Physiological And Molecular Mechanism Of Etiolation-Induced Adventitious Root Formation In Micro-Shoot Cuttings Of Tetraploid Robinia Pseudoacacia L.

Tetraploid black locust(Robinia pseudoacacia L.)is a leguminous,deciduous,ornamental tree species produced artificially by doubling the chromosomes in diploid cells of the congeneric black locust.In the present study,the developmental stages,physiological,phytohormones,and transcriptional regulations of adventitious root formation during in-vitro conditions by applying etiolation as a dark pretreatment were investigated in micro-shoot cuttings of tetraploid R.pseudoacacia at different time points 0 h,12 h,36,48,and 72 hours after excision(HAE).Adventitious root(AR)formation is a post-embryonic developmental process during the propagation of difficult to root plant species,and it is regarded as the most significant step in vegetative propagation,especially in forestry and horticultural plants species.Adventitious root formation in tetraploid R.pseudoacacia is a difficult task as it’s a hard rooting species.To cope with this critical problem,etiolation as a dark pretreatment was used to induce adventitious rooting in this specie.Etiolation has been used to improve adventitious rooting in many woody species.However,the parameters such as the time ratio of light and dark culture in the etiolation treatment of in-vitro cultured tetraploid R.pseudoacacia are unclear,and the mechanism of its occurrence is still unrevealed.Therefore,taking tetraploid R.pseudoacacia micro-shoot cuttings as plant materials,this paper studied the time points of adventitious rooting,the ideal light and dark ratio parameters,and the suitable medium for adventitious root induction.The major conclusions were described as follows:To elucidate the significance of etiolation in tetraploid R.pseudoacacia,the best treatment and best time points were selected by phenotypic and histological observations.Phenotypically our results confirmed that five days of dark pretreatment upon shifting to a subsequent light period could significantly enhance AR efficiency three days prior in etiolated micro-shoot cuttings compared to non-etiolated.The number of days was reduced from eight to five days using etiolation as pretreatment compared to control.Furthermore,light microscopy(LM)was used to confirm the particular time point of AR induction through histological observation.It was found that the critical time of AR induction was between 36 and 72 HAE because the initial cell division with the first meristemoids consists of meristematic cells with dense cytoplasm,and a large nucleus was discovered during this phase(induction phase)in the etiolated micro-shoot cuttings.Moreover,transmission electron microscopy(TEM)was used to examine the subcellular features of the presumptive AR initials located in the vascular cambium during AR formation in etiolated and control micro-shoot cuttings.We observed the abundance of starch grain(SG)in the cells(founder cells)of etiolated micro-shoot cuttings compared to the control in the plastids at HAE0 and HAE12 time points.However,the number and density of the starch grains rapidly decreased from the plastids at HAE36 to HAE72.Furthermore,in etiolated founder cells,the number of mitochondria,Golgi apparatus,and endoplasmic reticulum increased significantly from 48 to 72 HAE,while the number of starch grains decreased,implying that the starch grains were hydrolyzed and released as an energy source for mitochondria to provide "cash energy" during AR development.In addition,in the etiolated microshoot cuttings,the number of elongated founder cells was observed at 72 HAE compared to the control.The physiological analyses revealed that peroxidation in the basal region of the micro-shoot cuttings during the early phases(12 to 72 hours)of AR formation might be caused by reactive oxygen species(ROS)because activities of enzymes involved in ROS detoxification such as peroxidase(POX),polyphenol oxidase(PPO),and catalase(CAT)increased in the etiolated micro-shoot cuttings during the early phases of AR formation.Moreover,malondialdehyde(MDA)levels were also increased in etiolated micro-shoot cuttings in36 and 72 HAE as regarded as those of control.Auxin is the critical component that controls AR formation.To understand the hormones crosstalk,the endogenous content of several hormones in etiolated and control micro-shoot cuttings were analyzed.The results unveiled that abscisic acid(ABA)content significantly decreased in etiolated samples regarding the high ABA level found upon excision in control.Trans-zeatin(TZ)levels steadily increased upon excision.On the other hand,endogenous 24-epibrassinolide(BL)levels remained unchanged in the etiolated and control microcuttings.Moreover,salicylic acid(SA)levels considerably increased in etiolated samples compared to control.Therefore,a low level of ABA in etiolated treated micro-cuttings suggests that it may facilitate AR formation in tetraploid R.pseudoacacia.The initial TZ levels in the dark pretreated micro-shoot cuttings did not differ from control,suggesting that TZ level increases with IBA treatment during the subsequent light period and regulates AR development during the induction phase.In addition,gradual increases in SA level suggested that etiolation might promote SA level during the dark period compared to control.The Indole acetic acid(IAA)and gibberellic acid(GA)levels significantly increased from HAE12 to HAE36,respectively,though IAA level sharply decreased from HAE48 to HAE72 in etiolated micro-shoot cuttings.In contrast,IAA content sharply decreased from HAE12 to HAE72 in the control micro-shoot cuttings during the AR formation in the first 72 hours period.However,the GA level remains unchanged in control throughout 72 hours.The molecular interaction of various factors for etiolated in-vitro AR formation in tetraploid R.pseudoacacia is still unclear.Therefore,the samples were analyzed using Illumina high-throughput sequencing technology for the identification of differentially expressed genes(DEGs)at the mentioned time points(0,12,36,48,and 72 HAE).A total of 671 million clean reads were generated.In addition,11,567(DEGs)were identified and found to be involved in starch and sucrose metabolism and plant hormone signal transduction signalling.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were analyzed to monitor the changes at the transcriptomic level and detected 117 and 69 co-expressed genes related to phytohormone signal transduction and starch and sucrose metabolic pathways,respectively.The data exhibited dominance of auxin response factors-3(ARF3),gretchen hagen-3(GH3.3),and small auxin upregulated RNA(SAUR70)in the auxin,scarecrow-like1(SCL5)in gibberellin,ethylene-responsive transcription factor(ERF1,ERF96),and ethylene insensitive-3(EIN3)in ethylene,squamosa promoter-binding protein-like-12(SPL12)in brassinosteroid,MYC2 in Jasmonic acid and motif-binding protein-9(TGA9)in salicylic acid signalling pathways exhibited higher expression than control.Furthermore,ADP-glucose pyrophosphorylase large subunit(APL4),starch synthase-3(SS3),probable starch synthase-3(SPS4F),and vacuolar invertase(VI1)in sucrose biosynthesis and degradation pathways significantly upregulated in all-time points compared with control.Moreover,quantitative reverse transcription-polymerase chain reaction(qRT–PCR)analysis was performed on the expression patterns of eighteen genes associated with hormone signal transduction and carbohydrate metabolism.Eventually,the present study further uncovered the role of differentially expressed(DE)microRNAs(miRNAs)and their targeted genes.The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points.Seven DE miRNA libraries were constructed and sequenced.The DE number of 81,162,153,154,41,9,and 77 miRNAs were upregulated,whereas 67,98,84,116,19,16,and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12,0-vs-36,0-vs-48,0-vs-72,12-vs-36,36-vs-48,and 48-vs-72 HAE respectively.Furthermore,we depicted an association between ten miRNAs(novel-m0778-3p,miR6135 e.2-5p,miR477-3p,miR4416c-5p,miR946 d,miR398 b,miR389a-3p,novel m0068-5p,novel-m0650-3p,and novel-m0560-3p)and important target genes(auxin response factor-3,gretchen hagen-9,scarecrow-like-1,squamosa promoter-binding proteinlike-12,small auxin upregulated RNA-70,binding protein-9,vacuolar invertase-1,starch synthase-3,sucrose synthase-3,probable starch synthase-3,cell wall invertase-4,and trehalose phosphatase synthase-5),all of which play a role in plant hormone signalling and starch and sucrose metabolism pathways.The quantitative polymerase chain reaction(qRT-PCR)was used to validate the relative expression of these miRNAs and their targeted genes.The results presented here will provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in tetraploid R.pseudoacacia and will help to understand key regulators of in-vitro adventitious root development in other woody species,which may be deployed in the future for vegetative production at a commercial scale.