| Stevia rebaudiana Bertoni is a natural source plant of a novel sweetener with low calorie and high sweetness.The sweetness of stevioside in its leaves is about 200-300 times that of sucrose,wherein RA,RD and RM are high quality glycosides;S.rebaudiana also has pharmacological effects on lowering blood pressure,inhibiting bacteria,and resisting tuberculosis.Breeding S.rebaudiana varieties with salt tolerance and high quality glycoside content can contribute to the development of S.rebaudiana industry.In vitro culture,creating mutants by radiation mutagenesis and other techniques is one of the important ways for plants to obtain excellent varieties.Based on the previous research of the research group,this experiment carried out the creation and screening of high-quality stevia mutants resistant to salt,boron and zinc-like,and initially established the drying process of stevia mutants with high RA glycosides.Meanwhile,the mutation mechanism of high RA glycosides mutants resistant to boron and zinc was preliminarily discussed based on transcriptome sequencing.It laid a material foundation for the breeding and processing of S.rebaudiana and provided certain theoretical and technical support.The main research results are as follows:1.After screening,the callus induction rate of S.rebaudiana leaves on T1 medium with hormone ratio of 6-BA1.0mg/L+NAA0.5mg/L reached 100%,significantly higher than other explant induction media.Y6 medium with hormone ratio of 6-BA2.0+IAA0.5 has the best callus growth state and no obvious browning compared with other media,and is the best medium for callus induction and bud formation.Using callus radiation mutagenesis and screening with high salt,boron and zinc medium,7 salt tolerant mutant plants were obtained,among which 1-3 was Na Cl tolerant plant,4-7 was Na2SO4 tolerant plant,and no Na HCO3tolerant mutant plant was obtained.In addition,mutant plant GP was obtained by screening on high concentration boron medium and mutant plant GX was obtained by screening on high concentration zinc medium.2.The salt-tolerant mutant strains are obtained by tissue culture and propagation technology.The content of glycosides in leaves of mutant lines were determined and their main agronomic traits were investigated.The results showed that the agronomic traits and glucoside content of the 7 salt-tolerant mutant lines were varied to different degrees.Among them,the variation range of plant height is 35.00-66.33cm,with the No.3 strain the highest,which is 28.37%higher than the control.The variation range of stem diameter is 2.31-5.48cm,and the stem of No.3 strain is the thickest,which is 22.67%thicker than the control.In addition,the internode length varied from 19.00-36.00cm to 36.00 cm,and the internode length of line 6 was the shortest,which was 45.17%shorter than that of the control.The variation range of leaf length and width is 50.62-63.73cm and 8.89-12.16mm respectively.The variation range of main yield agronomic traits stem fresh weight and leaf fresh weight are 1.80-24.34g and 2.80-26.06g respectively,with stem fresh weight and leaf fresh weight of No.3 gaining the most weight,175.65%and 67.06%respectively compared with the control.However,there was no obvious change in stem folding rate.Except for the increase of 37.41%in leaf folding rate of No.6,there was no obvious change in other salt-tolerant mutant lines.Meanwhile,the change ranges of RA,RD and RM glycosides are 5.00-6.95%,1.21-3.03%and 0.64-0.87%respectively.Compared with the control plant without mutation,RA glycoside of mutant line 7increased by 45.63%;RD glycoside No.3 increased by 32.03%,and its main agronomic traits such as fresh leaf weight,fresh stem weight,plant height and stem diameter were significantly higher than those of the control.it was a salt-tolerant mutant with good comprehensive properties.3.The RA glycoside content of GX and GP mutant strains reached 10.93%and 10.56%respectively,nearly 2 times higher than that of the control.The dry weight yield per plant of GX and GP mutant strains reached 41.42g and 44.17g respectively,which were more than2.5 times of that of the control.The other main agronomic traits of GX and GP mutant strains,such as stem diameter,leaf length and leaf width,were significantly higher than that of the control.Both were new strains of high RA stevia with high yield and high quality.In addition,the boron content of GP mutant strain was 25.77mg/kg,with no significant difference from the control.The zinc content of GX mutant strain was 41.94mg/kg,significantly higher than that of the control.4.Under the temperature of 45℃,60℃,80℃ and 100℃,respectively,the GP plant leaves collected from field planting were dried according to the de-enzymed and non-de-enzymed groups and shade drying treatment,and their glycoside content were determined by high phase liquid chromatography HPLC.The results showed that the time for drying in the shade to reach constant weight was 48 hours,which was significantly longer than that for other drying treatments.However,the RA glycoside content in GP leaves was 11.37%under shade drying condition,which was the highest among all treatments.The second is drying at 45℃with RA glycoside content of 10.53%.It is suggested to spread it thinly in the shade or dry it under the sunlight after harvesting.If it is planted in large areas or in rainy seasons or weather,it is appropriate to dry it at 45℃.In addition,the content of RD glycoside was highest in the 100℃constant temperature treatment,and was significantly higher than that in other drying treatments.It was considered that the drying of RD glycoside S.rebaudiana leaves should be treated at 100℃constant temperature.5.Transcriptome sequencing was carried out on 023 leaves of GP and its original source plant respectively to screen differential expression related to boron transport and transcription factors specifically expressed in GP.As a result,a candidate transcription factor of SrNIP family related to boron transport to extracellular was screened out from GP mutant,and its expression increased by 3.324 times compared with the control.Five transcription factors specifically expressed in GP mutants were screened out,including one SrGGCT family,one P450 family,two SrPKC families and one SrPAP family.Eight UDP-glucosyltransferase genes with obvious differences between GP and 023 were also screened out in this experiment.The highest expression level of UDP-glucosyltransferase genes was8.4224 times higher than that of control 023,while the gene expression level of glucosyltransferase SrUGT76G1 directly involved in catalyzing ST glycoside to form RA glycoside was 1.575 times higher than that of control plants.In addition,the key enzyme genes of stevioside biosynthesis pathway other than glycosyltransferase gene were compared.The results showed that the expression of one SrKAH gene was 1.403 times higher than that of the control plant.The expression levels of SrCDPS1,SrKS and SrDXS2 genes were 2.379,5.032 and 2.127 times higher than that of the control plants respectively.6.Leaf transcriptome sequencing of GX mutant was carried out with original source plant 023 as control.The results showed that there were two candidate transcription factors of SrZIP family in GX mutant,which were responsible for zinc translocation into cell,and the expression level was 1.377 and 1.307 times of that of control plant respectively.Twelve transcription factors specifically expressed in GX mutants were also screened out,including3 AP2/ERF families,1 cullin family,1 P450 family,2 SrPKC family,1 SrSAMS family,2glco32 family,1 SrDIR family and 1 SrGlu-R family.Eight UDP-glucosyltransferase genes were also screened out in this experiment,and the expression level was increased compared with the control plant,with the highest expression level being 16.855 times that of the control plant.Among them,the gene expression level of glycosyltransferase SrUGT76G1,which directly catalyzes ST glycoside to RA glycoside,was 1.908 times that of the control plant.As for the stevioside biosynthetic pathway key enzyme genes other than glycosyltransferase genes,it was found that the expression of one SrKAH gene was 4.270 times that of the control plant,while the expression of SrCDPS1,SrDXS2 and SrGGPPS2 genes were 13.821,4.270and 3.134 times that of the original plant,respectively.In addition,two genes SrCDPS2 with significant expression differences and participating in the synthesis of other terpenoids were screened out,of which one expressed 25.191 times as much as the control plant,and the other expressed specifically in GX mutant.In summary,compared with the control original plant,the expression levels of one transcription factor related to boron transport in GP mutant and two transcription factors related to zinc transport in GX mutant,as well as some key enzyme genes and glycosyltransferase gene SrUGT76G1 in their respective stevioside biosynthesis pathway,have significantly changed,which should be closely related to their boron tolerance,zinc preference and high RA glycoside content.In addition,some specific transcription factors have also been found in GP and GX mutants,which are presumed to be closely related to the changes in agronomic traits,stress resistance and other secondary metabolites content of GP and GX mutants.However,the specific roles of these candidate transcription factors in GP and GX mutants need further experiment to verify. |
