| Rose(Rosa hybrida)has a long history of planting and cultivation,and is an indispensable ornamental plant in gardens.Ploidy breeding is an important breeding method,which plays an important role in obtaining new rose varieties and breeding intermediate materials.In this study,different plant materials were used to investigate the effect of different reagents and different treatment conditions on the polyploid induction effect of modern rose cultivar ’ Eyeconic Lemonade ’(2n=4x).The morphology,physiological indicators and transcriptome of ploidy and tetraploid were compared,and the genes and experimental methods that could be used for q PCR ploidy identification were analyzed.The result is as follows:1.Colchicine was used to induce polyploidy in tissue culture seedlings and field plants of ’ Eyeconic Lemonade ’ with different concentrations and treatment times,and the induced plants were identified by flow cytometry and chromosome counting.The results showed that the homozygous octaploid was successfully induced in the tissue culture seedlings,and only the chimera was induced in the field plants.The chromosome number of the tetraploid’ Eyeconic Lemonade ’ was 2n=4x=28,and the number of induced octaploid chromosomes was 2n=8x=56.The experimental results showed that 2g/L colchicine had the best effect on tissue culture seedlings treated for 48 h,and the mutagenesis rate was 17.85%.Colchicine has a strong toxic effect on tissue culture seedlings,and the treated plants can be transplanted after 8 months.2.Trifluralin was used to induce polyploidy in tissue culture seedlings of ’ Eyeconic Lemonade ’ with different concentrations and treatment times.The ploidy of the induced plants was also identified by flow cytometry and chromosome counting.Results indicated that the homozygous octaploid was successfully induced.The experimental results showed that the mutagenesis rate of 0.6g/L trifluralin for 24 h treatment of tissue culture seedlings was the highest,reaching 52.7%,but 60% of the mutagenized plants were chimeras.The toxic effect of trifluralin on rose plants is lower than that of colchicine,3 months can be transplanted.3.In the experiment,we also tried to use q RT-PCR to identify the ploidy of the induced plants.A total of 5 genes were selected,18 s r DNA,28 s r DNA,Chr2g0122871,LOC112181550,LOC112175411.18 s r DNA and 28 s r DNA are ribosomal DNA;Chr2g0122871 is a gene that regulates nuclear components;LOC112181550 is related to centromere assembly;LOC112175411 is related to DNA replication.The results of q RTPCR experiments found that Chr2g0122871,LOC112181550 and LOC112175411 could be used for the identification of autopolyploid induction.4.To observe the morphology and histology and compare the physiological indexes between the tetraploid ’ Eyeconic Lemonade ’ and the induced octaploid plants.The study found that the size of the guard cells of the octaploid was significantly increased(p<0.05),which was about 1.36 times that of the tetraploid,and the stomatal density of the octaploid was also significantly reduced(p<0.05).The chlorophyll content,superoxide and peroxidase activities of the octaploid plants were significantly higher than those of the tetraploid plants(p<0.05);the difference in the content of malondialdehyde was not significant.5.Transcriptome analysis of tetraploid ’ Eyeconic Lemonade ’ and induced octaploid plants.A total of 3834 differentially expressed genes were obtained.Through GO enrichment analysis,it was found that the most significant enrichment in biological processes was photosynthesis;the most significant enrichment in cell composition was thylakoid;the most significant enrichment in molecular function was oxidoreductase activity.KEGG enriched most notably in starch and sucrose metabolism,phytohormone signaling,and phenylpropane biosynthesis.Eight functional genes with significant differences were selected for q RT-PCR,and the experimental results were basically consistent with the transcriptome sequencing results. |
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