Establishment Of Genetic Transformation System Using Embryogenic Callus Of Fraxinus Mandshurica

Fraxinus mandshurica is a deciduous tree of Fraxinus in Oleaceae.Because of its strong adaptability,drought resistance,cold resistance,smoke resistance and pest resistance,it is a precious afforestation and greening tree species widely used in Northeast China.Traditional forest tree breeding methods have the disadvantages of long cycle,low breeding efficiency and slow effect.As a common forest tree genetic breeding method,plant somatic embryogenesis can accelerate the reproduction speed of trees and effectively shorten the breeding cycle.Agrobacterium-mediated genetic transformation has been widely used as an important means of genetic improvement,but the low transformation efficiency is still an urgent problem to be solved in the process of genetic transformation of Fraxinus mandshurica.In this study,the embryonic callus of Fraxinus mandshurica was used as the transformation material to carry out genetic transformation research,aiming at improving the genetic transformation efficiency of Fraxinus mandshurica.The main results are as follows:1.Optimization of embryogenic callus proliferation and transition culture conditions of Fraxinus mandshurica.The results showed that the optimal medium for the proliferation of embryogenic callus was WPM+0.15 mg·L^-1 NAA+0.1 mg·L^-1 6-BA+20 g·L^-1 sucrose+400mg·L^-1 CH+3.5 g·L^-1 gellan gum.When the culture period was 15 d,the proliferation rate could reach 212%,and the embryogenic callus was in good condition without browning.The proliferation rate of embryogenic callus in liquid culture was much higher than that in solid culture,but the amount of somatic embryogenesis in solid culture was significantly higher than that in liquid culture.The addition of 60 g·L^-1 PEG₄₀₀₀ in somatic embryo induction can increase the amount of somatic embryos.2.Genetic transformation of embryonic callus of Fraxinus mandshurica.The results showed that embryogenic callus was extremely insensitive to Kan,and 10 mg·L^-1 Hyg could significantly inhibit the proliferation of embryogenic callus.The concentration of infection solution,the addition method of acetosyringone and the co-culture time had significant effects on the transformation efficiency.At the same time,it was found that the somatic embryogenesis ability of different transformed cell lines was different.Among them,the somatic embryogenesis rate of cell line Line 7 was relatively high,and no somatic embryogenesis was found in cell line Line 3.The germination medium was 1/2 MS+0.2 mg·L^-1 6-BA+5.0 mg·L^-1 GA.3.GUS histochemical staining and PCR molecular detection of resistant callus.The results showed that the transformed embryogenic callus changed from light yellow to dark blue after GUS staining,and the positive rate was as high as 88.23%.Nine embryogenic calluses with positive GUS staining were randomly selected for PCR molecular detection,and it was found that all of them contained the target band.Subsequently,the Lob HLH34 gene of Larix olgensis was transferred into the embryogenic callus of Fraxinus mandshurica,which verified the stability and efficiency of the system.4.Physiological difference analysis of transformants with different somatic embryogenesis potential.The results showed that the contents of starch and total sugar in CK and Line 7 were significantly higher than those in Line 3 at the same culture time,and the accumulation of starch and sugar might promote the somatic embryogenesis of Fraxinus mandshurica.The CAT activity of Line 3 and CK showed an upward trend,while the CAT activity of Line 7 showed a trend of decreasing first and then increasing.It was speculated that the somatic embryogenesis ability of different cell lines was related to the level of these physiological indicators.