| Garlic(Allium sativum L.)is native to central Asia and southern Europe,and has a history of more than 2,000 years of cultivation in China.Due to the loss of fertility of garlic,long-term reliance on bulbs for asexual reproduction has resulted in the accumulation of viruses and serious degradation of its species,which seriously affected genetic improvement and germplasm innovation.Compared with other tissue culture approaches such as organogenesis,the somatic embryogenesis pathway has the potential advantages of rapid occurrence,high reproduction coefficient,strong genetic stability and neat regenerated individuals,which opened up a new way for the breeding of garlic virus-free seedlings.However,the research on garlic somatic embryogenesis mainly focuses on the optimization of culture conditions,the observation of morphological and anatomical characteristics,and the analysis of physiological and biochemical mechanisms.The molecular mechanism of garlic somatic embryogenesis is unclear.Therefore,clarifying the molecular mechanism of garlic somatic embryogenesis can not only lay the foundation for garlic germplasm resource innovation,genetic transformation and artificial seed preparation,but also discover new ways to solve the problem of garlic species degradation and new varieties selection.In this study,the bulb(explant,EX)of the garlic variety ’Ershuizao’ and its induced callus(CA),embryogenic callus(EC)and globular embryo(GE)were used as materials.Transcriptome sequencing technology was used to obtain differentially expressed genes(DEGs)in the above four stages.Through bioinformatics analysis to construct a regulatory network at the transcriptome level of garlic somatic embryogenesis,explore the molecular mechanism of garlic somatic embryogenesis.It provides a theoretical basis for constructing an efficient regeneration system of garlic somatic embryos.The main findings are as follows:1.A total of 2071 CA-stage DEGs,1233 EC-stage DEGs and 715 GE-stage DEGs were screened out;the GO classifications of the above-mentioned three-stage DEGs were related to the binding and catalytic activity,and the KEGG pathway was enriched in the pathways of MAPK signal transduction,plant hormone signal transduction,benzoic acid biosynthesis,sugar and starch metabolism and other pathways;Compared with CA and EC stages,DEGs in GE stage were enriched in fatty acid degradation,nitrogen metabolism and ascorbic acid and alginate metabolism.Transcriptome analysis results indicated that genes related to cell wall synthesis,stress response,carbohydrate metabolism and plant hormone signal transduction may play an important role in garlic somatic embryogenesis.2.In the CA,EC and GE stages of garlic somatic cells,70 DEGs related to auxin,cytokinin,substituted acid and jasmonic acid were found,including 23 related to auxin and14 related to substituted acid related genes.In the primary embryonic callus(PEC)stage,the expression of the IAA synthesis genes As YUCCA1,As TA1,and the extra-auxin transport gene As ABCB1 increased.In the late embryonic callus(LEC)stage,the expression of auxin transport-related gene As AUX1 increased.and the expression of auxin response genes As ARF1 and As ARF2 decreased.The expression of the c Z synthesis genes As LOG1 was the highest in the CA stage.The expression of classifier transport-related gene As ENT2,signal transduction-related genes As ORR-A1 and As ORR-A2 were the highest in the PEC stage.The expression of ABA synthesis rate-limiting enzymes As NCED1 and As NCED2 were lower than its EX stage.The expression of ABA signal transduction-related genes As PP2 C increased in PEC stage.,As ABI1 increased in LEC stage,and As PYL1 increased in GE stage.It shows that hormone synthesis-related genes regulated the changes of endogenous hormone content,and hormone signal transduction-related genes were involved in the occurrence of garlic somatic embryos.3.As AUX22 D encoded 179 amino acids and was located in the cytoplasm;As AUX22 D was a neutral hydrophilic protein with random coils and α-helix as the main structure,which has high homology with Ao AUX22 D of Asparagus(Asparagus officinalis);The promoter region contained response elements for light,auxin,and abscisic acid.The results of q RT-PCR showed that the expression level of As AUX22 D was higher in garlic leaves and roots,and the lowest in storage leaves;The expression of As AUX22 D was the highest in GE stage with bulb disc as explant,and was higher in garlic with inflorescence axis as explant.The expression of As AUX22 D was significantly increased on the 60 th day after somatic embryo induction in ’Ershuizao’ and ’Sichuan’. |
