Study On Biological Effects Of Different Types Of Ionizing Radiation On Industrial Crop Taraxacum Kok-saghyz Rodin

Natural rubber（NR）is a non-fungible natural polymer,is widely used in fields such as national defense,industry,medical,and transportation due to its unique physical characteristics.In recent years,the demand for NR has increased year by year worldwide.The diversification of NR sources is an important factor for the sustainable development of the industry.However,currently,NR is mainly sourced from the Hevea brasiliensis（HB）.Taraxacum kok-saghyz Rodin（TKS）,also known as rubber dandelion,is a perennial herb of the dandelion in the composite family,containing 2～10%NR in its roots.Its structure and properties are similar to NR sourced from HB,making it an important alternative source of NR.However,TKS germplasm innovation faces significant challenges due to self-incompatibility.Ionizing radiation（IR）technology is an effective and widely applicable physical mutagenesis method,not limited by species genetic transformation systems or genomic information.However,IR has not yet been applied to TKS.The mutagenic effects of radiation are affected by many factors,including the mutagenic source,radiation parameters（Linear energy transfer（LET）,dose）,and physiological status of plant materials（seeds,seedlings,nutritional organs,and tissue culture materials）.To better utilize IR technology for TKS germplasm innovation and provide reference for mutagenesis breeding practice,this paper studied the radiative effects of TKS from different physiological states（leaf explants,adventitious buds,and seeds）using high-LET carbon ion beam（CIB）and low-LETγ-rays and X-rays radiation.The effects of TKS radiation on M₁ growth and development,physiology and biochemistry,and transcriptome regulation were systematically studied.The main research results are as follows:1.IR combined with tissue culture for radiation mutagenesis system and study on radiation effects of explants.Optimal disinfection schemes and plant hormone（6-BA and NAA）ratios were determined to establish an efficient and stable tissue culture system for TKS.CIB（3 LET gradients and 8 dose gradients）and X-rays（9 dose gradients）were used to irradiate TKS leaf explants,and their morphological indicators（survival rate,fresh weight increase（FWI）,adventitious bud number,and aberration rate,etc.）were measured to create radiation response curves.The results showed that the 50%lethal dose(LD₅₀)of TKS leaf explants irradiated by CIB with LET of 31,65,and 96 ke V/μm were 10.5,7.6,and 5.8 Gy,respectively,while the LD₅₀ of TKS leaf explants irradiated by X-rays was 20 Gy.The corresponding relative biological effectiveness（RBE）were 1.9,2.6 and 3.4,respectively.The FWI of TKS leaf explants was severely inhibited by CIB at LET of 31,65,96 ke V/μm and X-rays at 3 ke V/μm.50%inhibition dose(ID₅₀)for FWI was 11.7,8.1,6.3 Gy for CIB,19.8 Gy for X-rays,respectively,with corresponding RBE of 1.7,2.4,and 3.2,respectively.In addition,the phenotype distortion rate induced by CIB is higher than that of X-rays.The dose points with the highest phenotype distortion rate induced by radiation are near the LD₅₀ for both CIB and X-rays.Considering all factors,CIB irradiation will be used primarily in future research.2.M₁ biological effects and transcriptomic responses of TKS adventitious buds to CIB irradiation.TKS adventitious buds were treated with different doses（0,5,10,15,20,30,and 40 Gy）of CIB irradiation to elucidate their mutagenesis mechanisms at the morphological,physiological,and transcriptomic levels.The results showed that CIB irradiation at doses of 5-40 Gy had inhibitory effects on the fresh weight（FW）,regeneration bud number,and number of adventitious roots of TKS adventitious buds after 15 d or 30 d of culture.Therefore,considering the growth parameters（FW,regeneration bud and root number）after CIB irradiation,the 15 Gy CIB irradiation treatment group was selected for subsequent physiological and gene expression studies.Physiological results showed that the activity of hydroxyl radical（OH^·）and malondialdehyde（MDA）content were significantly increased after 6 h of CIB irradiation treatment,indicating oxidative damage stress on TKS adventitious buds.Meanwhile,at the corresponding time points,the free radical scavenging ability of 1,1-diphenyl-2-picrylhydrazyl（DPPH）and the activities of antioxidative enzymes（（Superoxide dismutase,SOD）,（Catalase,CAT）and（Peroxidase,POD）and Ascorbate peroxidase（APX））also increased.Based on RNA-sequencing technology,5131,2464,472,and 421 differentially expressed genes（DEGs）were detected after radiation treatment for 2,6,24,and 72 h,respectively,compared with the control group.Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis results showed that DNA replication and multiple damage repair pathways（38up-regulated,16 down-regulated）,cell death（25 up-regulated,17 down-regulated）,auxin and cytokinin related to plant morphology（21 up-regulated,33 down-regulated）,photosynthesis（2 up-regulated,62 down-regulated）,and reactive oxygen species（ROS）（1 up-regulated,7 down-regulated）were all involved in the TKS response to CIB irradiation.Among them,DNA replication and repair are closely related to gene mutations,and ROS can also produce mutation sites.Additionally,this study found that CIB irradiation up-regulated genes related to NR metabolism.3.The IR treatment has significant biological effects on the dry seeds of TKS and exhibits dose-dependent responses.Using different types of IR（CIB andγ-rays）to treat dry TKS seeds,the morphological and physicochemical endpoints of the M₁ generation were recorded to obtain radiation dose-response curves for various endpoints.In terms of growth and development,when the CIB is≥250 Gy and theγ-rays is≥600 Gy,the germination rate of TKS is significantly inhibited.When CIB is≥100 Gy andγ-rays is≥200 Gy,the survival rate is significantly inhibited.Based on the survival curve,the LD₅₀ of CIB andγ-rays for TKS is 141 Gy and 333 Gy,respectively,which can be used as a reference dose for seed mutagenesis.The sensitivity of root growth and development,size of plant and leaf number to radiation is higher,except that the plant size and leaf number of the CIB 50 Gy treatment group did not show significant differences from the control.The other indicators were significantly lower than the control in the lowest dose group.The aboveground and underground biomass exhibited a certain degree of stimulation effect at low doses,and the inhibition degree of biomass increased with the dose.In addition,the treatment group showed phenotypic changes such as reddening of cotyledon and leaf curling.At the physiological level（30 d after sowing）,low doseγ-rays（100 Gy）showed a clear low dose stimulation effect on chlorophyll content.In the CIB≥150 Gy andγ-rays≥200 Gy treatment groups,malondialdehyde（MDA）content significantly increased,indicating that radiation-induced oxidative damage can persist after 30 d of sowing.Meanwhile,the activities of antioxidant enzymes（SOD,CAT and POD）in the radiation treatment group were higher than those in the control group.4.The content of NR in the roots of M₂ plants of TKS varied greatly,and some strains showed an increase in NR content of more than 20%.Using Fourier transform infrared spectrometry（FTIR）to determine the NR content of roots of 416 TKS M₂plants（control:139 plants,radiation treatment group:277 plants）,it was found that the changes in NR content of the radiation treatment group ranged from-30.80%to 56.06%.Among them,the NR content of A-104,A-41,and A-37 is 4.75%,4.54%,and 4.30%,respectively,which increased by 57.16%,50.29%,and 42.46%compared to the control.In summary,this study is the first to investigate the mutagenic effects of IR on TKS,covering various physical parameters,physiological states of plant starting materials,and multiple levels（growth and development,physiological and biochemical,and gene transcription）.An“IR+tissue culture”and“IR+seed”radiation technology system for TKS has been established,and superior individual plants with increased NR content have been obtained,and also an RNA-seq dataset for radiation response of TKS has been accumulated.The above research results provide basic data and strategies for future scale-up radiation mutagenesis breeding of TKS and plant growth promotion based on low dose stimulation effect.