Establishment Of Liquid Suspension Culture System Of Torreya Grandis

Torreya grandis is a rare dried fruit and woody oil plant unique to China,belonging to the evergreen tree Torreya genus of Taxus family of gymnosperms.It has important social,ecological and economic values.Because traditional grafting breed is difficult to meet the huge demand of the market at present,in recent years,the researchers conducted Torreya grandis embryonic callus induction,somatic embryogenesis,such as research,reveals the origin of Torreya grandis somatic embryogenesis and development process,thus to further optimize the somatic embryogenesis system provides a theoretical basis.However,at present,the synchronization rate of Torreya grandis embryo generation is low,and the research on Torreya grandis liquid suspension culture has not been reported.In this study,the conditions of Torreya grandis cell embryo maturation were optimized.Using Torreya grandis embryo culture as material,the suspension culture system was established and the conditions of suspension culture were optimized.The main findings of this study are as follows:（1）Optimized Torreya grandis solid somatic embryo maturation systemBy studying the effects of different concentrations of PEG6000,different medium components and different concentration ratios of ABA and GA₃ on Torreya grandis embryo maturation,the optimal medium for Torreya grandis embryo maturation was determined as follows:SH+PEG 6000 160 g·L^-1+ABA 3 mg·L^-1+GA₃ 1 mg·L^-1+S 30 g·L^-1+C 2 g·L^-1+agar 4 g·L^-1.Under these conditions,Torreya grandis embryos were the most mature,the granulae were full and the development state was the best.Morphological observation of somatic embryogenesis was carried out by means of fluorescence microscope.It was found that somatic embryogenesis underwent four stages:rod embryo,heart embryo,torpedo embryo and cotyledon embryo.（2）Constructed the liquid suspension culture system of Torreya grandisTorreya grandis embryo culture was used as the starting material to screen the concentration ratio of ABA and GA₃ in liquid suspension culture.The results showed that when the concentration of ABA and GA₃ were 1 mg·L^-1and 0.5 mg·L^-1,the growth rate of Torreya grandis embryonic cells reached the highest,52.47%.Under these conditions,the optimal liquid culture conditions of Torreya grandis calli were selected,and the results showed that the optimal liquid suspension culture of Torreya grandis embryogenic calli was 3%inoculation amount,110 r·min^-1 rotation speed,5.7 p H and 30 g·L^-1 sucrose concentration,and the optimal suspension cells were obtained under the dark culture condition for 18d.（3）Analyzed the related genes of GA₃ promoting the growth of Torreya grandis embryonic cells under the condition of liquid cultureThrough transcriptome analysis of Torreya grandis embryogenic calli treated with exogenous GA₃,a total of 428 differentially expressed genes were obtained,among which 236 differentially expressed genes were up-regulated（55.14%）and 192 differentially expressed genes were down-regulated（44.86%）.In the GO enrichment analysis of differential genes,the up-regulated genes after GA₃ treatment were mainly involved in catalytic activity,oxidation-reduction process,oxidoreductase activity,etc.,indicating that the removal of reactive oxygen species in Torreya grandis embryogenic callus may be an important way of its proliferation and development.In KEGG metabolic pathway,differentially expressed genes were mainly concentrated in phenylpropanoid biosynthesis,starch and sucrose metabolism,pyruvate metabolism and other pathways.Among them,the most differentially expressed genes were annotated in phenylpropane biosynthesis pathway,which indicated that the secondary metabolic pathway of Torreya grandis embryogenic callus had significant changes after GA₃ treatment,and it was found that HCT,COMT,bglX and POD might be the key genes in exogenous GA₃ regulation of phenylpropane biosynthesis metabolic pathway.