Culture Medium Optimization Of Ginkgo Biloba Callus

Ginkgo biloba L.is an ancient gymnosperm that has survived for 280 million years.As the "living fossil" plant,G.biloba is not only known as the famous landscape tree,but also for the important medicinal plant.The medicinal active ingredients in ginkgo leaves are mainly contributed by secondary metabolites of flavonoids,which industrial extraction is from ginkgo leaves through drying,processing and extraction.However,active ingredient content in ginkgo leaves is fluctuated by the growth environment,cultivation and tree ages,restricting the ginkgo extraction industry.Therefore,how to produce ginkgo flavonoids by tissue culture in a controlled environment,as well as uncover the biosynthesis pathway and metabolic regulation of ginkgo flavonoids are new directions worth studying.In this study,G.biloba callus was used as the experimental material,and the most suitable explants and inoculation methods were screened according to the observation of callus and the determination of physiological indexes.Based on the above,the most suitable medium ratio for callus growth in each stage of primary culture,secondary culture and exogenous treatment were gradually selected.The main results are as follows.?1?In the initial culture of G.biloba,explants such as leaves,stem segments and mature embryos were evaluated,and the ratio of hormones were adjusted to the callus induction by the orthogonal test.Callus induction time was the shortest was stem segment,followed by mature embryo and leaves.Callus status of leaf callus showed loose and fragile,while stem segment and embryo callus were compact.The most suitable way for leaf tissue culture was to use the middle or margin parts of the leaves and inoculate them with the upper blade upward.The most optimum medium for callus induction in initial culture are as follows.Stem segments:1/2MS+1.0 mg·L-1 NAA+1.0 mg·L-1 6-BA+0.5 mg·L-1 KT;leaves:MS+4.0 mg·L-1 NAA+2.0 mg·L-1 KT;mature embryo:MS+2.0 mg·L-1 NAA+1.0 mg·L-1 KT.?2?Leaf callus in the initial culture were selected for subculture,and single factor experiment was carried out to investigate the effects of AC,PVP and VC on preventing browning in the leaf calluses.The results showed that the browning preventing reagents with lower concentration had significant anti-browning effects.Among them,VC0.002 g·L-1 and AC0.5 g·L-1 had the best green preserving effect and good callus viscosity.PVP0.5 g·L-1 could proliferate callus in a short term,but the callus tissue was dry,loose and unsuitable for subculture.?3?Leaf callus in subculture was selected,and the effect of phenylalanine,salicylic acid and naringin on the accumulation of total flavonoids in callus was investigated by single factor test.The results showed that three substances had no significant effect on callus weight and inhibited its growth at their higher concentrations.However,the total flavonoids content was significantly increased when naringin was added,and the highest total flavonoids content was cultured by 100 ?g·L-1 naringin.?4?Transcriptome sequencing was performed on the callus from exogenous treatments.Sequencing samples were from three groups:control,salicylic acid 200 ?g·L-1 and naringin 100?g·L-1?three biological replicates each groups?.In all,we obtained 44,701,740,46,130,540,39,390,334,44,476,742,42,503,074,42,164,488,43,950,350,47,843,736,45,050,566 clean reads.The sequencing error rate was lower than 0.03%,Q20 was higher than 97.21%,Q30 was higher than 92.32%,and GC content was between 44.2%and 45.55%.?5?A total of 5,595 differentially expressed genes were detected.Through cluster analysis,differentially expressed genes in GO functional annotation were mainly enriched in‘iron ion binding','transmembrane transport' and 'coenzyme binding',etc.KEGG gene functional annotation differences mainly enriched in 'Galactose metabolism','Pyruvate metabolism','Propanoate metabolism',or other metabolic pathways and 'Plant hormone signal transduction'.In flavonoid synthesis pathway,some key enzyme genes such as PAL?Gb₀1672?,4CL?Gb₁9734?,DFR?Gb₂9686?,LAR?Gb₀8047?,ANR?Gb₂4242?,CHS?Gb₁9005?were identified as up-regulated expression.