| Cinnamomum camphora L.has high ornamental value,practical value and cultural value.However,as a subtropical tree,camphor tree has poor adaptability to low temperature.Increasing the cold resistance of the camphor can not only enrich the landscape trees in northern China,but also make it better to exert the value of landscaping and make it more robust to grow and reduce economic losses,and better play the advantages of economic tree species.The most effective way to improve the cold resistance of C.camphora is to breed new cold-resistant varieties,and the excavation of cold-resistant genes is the key to cold-resistant breeding.In the early stage of the laboratory,four CcCBF genes were isolated from the camphor.In order to study the function of CcCBFs genes in camphor.However,due to the particularity of the woody plants,the genetic transformation and regeneration cycle are long.It is impractical to study the function of CcCBFs by obtaining transgenic plants.Therefore,it is great significance to explore rapid and accurate gene function research materials.The embryogenic callus of C.camphora is solid in texture and has a tender yellow spherical granule.It is composed of cells of equal diameter.Its protoplasts have strong activity and possess embryogenic ability form somatic embryos.Early studies have found that the use of C.camphora embryogenic callus as a receptor can achieve better transformation results.Therefore,the C.camphora embryogenic callus may be a good material for genes function verification.In this study,the tissue culture technique was used to optimize the embryogenic callus-induced and proliferation system.Then,the embryogenic callus was used as a test material to analyze its ability to sense low temperature stress from the molecular and tissue levels.Under the strongest moments and physiological indexes of low temperature stress,the cold resistance identification system of the embryogenic callus was established in order to identify the cold resistance of different transgenic materials.The main resultsof this study are as follows:1.Optimization of Embryogenic Callus Induction and Proliferation for camphor treeInduction of C.camphora somatic embryos: The immature fruits of the camphor were used as the explants,and the effects of explant disinfection method,explant sampling period,plant growth regulator combination and hydrolyzed casein concentration on the induction of C.camphora somatic embryo were investigated.The results showed that the disinfection effect was better when 1% sodium hypochlorite was disinfected for 25 min.The induction rate of somatic embryos of fresh explants harvested in late July was higher than that of early and mid-August.Plant growth regulator 6-BA and 2,4-D(1.0 mg/L 6-BA+0.1 mg/L 2,4-D)was beneficial to the induction of C.camphora somatic embryos;hydrolysis casein added has a promoting effect to somatic embryos the induction.When the concentration of hydrolyzed casein was 750 mg/L,the somatic embryo induction rate was relatively high.The suitable induction formula obtained in this study was D4:MS+1.0 mg/L 6-BA+0.1 mg/L 2,4-D+0.8% agar+3%sucrose+750 mg/L hydrolyzed casein,somatic embryo the induction rate was 63.3%.Proliferation of C.camphora somatic embryos: The induced somatic embryos were subsequent preservation in fresh D4(reserved once every 2 weeks)to maintain high proliferation efficiency,and the somatic embryos obtained by proliferation have strong vitality.A total of three somatic embryo clones were screened in this study,named SE1,SE3 and SE5.Induction of C.camphora embryogenic callus: The somatic embryos of SE3 with the same source obtained by proliferation was used as a test material to explore the effects of plant growth regulator combination and sucrose concentration on the induction of embryogenic callus.The results showed that when the ratio of 2,4-D to 6-BA was 8(4.0 mg/L 2,4-D+0.5 mg/L 6-BA),the induction rate of embryogenic callus was relatively high at 18.3%,followed by a ratio of 2,4-D to 6-BA of 12(6.0 mg/L 2,4-D+0.5mg/L 6-BA),the induction rate was 13.3%;when the sucrose concentration was 5% The induction rate of embryogenic callus was 28.3%,and the embryogenic callus was moreviable and showed a tender yellow color.It can be seen that 2,4-D plays a leading role in the induction process of the embryogenic callus.The suitable sucrose concentration was beneficial to the adjustment of the embryogenic callus state.In this study,the suitable embryogenic callus induction formula was F3:MS+0.5 mg/L 6-BA+4.0 mg/L 2,4-D+2.0mg/L Vc+0.8% agar+5% sucrose+1000 mg/L hydrolyzed casein,the induction rate was28.3 %.Proliferation of C.camphora embryogenic callus: The induced embryogenic callus was used as a test material to explore the effects of plant growth regulator combination,hydrolyzed casein concentration and subculture frequency on the proliferation of embryogenic callus.The results showed that with the increase of 6-BA and 2,4-D mass concentration,the growth rate of the embryogenic callus increased first and then decreased,and the plant growth regulator was 1.5 mg/L 6-BA and 0.15 mg/L 2,4-D combined,a peak occurs,and the proliferation rate of the embryogenic callus was relatively high.The addition of hydrolyzed casein utilizes the proliferation of the embryogenic callus;The generation frequency was conducive to the preservation and proliferation of the embryogenic callus,and its vigor and proliferation ability were strong every 7 days.The suitable proliferative formula obtained in this study was J5: MS + 1.5mg / L 6-BA + 0.15 mg / L 2,4-D + 0.8% agar + 3% sucrose + 1000 mg / L hydrolyzed casein;Replace the subculture formula once every 7 days.In addition,four embryogenic callus clones were screened and named as EC1,EC3,EC5 and EC8.2.Establishment of Cold Resistance Identification System for C.camphora Embryogenic CallusThe EC5 embryogenic callus of C.camphora were used as test material,and CcCBFs expression at low temperature(4°C)were analyzed by real-time PCR.The results showed that the relative expression of CcCBFs was significantly higher than that of the control(0 h)at 1 h after stress treatment.The relative expression of CcCBFs increased first and then decreased with the stress time,and the relative expression increased significantly after 24 h of stress treatment.The physiological and biochemical indexes of the EC5 embryogenic callus of C.camphora under low temperature stress at 4°C were determined.The results showed that with the prolongation of stress time the content of proline and soluble sugar,firstly increased and then decreased.The peaks appeared at 8 h and 2 h.The content of malondialdehyde decreased firstly and then increased with the increase of stress duration.At the 12 h of stress,the peaks appeared.The activities of SOD and CAT firstly increased and then decreased with the prolongation of stress time.The trend was peaked at 12 h and 8 h after stress treatment.The results showed that the embryogenic callus of C.camphora could perceive 4°C low temperature stress at the cellular level.The physiological and biochemical indexes of the EC5 embryogenic callus of C.camphora under the low temperature stress at-4°C were determined.The results showed that the contents of proline and soluble sugar firstly increased and then decreased with the prolongation of stress time.The peaks appeared at 8 h and 2 h.The content of malondialdehyde decreased first and then increased with the increase of stress duration.The low peak appeared at 2 h after stress.The activity of CAT and POD firstly increased and then decreased with the prolongation of stress time.The trend peaked at 8 h and 2 h after stress treatment.The results showed that the embryogenic callus of C.camphora could perceive-4°C low temperature stress at the cellular level.In this study,the cold resistance evaluation method of the C.camphora embryogenic callus was established.When the content of proline and soluble sugar was relatively high,the activities of SOD,CAT and POD were relatively strong,and when the MDA content was relatively low,the corresponding toona the cold resistance of embryogenic callus was relatively strong;on the contrary,it was relatively weak.This study not only confirmed that embryogenic callus could be used as a good material to identify the cold resistance of C.camphora,but also revealed the expression pattern and physiological and biochemical changes of the C.camphora embryogenic callus under low temperature stress.The material responds to physiological indicators and the most violent moments that are sensitive to low temperature stress.The cold resistance identification indexes obtained by screening at 4°C mainly included: soluble sugar,proline,SOD,malondialdehyde and CAT,and theircorresponding research value moments were: 2 h,8 h,8 h,12 h,12 h.The cold resistance identification indexes obtained by screening at-4°C mainly included: soluble sugar,malondialdehyde,POD,proline and CAT,and their corresponding research value times were: 2 h,2 h,2 h,8 h,8 h. |
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