The Optimization Of Open Tissue Culture And Study On Gene Family Of SOD In Rhododendron

Azalea（Rhododendron simsii Planch.）is one of the top ten famous flowers in China,with rich and gorgeous colors,and is often used in landscape gardening.The application of plant tissue culture technology in azalea factory production can not only shorten the seedling period,but also greatly improve the space utilization rate of seedling workshop.In the traditional tissue culture technology,mercuric chloride（HgCl₂）with highly toxicity is used as the surface disinfectant of explants and culture medium that must be sterilized by high pressure sterilization.The recovery cost of HgCl₂is high,which is easy to cause biological toxicity and environmental pollution.At present,it has been stopped or banned.The sterilization and aseptic operation need to consume a lot of electricity and purchase aseptic operation equipment,and the production cost is higher.The open tissue culture can not only save the cost of seedlings,but also improve the efficiency of tissue culture by adding antibacterial agents to control the contamination during the tissue culture process.Therefore,in this study,take Rhododendron pulchrum Sweet.as materials,H₂O₂,NaClO and ClO₂ were used to replace HgCl₂ as surface disinfectants of explants to optimize the open tissue culture and rapid propagation system of azalea.ISSR molecular marker was used to detect the genetic stability of test-tube plantlets of R.pulchrum obtained form each open tissue culture.6 members of SOD gene family of R.pulchrum were cloned by using azalea genome data with qRT-PCR technique,and the transcript levels of 6 members of SOD genes were analyzed in test-tube plantlets of R.pulchrum and Rhododendron hybridum‘Hong Shanhu’under the oxidative stress of open tissue culture antibacterial agents.The results of this study can provide technical support for large-scale factory production of azalea test-tube plantlet,and have important theoretical significance and practical value.The main results are as follows:1 Optimization of open tissue culture system of azaleaScreening of surface disinfectants for explants:The terminal buds and stem segments with axillary buds of the current year branches of R.pulchrum were disinfected by different concentrations of H₂O₂,ClO₂ and NaClO.The results showed that the optimum disinfection method for explants was 10%H₂O₂ for 10 to 15 minutes,and the survival rate of terminal bud explants was 61.67%and that of stem segments with axillary buds was 23.33%.The terminal buds were more suitadle as explants for azalea primordial culture than stem segments with axillary buds.Screening of inhibition agent combinations in open primary tissue culture:In the primary culture medium WPM+0.5 mg/L ZT+0.5 mg/L GA₃,different concentrations of NaClO,mancozeb and S106 were added as antibacterial agents to compare the survival rate and shoot quality,and the results showed that the optimum type and concentration of inhibition agent in open primary culture medium was 0.01%NaClO,and the survival rate of material was 62.22%and the test-tube plantlets grew well.Screening of inhibition agent combinations in open proliferation tissue culture:In the optimized optimal proliferation medium WPM+0.1mg/L NAA+3.0 mg/L ZT,different concentrations of NaClO and mancozeb were added as antibacterial agents,and sterile sprout stems from open primary tissue culture were used as materials to compare the proliferation coefficient and sprout quality,and the results showed that the optimal type and concentration of the open proliferation medium inhibitor was also 0.01%NaClO,the proliferation coefficient was 3.067,and the test-tube plantlets grew well.Open rooting culture and transplanting:The open cultured test-tube plantlets could grow normally in the strong and rooting medium added with 0.01%NaClO and induced rooting,and the azalea test-tube plantlets were transplanted after stepwise acclimatization could survive normally.2 Genetic stability assay of azalea open tissue culture test-tube plantletsThe genetic stability of azalea test-tube plantlets obtained from different treatments were identified by ISSR molecular marker technique.The results showed that different concentration treatments had certain effects on the genetic stability of test-tube plantlets produced by explants,and the genetic diversity of primary plantlets produced by explants disinfection was 0.0576,and the genetic similarity coefficient of different test-tube plantlets was from 0.952 to 0.995.Among them,H₂O₂ had the least effect on the genetic distance of materials and ClO₂ had the greatest effect on the genetic distance of materials;The analysis of genetic differences between the test-tube plantlets obtained from each open treatment and the mother plant showed that the genetic similarity coefficients of the test-tube plantlets with different concentrations of open medium added with different antibacterial agents ranged from 0.919 to0.995,and the genetic distance between the open test-tube plantlets and the mother plant was close,and the variation rate of azalea test-tube plantlets was low.3 Cloning and bioinformatics analysis of RsSOD family genesAccording to the azalea genome data,6 RsSODs genes were successfully cloned,which were identified as RsCSD1a,RsCSD1b,RsCSD2,RsMSD2,RsFSD1 and RsFSD3;the prediction of protein structure showed that the protein structure of 6 RsSODs member were inconsistent,RsCSD1a,RsCSD1b,RsCSD2 and RsFSD1 were classified as stable proteins,RsMSD2 and RsFSD3 were classified as unstable proteins;the results of protein subcellular localization showed that RsCSDs mainly existed in cytoplasm,RsMSD and RsFSDs mainly existed in chloroplast and mitochondria;the prediction of cis-acting elements of RsSODs family promoters showed that they responded in light,phytohormones,adversity stress,plant growth and development and metabolic regulation;the results of phylogenetic tree of RsSODs family showed that RsSODs were more closely related to dicotyledons evolution.4 Expression of RsSODs genes under oxidative stress of antibacterial agentsThe transcript levels of RsSODs family members in test-tube plantlats of R.pulchrum and R.hybridum‘Hong Shanhu’under oxidative stress of antibacterial agents in open tissue culture were analyzed by qRT-PCR technique.The results showed that the oxidative stress response of 6 RsSODs genes differed not only in the type and concentration of the antibacterial agents but also in the varieties.In open tissue culture with NaClO as antibacterial agents,the low concentration（0.01%）of NaClO significantly promoted the expression of RsCSD1b,RsFSD1 and RsFSD3genes of R.pulchrum in the pre-culture stage only,and had little effect or even inhibited the expression of RsSODs of R.hybridum‘Hong Shanhu’;The medium concentration（0.015%）of NaClO significantly promoted the genes expression of RsCSD1b of R.pulchrum and RsCSD1b,RsMSD2of R.hybridum‘Hong Shanhu’in the pre-culture stage,the genes expression of RsCSD1a,RsCSD1b,RsMSD2,RsFSD3 of R.pulchrum and RsCSD1a of R.hybridum‘Hong Shanhu’were significantly promoted in the mid-culture stage,and in the late culture stage,the genes expression of RsCSD1b,RsFSD1 of R.hybridum‘Hong Shanhu’were significantly promoted;The high concentration（0.02%）of NaClO significantly promoted the genes expression of RsCSD1a,RsCSD1b of R.pulchrum and RsCSD1a,RsMSD2 of R.hybridum‘Hong Shanhu’in the pre-culture stage,the genes expression of RsCSD2,RsMSD2,RsFSD1,RsFSD3 of R.pulchrum and RsMSD2 of R.hybridum‘Hong Shanhu’were significantly promoted in the mid-culture stage.In open tissue culture with mancozed as antibacterial agents,the low concentrations（75mg/L）of mancozed significantly promoted the genes expression of RsCSD1a,RsCSD1b,RsMSD2 of R.pulchrum and RsCSD1a,RsCSD1b of R.hybridum‘Hong Shanhu’in the mid-culture stage,the genes expression of RsCSD1a,RsCSD1b of R.pulchrum and RsCSD1b,RsFSD1 of R.hybridum‘Hong Shanhu’were significantly promoted in the late culture stage;The high concentration（100 mg/L）of mancozed promoted the genes expression of RsCSD1a,RsCSD1b of R.pulchrum in the pre-culture stage,the genes expression of RsCSD1a,RsCSD1b,RsMSD2,RsFSD1 of R.pulchrum and RsCSD1a,RsCSD1b,RsCSD2 of R.hybridum‘Hong Shanhu’were significantly promoted in the mid-culture stage,and the genes expression of RsCSD1a,RsCSD1b of R.pulchrum and R.hybridum‘Hong Shanhu’were significantly promoted in the late culture stage.