Study Of Somatic Embryogenesis Induction And Mechanism For Ormosia Henryi Prain

Ormosia henryi Prain is a precious tree species in China and has great economic and ecological potential as a raw material for making high-grade furniture and handicrafts,which is an important tree species of Chinese traditional medicinal crops with its roots,branches and leaves.As the wild resources of O.henryi are scarce and easy to be destroyed,and scattered in villages or nature reserves,it is listed as the second class of state-protected tree species.There are some factors that restrict the sowing and propagation of O.henryi,such as animal feeding,deleterious alternate bearing phenomenon,the seeds with hard seed coat,poor water permeability,difficulty in absorbing water and instability of excellent traits.Therefore,it is the basis of solving the shortage of seed sources and cultivating high-quality seedlings to seek the propagation methods of excellent seedlings of O.henryi.Somatic embryogenesis(SE)is a more efficient method of asexual reproduction than organogenesis and enables large-scale breeding and commercialization via bioreactors,which is a characteristic of plant cells with high plasticity and totipotency and has great significance for asexual reproduction,germplasm conservation and genetic transformation.In order to establish SE system and explore its mechanism in O.henryi,the internal and external factors affecting SE were screened,their physiological biochemistry,histology,histochemistry and molecular biology were explored,and production methods of artificial seed preliminary were explored preliminarily.These results were as follow.1.SE was affected by both external and internal factors in O.henryi.In the external factors,EC induction rate was greater than 30%on B5 medium supplemented with 1.0 mg/L6-benzylaminopurine(BA)and 0.5 mg/L naphthaleneacetic acid(NAA)or 0.2 mg/L BA and 2.0mg/L 2,4-dichlorophenoxyacetic acid(2,4-D).EC induction rate was higher and its texture was better on B5 medium supplemented with 0.5 mg/L kinetin(KT)and 1.0 mg/L 2,4-D.Suspension culture was a better method for EC proliferation and SE induction.The medium supplemented with 30g/L sucrose and 0.5 g/L glutamine could promote the induction of EC and somatic embryos.In internal factors,EC induction rate and SE induction rate was highest in genotype GZGL(41.07%)and GZPT(18.4%)respectively.Among the different explants,mature zygotic embryo was suitable explant for SE induction in O.henryi.SE induction rate of immature embryos was best with the seeding collecting stage in early September.2.The germination rate of somatic embryo was the highest(54.81%)on B5 medium supplemented with 0.5 mg/L BA and 0.2 mg/L NAA.The number of cotyledon embryo(CE)was more on medium supplemented with 0.5 mg/L thidiazuron(TDZ)and 0.2 mg/L NAA.The root induction rate of CE was highest(92.5%)on B5 medium supplemented with 0.5 mg/L IBA and0.5 mg/L NAA.The vigorous seedlings were acclimated in the bottle for a week,and transplanted into the substrate(perlite:vermiculite:peat soil=1:1:1),their survival rate was 97.2%.The physical properties of artificial seed capsules were affected by the manufacture method and additives.In the manufacture method of artificial seed capsules,the shape of artificial seed capsule of double-layer embedding method was complete,and its physical properties and comprehensive evaluation scores were better than that of the single layer embedding method.The physical properties of artificial seed capsules were best with 20g/L water-retaining agent.The germination rate of artificial seed in O.henryi was highest(96%)on capsules supplemented with B5 medium,1.0 mg/L 6-BA,0.1 mg/L NAA and 1.0 mg/L GA3;however,the germination rate of artificial seeds decreased gradually with the increase of storage time at 4?.3.Histology and scanning electron microscopy of SE in O.henryi were observed that mature embryo dedifferentiated into EC,and EC developed into globular,heart-shaped,torpedo-shaped and cotyledon stages,this process was similar to zygotic embryo.Compared with EC,the abnormal callus showed different cell structure and surface structure,such as NEC cells were larger and its surface structure was wrinkled.BC cells existed a fracture phenomena,SC in outer cells were covered with a layer of elongated tissue.Histochemical observation showed that starch granules staining gradually turned light with SE development,however,protein granules staining dark;compared with EC,starch and protein granules staining were darker in SC,and lighter in NEC and BC.4.The biochemical events at different SE stages showed that the contents of soluble sugar,starch,the activities of PPO,SOD,APX,POD,the ratios of indole-3-acetic acid/abscisic acid(IAA/ABA),IAA/gibberellins(IAA/GAs),auxin/GAs(AUX/GAs),AUX/ABA decreased gradually with SE development(EC-GE-CE);in contrary,the contents of soluble protein,H₂O₂,all kinds of endogenous hormones increased gradually;however,CAT activity and the ratios of IAA/cytokinins(IAA/CKs),AUX/CKs,ABA/CKs,GAs/CKs increased first and then decreased.The high contents of GAs,ABA,the high ratios of ABA/CKs,GAs/CKs and the low ratios of IAA/ABA,IAA/GAs,AUX/GAs,AUX/ABA were the reasons why callus could not form EC.The low enzyme activities,the low contents of energy substances and H₂O₂were related to the NEC formation;the high contents of soluble sugar,H₂O₂,AUX,CKs and PPO activity and the low conent of soluble protein were the basic reasons for BC formation;the high energy substances contents and the low activities of SOD,POD were easy to form SC.5.There were 38100 differentially expressed genes of RNA-seq during the four stages of SE in O.henryi.The number of differentially expressed genes in NEC vs EC,EC vs GE and GE vs CE was 11589,8999 and 27982,respectively.Among them,plant hormone signal transduction pathways were significantly enriched in different SE stages.In this study,the differential genes of auxin,cytokinin,gibberellin and abscisic acid in plant hormone signal transduction pathway and biosynthesis pathway were analyzed.The down-regulation of AUX/IAA,ARF,CRE1,GID1,DELLA,PYL KAO,GA3OX,ABA2,AAO3 genes were beneficial to EC induction.The up regulation of AUX/IAA,ARF,CRE1,B-ARR,GID1,DELLA,PYL,ABF,CPS,KAO,GA20OX,GA3OX,VED genes promoted somatic embryo maturation and differentiation in O.henryi.