Establishment Of Somatic Embryogenesis System And Mechanism Analysis Of Abnormal Embryoids In Vitis Vinifera Cv.'Chardonnay'

Somatic embryogenesis system is the basis for the improvement genetic transformation of grape.However,the application and development of somatic embryogenesis are seriously affected by the low induction efficiency,poor ability to maintain and propagate embryogenic callus and high frequency of abnormal embryoids.In particular,a large number of abnormal embryoids seriously restrict the normal growth of somatic embryos.Therefore,analyzing the mechanism of abnormal embryoids or recycling abnormal embryoids is one of the core issues in the somatic embryos regeneration system of grape.In this study,the white wine grape variety‘Chardonnay' was used as experimental materials,optimized an efficient and stable somatic embryogenesis by adjusting the concentration and ratio of plant hormones,and studied on the recycling of abnormal embryoids with four different embryoids in the regeneration pathway.Moreover,the mechanism of abnormal embryoids was analyzed from histological identification,content of endogenous hormone and miRNAs regulation.The main results are described as follows:1.Somatic embryogenesis was established of Vitis vinifera cv.'Chardonnay'.Result demonstrated that the preservation time of explants was significantly correlated with the contamination rate of inducted-callus by NaClO sterilization with unopened flower buds as explants.Embryogenic callus(EC)were successfully initiated on MC(NN69+0.55 mg·L-1 2,4-D+0.5 mg·L-1 NOA+1.24 mg·L-14-CPPU+30 g·L-1 Sucrose+3 g·L-1 Phytagel),PIV(NN69+1.0 mg-L-1 2,4-D+2.0 mg·L-1 6-BA+30 g·L-1 Sucrose+3 g·L-1 Phytagel)and MEL2 medium(NN69+2.0 mg·L-1 Melatonin+2.0 mg·L-1 6-BA+30 g·L-1 Sucrose+3 g·L-1 Phytagel),which the induction rates were 9.52%,8.63%and 5.06%.The best somatic embryo formation medium was MS medium which contain 4 ?mol·L-1 ABA.Using scanning electron microscope to observe the microstructure of different tissues and cells,it showed that the surface morphology of EC and non-embryogenic callus(non-EC)was obviously different,and the microstructure of proembryogenic masses(PEM)is similar to embryogenic callus.The best medium for normal plantlets regeneration was MS contained 0.2 mg·L-1 6-BA and 0.1 mg·L-1 NOA.2.Somatic embryonic reinduction was established of Vitis vinifera cv.‘Chardonnay'.The influence factors of the EC reinduction rate were investigated.Result demonstrated that CIM medium was beneficial to induce EC from somatic embryo,and the induction rate was 8.33%.Cotyledons and hypocotyls showed higher callus inducibility and the induction rates were over 80%.Normal embryoids(NE)and Fused cotyledonary embryoids(FCE)were successfully induced EC with different embryoids in the regeneration pathway,which the induction rates were 8.42%and 4.44%.Secondary embryogenesis was directly formed from browned vitrified embryoids(VE)on X6 medium(MS+60 g·L-1 Sucrose).The cell morphology of EC and non-EC were significantly differences by carbol fuchsin staining.3.The morphology and histology was observed of abnormal embryoids from Vitis vinifera cv.‘Chardonnay'.The results showed that abnormal embryoids did not have a complete vascular tissue system and apical meristems than the NE.By measuring the endogenous hormone content of different embryos,the results showed that the least CK content of VE is 0.061 ng·g-1 during the maturation of somatic embryos.The content of IAA and ABA in NE was higher than other abnormal embryoids.In particular,the ABA content of NE was 4 times higher than abnormal embryoids(ECE,VE,FCE).The highest endogenous SA content of ECE was 7.4 ng-g-1,the lowest SA content of VE was 1.99 ng·g-1.Different kinds of endogenous hormone ratios were determined to balance the endogenous hormone.The results indicated that ABA/IAA,ABA/CK and CK/IAA in NE were higher than abnormal embryoids.4.High-throughput miRNAs sequencing of the abnormal embryoids in Vitis vinifera 'Chardonnay'.A total of 51 significantly differentially expressed miRNAs were identified from the three abnormal embryoids,and the number of miRNAs was mostly down-regulated;predicting potential targets,a total of 59 common target genes were identified,and most of the target genes were enriched in multicellular biological development and biological process,anatomical structure development and endogenous stimulus response pathway;and 2 target genes related to hormones and 12 target genes related to transcription factors were screened from the common target genes.Analysis of the regulatory relationship of specific miRNA-target genes of abnormal embryoids in 'Chardonnay' revealed that 16 differentially expressed miRNAs targeted 82 transcription factors to participate in the regulation of abnormal embryoids,of which vvi-miR2950-5p and vvi-miR156b,vvi-miR172d and vvi-miR3935-3p,vvi-miR166a and vvi-miR3630-3p are specifically expressed in ECE,VE,and FCE respectively,and the number of regulated transcription factors is the largest respectively.In addition,the number of hormone-related genes regulated by vvi-miR156b,vvi-miR2950-5p,and vvi-miR397a in ECE is the largest,and the number of genes targeted by VE and FCE is relatively small.Among the three embryos,Auxin(AUX)and ABA have the largest number of genes.