Inducement And Screening Of Cyclocarya Paliurus Red Callus And Cell Suspension Culture To Produce Anthocyanin

Anthocyanins are a large variety of secondary metabolites in plants with important functions,more than 700 of which have been isolated and identified up to now.Rasearches showed that anthocyanins not only play an important physiological role in plants,but also have many health beneficial activities such as anti-diabetes,antioxidant and antitumor in human body,and therefore these compounds have been widely used in food,cosmetics and medicines because of their outstanding color,health benefits and safety.At present,anthocyanins are mainly extracted from roots,stems,flowers,fruits and other raw materials of plants.Although there are many kinds of available materials,the supply is easily affected by climate,environment,diseases and insect pests.At the same time,due to the complexity of raw materials,the extracted anthocyanins are difficult to purify,and thus has high production costs.However,the anthocyanin production by plant cell culture shows many advantages,such as short production cycle,good controllability,independence of natural environment,easy extraction and purification,etc.,and consequently is regarded as a promising way to produce anthocyanins.Nowadays,there are several studies on the anthocyanins production by cell culture of grape,sweet potato and potato,but this novel production method has not been popularized and applied for its instability and high cost.In the present paper,a unique and novel red callus line of Cyclocarya paliurus was induced and cultivated,whose morphological,growth and metabolic characteristics were fully studied as well,and the red cell suspension culture was further successfully developed to product anthocyanins.The main results and conclusions are as follows:（1）A unique and novel red callus line of Cyclocarya paliurus was induced and cultivated.The additive amounts of three hormones in the culture medium were optimized by orthogonal experiment of L₉（3⁴）,and the results showed that the hormones combinaton of 1.0 mg/L KT,0.5 mg/L 2,4-D and 0.3 mg/L NAA in MS medium was suitable for callus inducement,and the callus induction rate reached 100%under the optimal conditions.Five typical kinds of callus lines were got by inducing and screening.What’s more,a rare red callus line of Cyclocarya paliurus was successfully obtained through repeated screening and cultivation,which displayed a bright red appearance,metallic luster,loose texture,rapid growth and stable traits as well.（2）The main secondary metabolites in the red callus of Cyclocarya paliurus were identified.Using UPLC-MS/MS and HPLC,the main secondary metabolites in the red callus of Cyclocarya paliurus were identified,including four polyphenols（（+）-catechin,procyanidin B1,cyanidin 3-O-galactoside and cyanidin 3-O-glucoside）and three triterpenoids（betulinic acid,oleanolic acid and ursolic acid）.At the same time,HPLC quantitative determination methods of polyphenols and triterpenoids in the red callus of Cyclocarya paliurus were established respectively,which were validated to be stable and reliable.（3）The main differences in growth,morphology and metabolism between red and light yellow-green calluses were described by comparison.The growth curves of the two typical callus lines were both S-types,however the growth rate and summit biomass of the red calluses were slightly lower than that of the light yellow-green.Both of the two typical callus lines grew rapidly and showed traits in the long-term sub-culturing,but the red callus displayed a bright red color,loose texture and metallic luster,while the light yellow-green callus present with light yellow-green,granular,loose and fragile appearance.The following five secondary metabolites,including（+）-catechin,proanthocyanidins B1,betulinic acid,oleanolic acid and ursolic acid,were all detected in two callus lines,but its contents were significantly higher in the red callus than that in the light yellow-green except betulinic acid.Cyanidin 3-O-galactoside and cyanidin 3-O-glucoside were found in the red callus with high content,but were not detected in the light yellow-green.（4）Comparative transcriptome sequencing were performed to interpret the formation mechanism of secondary metabolism differences between red and light yellow-green callus lines of Cyclocarya paliurus.105,041 high-quality unigene sequences were obtained from RNA-Seq of the red and light yellow-green calluses of Cyclocarya paliurus by using Illumina Hi Seq 2500 sequencing platform.Through Blast X software,the assembled unigenes were searched against seven public databases such as NR,Pfam,String,Swissport,GO,COG and KEGG,and 62.21%unigenes were annotated in these public databases.A total of 21 significant differentially expressed genes were found by comparison analysis between two callus lines,including five genes related to anthocyanins synthesis（CHS,CHI,F3’5’H,3GT and 3Gal T）,eight genes related to triterpenoids synthesis（HMGS,HMGR,MVD,DXS,FPS,SS,SE andβ-AS）and eight transcription factors（MYB44,MYB7,MYB1,MYC2,b HLH122,WD40,ERF003 and ERF105）,whose expression levels were all validated by RT-q PCR determianiton.The above-mentioned gene differential expression analysis were further used to interpret the formation mechanism of secondary metabolites differences between two typical callus lines.（5）A red cell suspension culture system for anthocyanin production was established.By using the fine red calluses of Cyclocarya paliurus as material.The key parameters of the cell suspension culture were optimized,including inoculation quantity,liquid filling quantity,rotation rate,photoperiod and hormone concentration.The results showed that the optimum culture conditions were as following:10%inoculation quantity,40%liquid filling quantity,105 rpm/min and continuous photoperiod,and the optimal hormones combination was 0.3mg/L TDZ,0.3 mg/L 2,4-D,0.1 mg/L NAA and 1.0 mg/L IBA.Under the optimized culture conditions,the total content and production of anthocyanins were 1342.36μg/g and 20.81mg/L（dry weight）respectively on the 7^th day of cell culture.Meanwhile,the peak cell biomass was 15.75 g/L（dry weight）on the 8^th day after inoculation.In conclusion,a unique and novel red callus line of Cyclocarya paliurus was obtained by a long-term screening and cultivation,which was significantly different from the common light yellow-green callus in morphology and metabolism.The significant expression differences of transcription factors and key genes in the synthesis pathway of polyphenols triterpenoids led to the significant different contents of total anthocyanin,total polyphenols and total triterpenoids between the red callus and the light yellow-green callus.The established cell suspension culture using the red callus lines displayed a stable traits,high growth rate and high anthocyanin content,which gave it a good application prospect in anthocyanin production.