Study Of Seed Germination Promoting Factors With Inhibiting Damage And Bud Transcriptome For Astragalus Membranaceus Var. Mongholicus

Astragali Radix(Huangqi)is the dry roots of Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao(A.membranaceus var.mongholicus)or Astragalus membranaceus(Fisch.)Bge(A.membranaceus),which is one of the largest genus of seed plants and widely cultivated medicinal plants in China.However the two species Astragalus medicinal plants contain only a relatively few annual species,of which little is known with regard to seed dormancy and germination.A.membranaceus var.mongholicus is suited for high cold areas due to many forked roots and strong cold resistance.A.membranaceus var.mongholicus is relatively stable after introduced because of its excellent root types and strong drought resistance.Its planting in arid and semi-arid areas of Northwest China has increased in recently years.A.membranaceus var.mongholicus generally produces medicinal materials by seeds cultivation.Seeds nursed in the first year are transplanted in the second year,for medicinal material formation.However,the field emergence rate of seeds is less than 30%when sown direct in field,and has very low seedlings raising efficiency using the traditional methods.The seeds also have dormancy that necessitate increasing the sowing quantity.Research on seed dormancy mechanism and germination conditions of Astragalus medicinal plants is not indepth and systematic.This hampers its beneficial medicinal exploitation.The forked roots of A.membranaceus var.mongholicus is the reason for its poor appearance and processing quality,therefore decreases planting area year by year.At the same situation,native to Inner Mongolia of China,the A.membranaceus var.mongholicus was introduced to other ecological field,where it has excellent root type.At present,it has a large planting area and high benefit in the arid and semi-arid areas of Gansu Province.A.membranaceus var.mongholicus was therefore,taken as the research material in this study.A number of scarification methods were used to study the germination characteristics in order to explore effective ways to break the seed dormancy to improve germination quantity and quality.Hence,the effects of pre-treatments by sand rubbing,warm water soaking and incubation temperature to break the seed dormancy to enhance germination of the seeds of the medical plant and also study the effect of the pretreatments on antioxidant system,physiology and biochemistry,photosynthetic and morphological characteristics of the seedlings.Based on the above study,two genotypes of A.membranaceus var.mongholicus with green bud(GV)and purple bud(PV)were used.The seed dormancy-break and germination characteristics were investigated comparatively.Based on these,the transcriptomic differences of Astragalus mongholicus varieties with different bud color genotypes were analyzed in order to reveal the differences in seed dormancy and germination promoting factors and their transcriptional levels,so as to provide scientific and technical basis for the construction of the medicinal plant seedling base.The main results are as follows:1.The seeds pre-treatments by scrolling with sands for different times has a significant effect on the seed germination traits of A.membranaceus var.mongholicus.The degree of influence for germination quality varies with the varieties with different colour bud,which have different adaptability responsive to scrolling times.Seeds scrolling pre-treated for 10 mins can significantly improve seed emergence trait for GV,while the optimum pre-treatment time appeared in treatments 20 and 40 mins for purple variety(PV).In any case,sand rubbing for 10-50 min in a shaker all promoted the seed germination potential of the two Astragalus membranaceus var.mongholicus varieties in various degrees,improving uniformity of seed emergence.Sand rubbing has a good effect on breaking dormancy and inhibiting damage of A.membranaceus var.mongholicus seeds.2.Warm water soaking pretreatment was found to be the best and safer for the breaking of the dormancy of the A.membranaceus var.mongholicus seeds.The warm water pretreatment was applied to the seeds at different temperature levels including control(no treatment on the seed).In warm water treatment the seeds were soaked for 6 and 12 hours at the temperatures 30℃,40℃,50℃,60 oC and 70℃,respectively.The result showed that germination percentages did increase with warm water treatments as compared with the control.Scarified seeds germinated to nearly 80% at warm water temperature of 30℃ for 6hours for both genotypes.Seedling emergence was higher for GV seeds exposed to 30℃ for6 hours.Forty-three percent of the scarified seeds sown under laboratory conditions germinated within 10 days after sowing,and an additional 20% germinated in the additional5 days period.The seeds soaking pretreated at 30℃ and 40℃for 6 hours could significantly improve seed germination and seedling emergence characteristics for A.membranaceus var.mongholicus,however,when the soaking duration was extended to 12 h the best temperature of seed soaking pretreatment was only at 30℃.All the above study laid a good basis for improving efficiency of the medicinal plant standard cultivation.3.The seeds of Astragalus membranaceus var.mongholicus were germinated at constant temperature in an incubator to break the seeds dormancy.When the seeds of A.membranaceus var.mongholicus were placed at 5℃ and 10℃,the emergence rate,emergence potential and emergence index were low.When the culture temperature was more than 15℃,the emergence characteristics significantly improved.During the temperature extent of 15℃-30℃.The temperatures 15℃-30℃ are adaptive to the A.membranaceus var.mongholicus seed sowing and seedling cultivation.The temperature during the emergence of A.membranaceus var.mongholicus affects its emergence characteristics.The temperature conditions have a significant impact on the emergence quality of the plant.Etiolation was observed.The emergence quality was seriously affected when the temperature was 30℃ or higher.4.Comprehensive index evaluation for photosynthetic indicators revealed that the difference of photosynthetic characteristics of A.membranaceus var.mongholicus seedlings grown for 30 day after soaking seeds at different temperatures reached a significant level.The difference varied with varieties and soaking temperatures and duration.For green bud varieties,the photosynthetic characteristics of seedlings after soaking seeds at 6 h-40℃ were the best,followed by 6 h-50℃ and 12 h-30℃,while the best pretreatment of purple bud varieties was12 h-30℃,followed by 6 h-50℃ and 12 h-50℃.These show that varieties with different color bud have different physiological regulation mechanisms.5.The antioxidant enzymatic activities of the seedlings germinated by seed pretreatment at different temperatures for 6 h and 12 h also showed significant difference and the differences varied with the genotypes,GV and PV.Both temperature pretreatment and exposure time could induce increase antioxidant enzymes activities of superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)and ascorbate peroxidase(APX).Especially the activities of SOD and POD in seedlings were enhanced by 30℃ and 40℃ at exposure time of 6 h and12 h while CAT was not much affected.The two genotypes are recommended to grow under warm water soaking.This study further verified that the use of warm water to pretreat seeds to break dormancy of leguminous A.membranaceus var.mongholicus is an excellent method to improve plant density and reduce seed loss on the field as a results of dormancy.After soaking seeds in warm water at appropriate temperatures of 30℃-40℃,compared with the control the antioxidant enzyme activities of seedlings germinated from A.membranaceus var.mongholicus seeds were generally enhanced,and the enhanced degree varied with the treatment time and variety.The APX and POD activity of seedlings of A.membranaceus var.mongholicus with purple bud could be effectively enhanced after seed soaked at 30℃ for 6 h,while it was enhanced at 30℃ for 12 h for green variety.The CAT activity of the seedlings of A.membranaceus var.mongholicus with green bud could be effectively enhanced after seed soaked at 30℃ for 6 h and 12 h,while it was only enhanced at 30℃ for 12 h purple variety.The antioxidant capacity of green bud variety was stronger than that of the purple bud variety.6.After Illumina hiseq 2000 high-throughput sequencing,the corresponding q30 values of the three A.membranaceus var.mongholicus varieties with heterochromatic bud reached more than 94%,indicating that the quality of sequencing was better(no less than 20%),and the average proportions of GC of Ganqi 1,Ganqi 2 and Longqi 1 were 42.96%,42.94% and43.07% respectively.The sequence of differential gene expression was Ganqi No.1 > Ganqi No.2 > Longqi No.1.The number of differential genes between green bud Ganqi 1 and purple bud Ganqi 2 was 3011,while the number of differential genes between Ganqi 2 and mixed color bud variety Longqi 1 was 3940,while the number of differential genes between Ganqi1 and mixed color bud variety Longqi 1 was 4368.In summary,the suitable temperature for germination and emergence of A.membranaceus var.mongholicus seeds is 15℃-25℃.Rolling seeds mixed with sand and soaking seeds at appropriate temperature for a certain period of time can effectively improve the germination and seedling emergence characteristics of the plant seeds.Sand rubbing combined with seed soaking has a better effect on promoting germination and inhibiting damage.At the same time,it can protect the integrity of seed structure,improve the quality of seed germination and seedling emergence,and lay a high-yield foundation for stress resistance biochemistry and photosynthetic property of seedlings.The technology of breaking dormancy,inhibiting damage and promoting germination explored in this study is expected to solve the problem of low field emergence rate caused by the traditional sowing method of Astragalus medicinal plant seeds,improve the seedling raising effect,and provide a scientific basis for breaking the seed dormancy.The results provide a scientific and technical basis for the breeding of new varieties and the identification of germplasm resources.