| In order to further improve the regeneration system in vitro culture of processing tomato anthers.This experiment were used three hybrid combinations(P1×20040805,20040805×Diamond and P3×20040805) of processing tomato anthers as experimental materials.The effects of microspore development period,genotype,hormone type and hormone ratio on the regenerated plants of tomato anther culture were studied.The medium formula for the induction and proliferation of suitable anther callus was screened from two kinds of induction medium and thirteen kinds of proliferation medium.The callus of the processing tomato hybrid combination (P3×20040805) anther and the roots derived from callus differentiation were identified by flow cytometry and root tip chromosome counting.A technical system for the identification of callus pedigree induced by callus of processed tomato anthers was preliminarily established,and a higher rate of callus proliferation was obtained,which laid the foundation for the next step of using the in vitro culture technique of anthers to process tomato germplasm resources.The main test results were as follows:1.The microspore development period of processing tomato was closely related to the floral organ morphology.When the microspore development period of processing tomato was in the single-nuclear edge period,the length of 20040805×Diamond and P3×20040805 flower buds of hybrid combination was 5.00-5.99 mm,and the length of P1×20040805 flower bud was 6.00-6.99 mm;The floral organ morphology of different hybrid combinations was basically same during the same microspore development period.The nucleus of micronucleus in the single-nuclear edge,the microspore cells were round,the nucleus was close to the cell wall and the nucleus appeared in the nucleus.The flower shape was slightly expanded,the flower buds wear slightly open,the color of the scorpion was yellow-green,the anther color was yellow-green and the anther was easy to peel.2.Different genotypes were processed in tomato anther callus,and the induction medium was different.MS+0.5 mg/L 2,4-D+1.0 mg/L KT was a suitable medium for inducing callus induction of processing tomato hybrid combination P1×20040805;The highest induction rate of P1×20040805 was 13.33%;MS+1.0 mg/L NAA+1.0 mg/L 6-BA was a suitable medium for inducing callus induction of processing tomato hybrid combinations (20040805×Diamond and P3×20040805);The suitable medium for the propagation of callus from three genotypes of processing tomato anthers was MS+1.0 mg/L 6-BA+0.2 mg/L NAA.The callus proliferation rate of P3×20040805 reached 93.33%.The addition of a low concentration of TDZ to the proliferation medium didn't promote the proliferation of callus cells.However,it can induce the formation of dense tumor-like callus.When the concentration of NAA was 0.05 mg/L,the callus of the processing tomato hybrid combination P3×20040805 was induced to differentiate into roots,and the rooting rate was 6.67%.However,root differentiation was not observed in MS medium supplemented with different concentrations of IAA.3.The root tips obtained callus differentiation of processing tomato hybrid combination P3×20040805 were identified by apical chromosome counting method,and the number of chromosomes in tomato root tips was 24.In addition,a microscopic examination of the number of chromosomes naturally occurring in the field was performed,and the number of chromosomes was also found to be 24.The callus obtained by the processing tomato hybrid combination P3×20040805 was subjected to ploidy determination by flow cytometry.Most of the anther callus was diploid,and some cells of a few callus were mutated to produce chimera.Three types of chimeras were detected by flow cytometry,suggesting that they may be haploid,diploid and tetraploid chimeras,haploid and diploid chimeras,and diploid and tetraploid Chimera.No haploid callus was found by flow cytometry and root tip chromosome identification of the anther callus and differentiation of the tomato hybrid combination P3×20040805. |
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