| Astragali radix,better known as huang qi,is the dry root of leguminous plant Astragalus membranaceus（Fisch.）Bunge.Formononetin（FO）and calycosin（CA）are two important isoflavone active compounds.It possesses a wide range of biological properties and used to clinically treat cardiovascular,respiratory,and hepatic diseases.Due to medical market increased demand for FO and CA and low active ingredient content in astragali radix planting field,our laboratory has established stable astragalus membranaceus hairy root culture system（AMHRCs）to produce FO and CA,and successfully induced with bacteria,signaling molecules induction and UV radiation method to increase FO and the content of CA,but because the bacteria induced complex operation,signaling molecules is expensive,UV radiation has a harm to human body,finding a more simple,effective,convenient and inexpensive induction method to increase the content of CA and FO is still our research target.Light,in addition to being the main energy source for photosynthesis,also plays an important role in signal transduction in regulating plant development,morphogenesis,chemical composition content and antioxidant capacity.Given these characteristics,we attempt to elicit AMHRCs with LED light to promote growth and increase the content of CA and FOand to study the biosynthesis mechanism of increased CA and FO contents under the optimal light treatment.The main results are as follows:1.The effects of different LED light on the growth of AMHRCs and the content of CA and FOThe experiment was carried out under different LED light for 62 days.Compared with the control group（dark culture）,all light promoted the growth of AMHRCs at 62 days,and red,mixed,blue and green light promoted the growth of AMHRCs more obviously.Under blue,red and mixed light,the length and density of root hairs on the surface of AMHRCs increased significantly,suggesting that LED light could promote root hair growth by promoting root hair growth.The contents of CA and FO were analyzed and detected.The contents of CA and FO did not increase significantly under red,white and mixed light during 0-62 days of culture cycle.The content of CA and FO in blue and redlight was significantly higher than that in dark culture.The maximum value of CA was 166.26±21.57μg/g DW on the 55th day of blue light irradiation,which was 2.92 times of that in dark culture.FO content reached the maximum of86.95±10.41μg/g DW on the 55nd day of blue light irradiation,which was 2.27 times that of dark culture.From the perspective of growth and root hair,blue,red and mixed light are suitable,but from the perspective of the accumulation of CA and FO content,blue light is the best light.Comprehensively considering,blue light is the most suitable light for the accumulation of active components CA and FO under the condition of not affecting growth.2.The effects of initial inoculation amounton on AMHRCs growth and CA,FO contents under blue lightAMHRCs inoculated with 0.2%,0.4%,0.6%and 0.8%were cultured under blue light for76 days,and their biomass was measured.The results showed that FW and DW reached the maximum accumulation at about 62 days when inoculated with 0.2%and 0.4%,and their biomass did not increase with time.When the inoculation amount was 0.6%and 0.8%,FW and DW reached the maximum accumulation at about 55 days,and the biomass basically did not increase with the extension of time.From the perspective of culture cycle,0.6%and 0.8%had shorter incubation period and were more appropriate initial inoculation amounts.The changes of surface root hair were observed.Compared with 0.2%and 0.4%,the initial inoculated amounts of 0.6%and 0.8%showed dense surface root hair and obvious increase.The contents of CA and FO were measured at 0-76 days and CA and FO were the highest when the initial inoculum amount was 0.6%for 48 days.FO content reached 114.08±10.11μg/g DW at 48 days,which was 2.84 times of that in dark culture.The content of CA was higher from 48 to 62 days,and the content of CA was 160.53±17.91μg/g DW at 48 days,3.31 times of that in dark culture.In conclusion,blue light irradiation for 48 days and initial inoculation amount of 0.6%are the most suitable culture conditions to promote the growth of AMHRCs and the accumulation of CA and FO contents under the condition of shortening the culture time.3.The effects of blue light on AMHRCs physiology and related biosynthesis gene expressionThe content of SS and AA in AMHRCs decreased gradually under the optimal light condition（blue light and initial inoculation amount of 0.6%）,indicating the transformation from primary metabolism to secondary metabolism.The contents of H2O2,CAT,POD and OFRall changed significantly compared with those before light exposure,suggesting that blue light exposure did trigger the ROS system of AMHRCs.The expression levels of nine key enzyme genes related to CA and FO biosynthesis（PAL,C4H,4CL,CHS,CHR,CHI,IFS,IOMT and I3’H）were analyzed.The transcriptional levels of other CHS genes PAL,C4H,4CL,CHR,CHI,IFS,IOMT and I3’H increased under blue light irradiation,and the abundance of C4H,4CL,CHS,CHR,CHI and IFS reached the highest on day 48,increased to 5.99,7.05,2.48,9.17,10.40 and 2.94 times,respectively.These results suggest that blue light exposure promotes the biosynthesis of CA and FO by up-regulating the expression levels of key biosynthase genes.In conclusion,it is determined that blue light and initial inoculation amount of 0.6%for48 days are the most suitable conditions to promote the growth of AMHRCs and accumulation of CA and FO.Blue light exposure affects the nutrient allocation between primary and secondary metabolites,stimulates the ROS system of AMHRCs,and up-regulates the expression of CA and FO synthase genes,and ultimately increases the accumulation of CA and FO secondary metabolites.This study provides a theoretical basis for the large-scale production of CA and FOusing LED light in the future,and also provides a new method for increasing the content of secondary metabolites by using LED light treatment of other systems. |
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