Effects Of Exogenous Hormones On Cannabinoid Content In Cannabis Sativa L.and Transcriptome Analysis

Cannabinoids are unique secondary natural products of Cannabis sativa L.plant.Among many medicinal plants with therapeutic potential,industrial cannabis is the large-scale application plant.Cannabinoids are mainly distributed in the glandulars of the calyx female plant.The proportion of female plants directly affects the production of cannabinoids.So it is very important to increase the proportion of female plants.The synthesis of cannabinoids is very complex,which is affected by endogenous genes and external environment.Among them,the treatments of exogenous hormones is considered to affect the synthesis of secondary metabolites in many plants,but there are few studies on these research on cannabinoids.Therefore,it is urgent to study the synthesis mechanism and gene regulatory network of cannabinoids in response to exogenous hormones,in order to provide references for theoretical research and practical application in this field,and to optimize the germplasm resources of cannabis.In this study,Ethrel?ETH?,Gibberellin?GA₃?,Kinetin?KT?and Auxins?IAA?were used to soak the industrial cannabis DMG233 seeds.Exogenous ETH,GA₃ and KT were sprayed on the industrial cannabis DMG227 plants.And cannabis material DMG227 sprayed by 20 mg/L KT were used for transcriptome analysis.The results of the experiment can provide some references for the content and synthesis mechanism of the major cannabinoids cannabidiol?CBD?and tetrahydrocannabinol?THC?in industrial cannabis.The main findings are as follows:1?Seed soaking treatment:1,5,10 mg/L KT,1,50,100 mg/L ETH,10,50,100 mg/L GA₃ and IAA were used to soak seeds before sowing.Among them,under KT and GA₃ treatment conditions,the seed germination rate decreased significantly with the increase of hormone concentration;Under 100mg/L ETH treatment,the proportion of female plants increased most significantly in the cannabis plant group,and the ratio of male and female reached 1.57:1;At the same time,THC content decreased significantly after ETH and GA₃ soaking treatment.2?ETH spraying treatment:The 10 mg/L,100 mg/L and 200 mg/L concentrations of ETH were sprayed to treat industrial cannabis at two,four and six weeks of flowering.100 mg/L ETH was sprayed with industrial cannabis for two weeks of flowering can significantly increase the content of CBD and THC at 4 weeks sampling after the treatment,respectively by 50.24%and 52.94%.100 mg/L and 200mg/L ETH were sprayed with industrial cannabis for four weeks of flowering can significantly increase the content of CBD and THC at 2 weeks sampling after the treatment.CBD increased by 53.16%and54.43%respectively,and THC increased by 35.71%.Three concentrations of ETH sprayed with industrial cannabis for 6 weeks at flowering period can significantly reduce CBD and THC content.Exogenous ETH spraying had no effect on the biomass yield of cannabis plants.3?GA₃ spraying treatment:The 10 mg/L,100 mg/L and 200 mg/L GA₃ were sprayed to industrial cannabis in two,four,and six weeks three different periods after flowering.Treatment of industrial cannabis for two weeks at the flowering stage with different concentrations of GA₃,the content of CBD and THC increased significantly at 24 h after the treatment,and the content of CBD and THC decreased significantly at 4 weeks after the treatment;The treatment of industrial cannabis for four weeks at flowering stage showed no significant difference in CBD and THC content at 24 h after the end of the treatment.The CBD and THC content at the end of 2 weeks after the treatment decreased significantly.6 weeks of 10 mg/L and 100 mg/L GA₃ spraying can significantly reduce the content of CBD and THC.Therefore,GA₃ is not suitable as an exogenous hormone to increase the production of cannabinoids.4?KT spraying treatment:The 20 mg/L,40 mg/L and 100 mg/L concentrations of KT were sprayed to treat industrial cannabis at two,four and six weeks of flowering.20 mg/L and 40 mg/L KT were sprayed industrial cannabis for two weeks during the flowering period can significantly increase the content of CBD and THC at 4 weeks sampling after the treatment.CBD increased by 69.08%and50.72%,and THC increased by 41.18%and 35.29%.100 mg/L KT was sprayed with industrial cannabis for four weeks during the flowering period can effectively increase the content of CBD and THC at after 2 weeks sampling after the treatment.CBD and THC increased by 23.42%and 21.43%.Three concentrations of KT sprayed with industrial cannabis for 6 weeks at flowering stage can significantly reduce CBD and THC content.Spraying KT had no effect on biological yield.5?Transcriptome sequencing analysis:20 mg/L KT were sprayed on industrial cannabis strain DMG227 plants of 2 weeks at the flowering stage,samples were collected at 1,10,20,and 30 days after treatment for the content detection of CBD and THC,and samples at 30 days were used for transcriptome sequencing.The control group and the treatment group were taken,with 3 biological replicates in each group,6 samples in total.CBD and THC content were significantly higher in samples of KT treatment than the control.KEGG enrichment analysis of differential genes showed that 12pathways were significantly enriched after 30 days of treatment,including secondary metabolic pathways such as sesquiterpene and triterpene biosynthesis,linoleic acid metabolism,flavonoid biosynthesis,and phenylpropane biosynthesis.The content of CBD and THC was significantly higher than the control.