Polyploidy Induction And Identification Of Nanhong Pear And Pyrus Betulifolia

‘Nanhong’pear is a red spontaneous bud sport of‘Nanguo’pear（Pyrus ussuriensis Maxim.）,‘Nanhong’pear has bright colors,delicate pulp,refreshing plenty of juice and fragrant pulp.However,the small size of‘Nanhong’fruit affects its commercial value.Pyrus betulifolia has strong adaptability.It has strong photophilia,cold tolerance,drought tolerance,waterlogging tolerance and barren soil resistance.It is an ideal arborization rootstock for pear trees in the north which is widely used.Polyploid pear has the advantages of vigorous growth,high yield and huge organs.Polyploid induction is an important way to cultivate large fruit pear varieties and dwarf stress resistant pear rootstocks.In this study,taking‘Nanhong’pear and Pyrus betulifolia as materials,the leaf regeneration system of‘Nanhong’pear was established.Taking‘Nanhong’pear leaves and Pyrus betulifolia cotyledons as materials,a batch of polyploid germplasm resources were created after colchicine mutation.The main results are as follows:1.Establishment of Leaf Regeneration System of‘Nanhong’pearTaking the isolated leaves of‘Nanhong’pear as explants,the leaf regeneration system of‘Nanhong’pear was established by exploring the effects of hormone concentration,basic medium type,dark culture time and leaf orientation on the leaf regeneration of‘Nanhong’pear.The suitable medium for leaf regeneration of‘Nanhong’pear was NN69+2.0mg·L^-1TDZ+0.4 mg·L^-1NAA+20 g·L^-1sorbitol+7 g·L^-1agar,the suitable dark culture time for leaf regeneration of‘Nanhong’pear was 20 days,and the suitable inoculation method for leaf regeneration of‘Nanhong’pear was far axis face down.The leaf regeneration rate and the average number of regenerated buds were 94.67%and 3.58.2.Polyploid induction and identification of‘Nanhong’pearBased on the leaf regeneration system of‘Nanhong’pear,the polyploidy of‘Nanhong’pear was induced by impregnating the leaves of‘Nanhong’pear with different concentrations of colchicine.The suitable concentration of colchicine for polyploid induction of‘Nanhong’pear was 0.2%and the induction rate was 32.03%.The most suitable nuclear dissociation solution for the ploidy identification of‘Nanhong’Pear by flow cytometry was HEPES dissociation solution,and the induced suspected polyploid adventitious buds were identified by flow cytometry.Finally,95 tetraploid lines of‘Nanhong’pear were obtained,numbered N-4-1 to N-4-95.The induced tetraploids of‘Nanhong’pear were observed and identified.Compared with diploids,the tetraploid plants of‘Nanhong’pear showed the characteristics of thicker leaves,larger leaf area,wrinkled leaf surface,darker leaf color,larger stomata and lower stomatal density.3.Polyploid induction and identification of Pyrus betulifoliaIn this study,in vitro cotyledons of Pyrus betulifolia were used as materials,and cotyledons of Pyrus betulifolia were impregnated with different concentrations of colchicine to induce polyploidy of Pyrus betulifolia varieties.The suitable concentration of colchicine for polyploid induction of Pyrus betulifolia varieties was 0.1%and the induction rate was 34.70%.The most suitable nuclear dissociation solution for the ploidy identification of Pyrus betulifolia varieties by flow cytometry HEPES dissociation solution,and the induced suspected polyploid adventitious buds were identified by flow cytometry.Finally,34 tetraploid lines of Pyrus betulifolia were obtained,numbered D-4-1to D-4-34.Compared with diploid,tetraploid plants showed the characteristics of thicker leaves,larger leaf area,changed leaf shape,darker leaf color,larger stomata and lower stomatal density.4.Rooting of polyploid tissue culture seedlings of‘Nanhong’pear and Pyrus betulifoliaIn this study,the suitable rooting hormone concentration of diploid and tetraploid tissue culture seedlings of‘Nanhong’pear and Pyrus betulifolia was explored.The hormone suitable for the rooting of diploid and Tetraploid of‘Nanhong’pear was 0.2 mg·L^-1NAA and 0.2 mg·L^-1IBA.The hormone suitable for the rooting of diploid and Tetraploid of Pyrus betulifolia was 0.2 mg·L^-1IBA.The morphological observation of the induced roots showed that the roots induced by NAA were stronger than those induced by IBA.