2,4-D by histological analysis,while the three species show different stress tolerance to 2,4-D:Lilium'Sorbonne'>Lilium brownii var.giganteum>Lilium brownii.Transcriptome analysis supported this observation:expression profile of Lilium brownii var.giganteum and Lilium 'Sorbonne' were not significantly influeced by 2,4-D at early developmental stage,while large number of genes quickly responded to 2,4-D stress in Lilium brownii.4.Mining of key pathways and candidate genes of dedifferentiation and bulblet organogenesisAs described above,wounding is the decisive signal in bulblet de novo regeneration of lily.Abscisic acid response,carbohydrate metabolism and active oxygen metabolism were identified to be critical pathways in response to wounding via hybrid sequencing,among which 14 key candidate genes including serine/threonine protein kinase.2,4-D was found to deeply regulate pathways related to callus formation,embryogenic ceells identity maintenance and stem cell population maintenance,as well as influence carbohydrate metabolism,cell differentiation,mitochondrion function,RNA transcription and stress response.39 candidate genes in response to 2,4-D stimulus were identified including 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase,3'-phosphoadenosine 5'-phosphosulfate synthase,3'-phosphoadenosine 5'-phosphosulfate synthase,succinate dehydrogenase assembly factor 2,succinate dehydrogenase assembly factor 2 and protein regulator of cytokinesis 1,among which 6 candidates were assigned higher priority.Weighted gene co-expression network analysis across samples and treatments within each lily taxa investigated identified 13 candidate hub genes in response to 2,4-D,including exonuclease 1,replication factor A1,G2/mitotic-specific cyclin-B,cyclin-A,pectinesterase,histone H2A,translation initiation factor 4A,cleavage and polyadenylation specificity factor subunit 3,fanconi-associated nuclease 1,cofilin and integrin-linked kinase-associated serine/threonine phosphatase 2C.Besides,the importance of photosynthetic pathway in detached lily scale cultured under light was revealed for the first time,which was enriched in scale specifically compared to basal plate.