| Picea pungens var.glauca is native to North America,with beautiful tree shape and beautiful blue leaves of needles all year round.In recent years,the market demand for Picea pungens var.glauca seedlings has gradually increased.However,there are problems such as unstable progeny traits in the propagation of Picea pungens var.glauca seeds,which limit the quality of seedlings and are not conducive to large-scale production.Somatic embryogenesis technology is an efficient asexual reproduction technology,and has important research value for the propagation of Picea pungens var.glauca,which uses the same seed to generate a large number of embryogenic clones,and proliferates a large number of plants with the same genotype in a short time,ensuring the same genetic traits of the plants.In this study,mature zygotic embryos of Picea pungens var.glauca were used as explants to optimize the key technologies in somatic embryogenesis of Picea pungens var.glauca,including EC induction,EC proliferation and maintenance,SE maturation,SE germination,plant regeneration and EC cryopreservation technology;and using 4 cell lines with different SE differentiation abilities as test materials to conduct SE maturation experiments.In terms of cytological morphology,storage substances,antioxidant enzymes and endogenous hormones,this study revealed the dynamic physiological changes and differences of different somatic embryogenesis cell lines during somatic embryogenesis,and found out the key factors leading to the differences of callus SE differentiation capabilities.The main results are as follows:(1)In the process of EC induction culture of Picea pungens var.glauca,the optimal concentration of 2,4-D was 4.0 mg/L,the optimal concentration of 6-BA was 2.0 mg/L,the optimal concentration of sucrose was 10 g/L,and the highest EC induction percentage was57.16 %.(2)In the process of EC proliferation and maintenance culture of Picea pungens var.glauca,the optimal 2,4-D concentration range was 1.0-3.0 mg/L,the optimal 6-BA concentration range was 0.5-1.0 mg/L,and the optimal sucrose concentration was 10 g/L.Conditions in the proliferation stage of EC have an effect on both the cellularity of the callus and the subsequent maturation of SEs.Culture conditions with the best proliferation percentage may not be able to improve the yield of SEs and need to be considered comprehensively.(3)In the process of SE differentiation culture of Picea pungens var.glauca,the optimal basic medium is DCR medium,the optimal ABA concentration is 10.0 mg/L,the optimal carbon source and concentration is 30 g/L sucrose,the optimal Gelrite concentration is 4-6 g/L,and the optimal cell density is 50-80 mg per dish.(4)In the process of EC cryopreservation of Picea pungens var.glauca,the optimal cryopreservation conditions were: pretreating in m LV liquid medium with 0.4 mol/L sorbitol for24 hours,and then adding 5 % DMSO.The resuscitated EC obtained the highest SEs yield of1030 ± 164 /g FW callus through SE differentiation culture.(5)Cytological observation of 4 callus of Picea pungens var.glauca with different somatic embryogenic ability showed that the cell line without somatic embryogenic ability lacked early SEs,which may be the reason why it could not transfer from EC proliferation to SE differentiation stage.(6)In the process of SE differentiation,the physiological and biochemical changes of cell lines with different somatic embryogenic abilities are significantly different.The accumulation of MDA,low SOD activity in cells,and serious damage to cells by ROS lead to the decline of somatic embryogenesis ability and even the loss of somatic embryogenesis ability.Cell lines with somatic embryogenesis have active cell metabolism and significant changes in enzyme activity at different culture time.Cell line without somatic embryogenesis has no SE differentiation,and its enzyme activities gradually decreased or did not change significantly with culture time.SOD,POD activity and MDA content can be used as physiological markers for early identification of somatic embryogenesis.The turning points of TP content and POD and CAT activities can be used as physiological markers at different stages of SE differentiation.(7)In the process of SE differentiation,the changes of endogenous hormone contents among the cell lines with different somatic embryogenic abilities were significantly different.Endogenous ABA and IAA have high levels and play a key role in the development of SEs.ABA is the initiator of embryo differentiation,and IAA promotes the development of early embryos.ZR promotes cell division and increases the number of SEs produced.The content of endogenous NAA and GA is lower,but it still has a certain promoting effect on SE development.During the development and maturation of SEs,the changes of endogenous hormones in the cell lines without somatic embryogenesis were not obvious,and the changes of endogenous hormones in the cell lines with good somatic embryogenesis ability generally showed a trend of first increase and then decrease,and the changes of endogenous hormones in the cell lines with moderate or poor somatic embryogenesis abilities generally showed a gradual upward trend. |
