| Pinus koraiensis Sieb.et Zucc.,as the dominant species in mixed forest of Korean pine and broad-leaved tree species,is a precious native tree species that is extremely important high-quality timber forests and nut-production forest in the mountain areas of northeastern China.In this study,we optimized the Pinus koraiensis somatic embryogenesis system by screening and establishing the cryopreservation scheme of embryogenic callus,explored the morphology and physiology of different type calli and different developmental stage embryos of Pinus koraiensis to explain the relationship between the Korean pine somatic embryogenesis and the exogenous substances,and to reveal the morphological and physiological differences between different callus types.The main results are as follows:(1)During the induction process of the embryonic callus of Pinus koraiensis,the explant source stock trees significantly affected the induction rate of embryogenic callus.Among the 28 stock trees only 10 were successfully regenerated embryogenic callus,and the highest induction rate could be up to 22%,but just 2%for the lowest This means that the genotype may play a decisive role in the induction of embryogenic callus.(2)For maturation culture of Pinus koraiensis somatic embryos,the best culture method was dispersion culture(91·g-1FW callus);the best maturation medium is mLV?80?mol L-1 ABA+1.2%gellan gum+0.2 mol·L-1 sucrose;optimal cell density is 50 mg callus per dish.Among them,0.2 mol·L-1 sucrose treatment had the greatest influence on the number of somatic embryogenesis(9.3 times),and 1.2%gellan gum treatment had the second effect on promoting the number of somatic embryogenesis(7 times).The number of somatic embryogenesis was minimal(2.1 times).In addition,the number of somatic embryos had a strong genotype dependence on the maturation culture mode,medium type,ABA concentration and gellan gum concentration.At the same time,embryogenic callus was found to gradually lose its potential for somatic embryogenesis after 6 months of subculture.The results indicated that the genotype and physiological state of embryogenic callus were closely related to the ability of somatic embryogenesis,and certain exogenous substances could promote somatic embryogenesis.(3)During the maturation culture of Pinus koraiensis somatic embryos,the proembryony stage to the early single embryo was from the 0th to the 7th day,the period of early single embryo developing to the early stage of early single embryo was from the 7th to the 14th day,the period of thearly stage of early single embryo to late single embryo was from the 14th to the 21th day,the period of late single embryo developing to early cotyledonary embryo was from the 21th to the 28th day,the period of early cotyledonary embryo developing to middle cotyledonary embryo was from the 28th to the 35th day,the period of middle cotyledonary embryo developing to maturation cotyledonary embryo was from the 35th to the 60th day.During this period,the fresh weight and dry weight ratio increased with time,the water content decreased gradually,and decreased to the cotyledonary embryo stage;the soluble protein content reached the highest at 28 d(15.68 mg·mg-1 FW callus),soluble sugar content reached the highest at 14 d(10.40 mg mg-1 FW callus);starch content reached the highest at 35 d(12.51 mg mg-1 FW callus);The development of somatic embryos was bimodal,with accumulation peaks at 7 d and 28 d;POD and CAT activities were unimodal,reaching a cumulative peak at 14 d.It is indicated that the process of somatic embryogenesis is a process of energy storage,and the interaction of antioxidant enzymes regulated the occurrence and development of somatic embryos.(4)Embryogenic callus had larger nuclei than that of non-embryonic callus and callus that lost the potential for somatic embryogenesis,and the cells were arranged more closely to form a lump.Among them,embryogenic callus cells had high levels of soluble protein(3.98 mg g-1 FW callus),soluble sugar(2.54 mg·g-1 FW callus),starch(16.29 mg·g-1 FW callus)and proline(0.18 mg·g-1 FW callus);low level of polyphenol content(0.19 mg·g-1 FW callus),polyphenol oxidase(4.06 mol·min-1·g-1 FW callus),SOD(70.86 U·g-1·FW callus)and POD(0.05 U·mg-·FW callus·min-1)activity.It showed that larger nuclei and tight cell arrangement and high content of storage materials,low levels of polyphenols,polyphenol oxidase and antioxidant enzymes could be used as morphological and physiological markers of somatic embryogenic potential callus.(5)Pre-culture conditions,cryogen concentration and thawing temperature significantly affected the cell survival rate during the cryopreservation of Pinus koraiensis embryogenic callus.The optimal cryopreservation conditions were:0.4 mol·L-1 sorbitol solution in 18 h of incubation,10%DMSO cryoprotectant.After 14 days of resuscitation culture,the callus survival rate reached 90%.In conclusion,the results of this study further confirmed that the genotype played a decisive role in the induction of embryogenic callus and the maturation of somatic embryos,and the physiological state of the callus also significantly affected the potential of somatic embryos.Morphological changes during embryogenesis were closely related to physiological changes.These findings had important theoretical significance for accelerating the breeding and rapid and large scale propagation of Pinus koraiensis and understanding the molecular mechanism of Pinus koraiensis somatic embryogenesis. |
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