Study On The Mechanism Of Seed Dormancy Release And Germination In Relict Plants Eucommia Ulmoides And Bretschneidera Sinensis

Seed dormancy has been one of research hotspots in seed biology.Eucommia ulmoides Oliver belongs to Eucommia,Eucommiaceae and Bretschneidera sinensis Hemsl.belongs to Bretschneidera,Bretschneideraceae.They are both special rare relict plants in China,which are characteristic of single seed,monotypic genus.Their seeds have dormancy,but the dormancy reason and release mechanism are still unknown.Due to the unique phylogenic characteristics and application values of these two plants,reasons of seed dormancy and release mechanism of fresh mature seeds（natural spreading unit）of Eucommia ulmoides Oliver and Bretschneidera sinensis Hemsl.were elaborated through cold/warm stratification processing,pericarp scarification,after-ripening and hormone processing.The dormancy breaking schemes which are applicable to production were established.Seeds of these two plants at different time were processed by cold stratification processing at 5°C.The dormancy release and germination mechanisms（mainly Eucommia ulmoides Oliver）were analyzed preliminarily through methods of cell morphology,physiology and biochemistry,metabolomics and transcriptome.Some major conclusions are introduced as follows:（1）Fresh mature seeds of Eucommia ulmoides Oliver have superficial physiological dormancy and Type 3 superficial physiological dormancy,which is known as the composite dormancy.Seed dormancy is caused by restriction of pericarp in water absorption of embryo,inadequate growth potentials of embryo and existence of germination inhibitors.Fresh mature seeds of Eucommia ulmoides Oliver has a low germination rate（only 13.3%）under 10°C,but embryo in vitro has a high germination rate（>95%）under several temperatures.Pericarp inhibits water absorption of embryo significantly.According to cytological microscopic observation,pericarp of fresh mature seeds is rich of pectin which degrades gradually in the cold stratification processing.The GC-MS analysis and germination test results imply that phenolic substances in pericarp are very likely to be the inhibitor of germination.Pericarp scarification,after-ripening,cold/warm wet stratification and seed immersion in GA₃all can promote dormancy release and seed germination.Among them,the cold wet stratification under 5°C presents the best dormancy release effect.The final average germination rate of seeds at 90d under 10°C and alternate illumination conditions reaches as high as（93.7±0.3）%.Based on the above study,the seed dormancy release strategy of Eucommia ulmoides Oliver is proposed as follows.Seeds are collected in the last ten-day of November every year,screened by flotation and then dried in dark.Next,seeds are kept dry under room temperature for 1 month and then stored in sands outdoor for 3 months in winter.Seeds can be sowed directly in early spring of next year.（2）Seed dormancy release of Eucommia ulmoides Oliver is very likely to be regulated by endogenous plant hormones metabolism and anti-oxidative metabolism pathway,while energy metabolism mainly acts on the germination process after dormancy release.ABA and IAA drop quickly at 30d of cold stratification,while GA₃and c ZR contents increase significantly.This indicates that ABA and IAA are very likely to play an important role in maintaining seed dormancy,while GA₃and c ZR are very likely to promote germination.According to the data of transcriptome,a total of 241 differentially expressed genes（DEGs）are enriched to metabolism pathways of ABA,GA,IAA and CTK in the cold stratification process.The key genes of metabolism pathway of ABA are ZEP,NCED,and CYP707A.The key genes of metabolism pathway of GA are KO,CPS,and GA20ox1.The key genes of metabolism pathway of IAA are ALDH,and DAO.The key genes of metabolism pathway of CTK are UGT and IPT.Expressions of all these key genes are up-regulated（or down-regulated）significantly in dormancy release and germination.As the cold stratification processing continues,contents of superoxide anion,H₂O₂and MDA contents present a variation trend of“decreasing firstly and then increasing”,while contents of As A,GSH and GSSG decrease significantly at 30d of stratification.The activities of SOD,DHAR and GR also decrease gradually,while the activity of CAT increases continuously.These demonstrate that a lot of reactive oxygen substances（ROS）are produced in dormancy release and germination process of Eucommia ulmoides Oliver seeds,accompanied with lipid peroxidation.Moreover,CAT is very likely the major remover of excessive ROS.According to the data of transcriptome,162 DEGs are enriched to metabolism pathways of peroxidases,phenylpropane and As A-GSH.Expressions of POD in the metabolism pathway of peroxidase,PAL,4CL,CAD and CCR in the metabolism pathway of dimethylmetane,GST,DHAR and APX in the metabolism pathway of As A-GSH all upregulate significantly during stratification.As the cold stratification processing continues,differential expressions of glycometabolism-related genes are very significant,which mainly involve metabolism pathways of starch and sucrose metabolism,EMP,PPP,TCA and oxidative phosphorylation.A total of 205,48,30,79 and112 DEGS have been enriched.Expressions of key genes,such as SUS,Inv,β-AMY andα-AMY in the metabolism pathways of starch and sucrose,PK,HK and pfka of the EMP pathway,G6PD and PGLS of the PPP pathway,CS and OGDH of TCA pathway as well as ATPase of the oxidative phosphorylation pathway,are all upregulated significantly in the cold stratification.（3）Fresh mature Bretschneidera sinensis Hemsl.seeds have superficial physiological dormancy and cold stratification under 5°C can break dormancy effectively.The dormancy release also involves significant changes in hormones,anti-oxidation and energy metabolism,indicating that dormancy release and germination are also very likely to be regulated by these ways.As the cold stratification processing continues,content of ABA in embryo decreases continuously,while contents of GA₃,IAA and SA“increase firstly and then decrease”.The activities of SOD,POD,CAT and APX as well as contents of soluble sugar also present the variation trend of“increasing firstly and then decreasing”.However,contents of MDH and G-6-PDH present the opposite variation trend.In a word,although seed dormancy of two rare relic plants has different reasons,the dormancy release and germination both involve endogenous hormones,antioxidation and energy metabolism.The classical hormone regulation theory of seed dormancy&germination and the embryo germination stress hypothesis are further proved by expressions of physiology and gene expressions.The research conclusions in this study can provide theoretical references to forestry production and species protection.