Effects Of ⁶⁰Co-γ Rays And Chemical Mutagens On Rosa Roxburghii Seeds

Rosa Roxburghii Tratt has become the characteristic industry of Guizhou Province because its fruits are rich in nutrition and active substances.The economic cultivation area of the province has exceeded 130,000hm².Although a few excellent germplasm resources have been discovered in the early stage,the genetic rules of main agronomic traits and variety breeding of R.Roxburghii were revealed slowly.At present,the main cultivar is‘Gui nong 5’,accounting for more than 90%of the total area and yield,which objectively can not meet the needs of processing industry and market diversification.Germplasm innovation is the basis of breeding new varieties.Through radiation and chemical mutagenesis,a large number of mutant populations can be obtained in a short period of time.After selection and identification,it is possible to screen out the heritable variation that meets the demand,and then directly or indirectly breed new varieties that meet the production demand.The essay focuses on the seeds of R.Roxburghii as experimental material,the system explores the ⁶⁰co-γrays,colchicine and EMS mutagenesis effect of R.Roxburghii seeds,screening suitable dose,and combined with morphology,cell and molecular identification of mutations in plant level,in order to R.Roxburghii germplasm innovation,and new variety breeding and large-scale radiation and mutation breeding in the future to lay the material and technical basis.The main results are as follows:1.Seven ⁶⁰co-γrays irradiation doses of 10 Gy,40 Gy,70 Gy,100 Gy,130 Gy,160 Gy and 190 Gy were set to irradiate R.Roxburghii seeds respectively.After sand storage,seeds were sown in spring,and the seedling rate was calculated one month later.The results showed that with the increase of irradiation dose,the seedling formation rate of R.Roxburghii seeds decreased continuously,and the seedling survival rate of R.Roxburghii seeds was significantly lower than that of non-irradiated seeds（control）under the lowest irradiation dose（10 Gy）.When the irradiation dose was 100 Gy,the seed mortality was 100%,indicating that R.Roxburghii seeds were sensitive to ⁶⁰co-γrays.When the radiation dose was 40 Gy,the survival rate of seedlings was also lower than that of the control half,indicating that the lethal dose of R.Roxburghii seeds should be between 10 and 40 Gy.The seedling growth indexes showed that the plant height and internode length decreased with the increase of the dose in the range of 10 Gy-70 Gy,but the stem diameter generally increased.The analysis of physiological indexes showed that the content of soluble sugar and SOD decreased with the increase of irradiation dose,while the content of malondialdehyde decreased,while the content of soluble protein and free proline increased first and then decreased with the increase of irradiation dose.2.Three colchicine concentration gradients（0.05%,0.1%and 0.2%）and two time gradients（12 h and24 h）were set to soak R.Roxburghii seeds which had just become white after being stored in sand,and the seedling rate was calculated after one month of spring sowing.The results showed that the seedling rate decreased significantly with the extension of colchicine concentration and soaking time.The seedling rate of the treatment with 0.1%colchicine for 12 h was about 32.5%,and the relative seedling rate（54.2%）was nearly half of that of the control.3500 R.Roxburghii seeds were treated with the combination of concentration and time.After 10 months,1 diploid and tetraploid chimera and 2 diploid and octaploid chimera were screened by flow cytometry.Compared with the seedlings without colchicine treatment（control）,chimeras showed significant morphological differences such as decreased stomatal density,increased stomatal length,and increased length and width of guard cells.3.R.Roxburghii seeds with improved permeability were used as experimental materials after the seed coats were mechanically worn by sandpaper,and the seeds were soaked with EMS with four concentration gradients of 0.1%,0.3%,0.6%and 0.9%for 12 h and 24 h,respectively,before sowing.The results showed that low EMS concentration of 0.1-0.3%could increase the seed emergence rate and seedling formation rate,but with the increase of EMS concentration or the extension of treatment time,the seed emergence rate and seedling formation rate decreased significantly.When the EMS concentration increased to 0.9%for 12h or0.6%for 24h,the seed mortality rate was 100%,and the semi-lethal dose was 0.3%.At the same time,R.Roxburghii seeds were used as experimental materials and sowed after soaking for 12 h and 24 h by 0.1%,0.3%,0.5%and 0.7%EMS concentration gradients,respectively.The results showed that 0.1%EMS treatment could improve the seed emergence rate and seedling formation rate,but with the increase of EMS concentration and the extension of soaking time,the seed emergence rate and seedling formation rate decreased significantly,and when the concentration was above 0.7%,the seeds almost died.When EMS concentration was 0.3%-0.5%for 12h,the relative seedling formation rate was 46.7-56.0%,indicating that the lethal concentration was between 0.3%-0.5%（12 h）.The growth traits of surviving seedlings showed different phenotypic variation in plant height,plant type,leaf type and branch,and the variation rate increased with the increase of treatment concentration within a certain range.Taking into account the seedling rate and phenotypic variation rate,it was found that 0.3%-0.4%EMS soaking for 12 h was the suitable dosage condition for R.Roxburghii seeds.The results can provide technical reference for constructing a large number of R.Roxburghii mutant population in the later stage.4.Using the suitable mutagenesis dose conditions obtained by the above screening,we expanded the number of treated seeds for ⁶⁰co-γrays and EMS mutagenesis respectively,and obtained 50 and 153candidate progenies of phenotypic variation respectively.PCR detection using SSR primers selected by the research group in the early stage found that,With 32 plants randomly selected from 100 non-treated seed progenies as control,18 plants from ⁶⁰co-γrays induced progenies and 123 plants from EMS induced progenies were stably detected with different bands at the DNA level,with a total difference rate of about1.00%and 1.92%,respectively,which could lay a technical and material foundation for physical and chemical induced breeding of ⁶⁰co-γrays in the future.