| Somatic embryogenesis(SE)is the developmental reconstruction process of somatic cells from somatic cells to embryoids under certain hormone or stress conditions,which is the best interpretation of totipotency of plant cells.It has broad application prospect and great economic value in many fields such as large-scale propagation,artificial seed preparation,germplasm conservation,genetic transformation and genetic engineering.Sweetgum(Liquidambar spp.)is one of the most important tree species in the world.Liquidambar styraciflua,as important as eucalyptus and poplar,is a commercially fast-growing wood species.L.formosana is widely distributed in China and has strong ecological adaptability.The hybrid sweetgum(L.formosana × L.styraciflua)has both characteristics and obvious heterosis.At present,the SE of hybrid sweetgum still has some problems,such as low somatic embryogenesis rate and asynchronous development,which need to be optimized and improved.Moreover,the physiological and molecular mechanisms of hybrid sweetgum SE remain unclear.Hence,based on previous work,this paper aims to establish an efficient SE system,optimize the conditions at each stage of SE,and significantly improve the somatic embryogenesis efficiency.Combined with transcriptome and physiological and biochemical studies,the mechanism of SE was analyzed while improving the SE system.The results are as follows:(1)Induction stage: Immature zygotic embryos of 8 cross combinations of hybrid sweetgum were used as explants to study the induction stage of SE.The results showed that the initiation frequencies of No.3,4 and 7 was over 75%.In addition,different types of tissue(torpedo embryos,cotyledons and roots)of cell line SF15SH-5a were used as explants to study the reinduction of embryogenic callus.The results showed that torpedo embryo as explants,modified Blaydes medium as basic medium,2,4-D 1.0 mg/L+6-BA 0.5 mg/L as hormone concentration of embryogenic callus induction rate was the highest,up to95%,and the state of callus was the best.Proliferation stage: SF15SH-5a cell line was used as material to establish a stable and efficient suspension culture system.The optimal conditions were as follows:inoculation dose 1.0 g,subculture cell density 1/10,sucrose,2,4-D 1.0 mg/L,6-BA 0.5 mg/L,and the maximum multiplication factor was 7.85.Somatic embryo maturation stage: SF15SH-5a cell line was used as material to study the effects of phytagel,agar and activated carbon on somatic embryo maturation,and the optimal concentration of phytagel 6 g/L,agar 8 g/L and activated carbon 1 g/L was obtained by comprehensive balance method,and the maximum number of somatic embryos reached 946 per gram of fresh weight.In addition,SF15SH-5a cell line was used as the material,and synchronous development of hybrid sweetgum somatic embryos was established in combination with the above optimal culture conditions and the method of fractional sieving and filtration.The results showed that the maximum maturation efficiency of somatic embryos reached 2603.33 per gram of fresh weight after 40 mesh-sizesieve,and the synchronization rate of torpedo-shaped embryos was 77.8%.Germination stage: the effect of basic medium type and osmotic pressure on somatic embryo germination was studied.The results showed that the best germination medium was WPM basic medium +3g/L agar +2g/L phytagel,with the highest germination rate of 94 %,and the survival rate of plantlet conversion was 84%.(2)The suitable cryopreservation conditions for embryogenic callus and somatic embryos of hybrid sweetgum were explored using the callus subcultured for 7-10 days and torpedo embryo of SF15SH-5a cell line.Programmed cooling cryopreservation method was used to study the effects of cryoprotectant concentration and preculture time on the cryopreservation effect of embryonic callus.The results showed that the cell survival rate was 44.90% and the recovery growth rate was 76.67% when 0.5 M sorbitol and10% DMSO were used as the combined cryopreservation agent at 4℃ for 3 days.The effects of loading time,dehydration time and preculture time on the freezing effect of torpedo embryos were studied by vitrification cryopreservation method.The results showed that the survival rate of torpedo embryos was43% when loaded for 80 min,dehydrated for 60 min and precultured for 0 d,and the induction rate of surviving torpedo embryos was more than 90%.Therefore,the establishment of these two systems not only ensured the long-term preservation of embryogenic ability of hybrid sweetgum embryogenic callus,but also enriched the selection of materials for cryopreservation.(3)Using the SF15SH-5a cell line as the material,the cytological observation of the whole process of hybrid sweetgum somatic embryogenesis was carried out.The results showed that the embryogenic callus of hybrid sweetgum was directly differentiated from cortical cells or cambium cells,and a small part was transformed from non-embryonic callus;The somatic embryos of hybrid sweetgum have the characteristic of single embryogenic cells,which were in exterior of embryogenic calli in most cases and in inner of embryogenic calli in a few cases;Somatic embryo development is similar to zygotic embryo development,and both develop from problastoid mass to globular,heart-shaped,torpedo and mature cotyledonary embryo stages.The developmental process of hybrid sweetgum somatic embryos is the process of establishing polarity.The internal equal diameter cells and pre-cambial cells of spherical embryos elongate axially,and are accompanied by the decline of embryonic stalk cells.These are important factors for the transition from spherical embryos to heart-shaped embryos.The formation of torpedo embryo was accompanied by the gradual formation of bud apex meristem,root apex meristem and vascular bundle,then bud and leaf primordium torpedo embryo apex meristem began to form,and vascular bundle formed in mature cotyledon embryo,and presented a V-shaped distribution.Abnormal embryos had abnormal vascular tissue growth and loss of polarity,which resulted in somatic embryo development stagnation or variation.This indicated that the development of hybrid sweetgum somatic embryos is the process of polarity establishment,and the successful establishment of polarity is the premise of normal germination of somatic embryos.(4)Soluble sugar,starch and soluble protein content and SOD activity in somatic embryogenesis of SF15SH-5a cell line were measured.The study showed that embryonic callus soluble sugar content was the highest,and decreased gradually with the development of somatic embryos,while the soluble sugar content in cotyledons was the lowest,and then gradually increased;Starch content was highest in early somatic embryo and lowest in cotyledon embryo.The contents of soluble protein in torpedo embryo were the highest.These three kinds of contents all showed the trend of increasing first and then decreasing and then increasing.SOD activity increased gradually from embryogenic callus to mature somatic embryo seedlings,and SOD activity was the lowest in non-embryogenic callus.The results indicated that the somatic embryo of the hybrid sweetgum was a process of continuous energy consumption and material accumulation.The contents of soluble sugar starch and total protein in the normal somatic embryo of the hybrid sweetgum were higher than those in the vitrified somatic embryo.Compared with normal cotyledon embryos,hybrid sweetgum malformed cotyledon embryos have lower soluble sugar content and higher starch and total protein content.These results indicate that the occurrence of vitrified somatic embryos and the formation of malformed seedlings are related to the difference of physiological and biochemical characteristics.(5)Transcriptomic studies on induction phase and morphogenesis of hybrid sweetgum were carried out using embryonic cell line cultures and roots,stems and leaves of somatic embryo seedlings as materials.Comparative transcriptome analysis showed that 19,957 genes were differentially expressed in10 pairs of SE.Genes related to plant hormone signal transduction,especially auxin and cytokinin signal transduction components,were significantly enriched.HB-WOX,B3-ARF,AP2/ERF,GRFs and other transcription factors were identified by K-means.In combination with K-means and weighted gene coexpression network analysis(WGCNA)algorithm,the co-expression modules related to somatic embryo development,embryogenesis and non-embryogenesis were identified,and a number of new somatic embryogenesis genes,including AIL1,AIL5,ARF3 and MYB36,were discovered.These molecular marker genes may be the key factors determining SE potential of hybrid sweetgum.It needs further study and discussion in the future.The results will lay a foundation for the large-scale asexual propagation and further genetic improvement of the hybrid sweetgum.Meanwhile,the essence of SE of the hybrid sweetgum will be revealed from the physiological,biochemical and transcriptome levels,which will provide important theoretical guidance for the better application of SE technology in forestry production. |
