Isolated Microspore Culture And Karyotype Analysis In Radish (Raphanus Sativus L.)

The development and improvement of biotechnology continuously,which offer some new ways and means for radish breeding.Microspore culture accelerates the process of elite cultivar development.In this study,13 genotypes of radish were used,to optimize the technique system of isolated microspore culture.At the same time,plantlet regeneration and ploidy identification were studied as well as karyotype analysis.The main results were as follows:1.Of 13 genotypes,only nine could produced embryoids with a various production rate ranging from 17.03 embryoids per bud in NAU-SPZ×NAU-HP to 0.12 in NAU-ZSDG.2.Heat shock treatment for 48h at 32.5℃was necessary to induce microspore division and initiate embryogenesis.3.The developmental stage of microspores could be estimated exactly by petal/anther ratio. The ratio at 0.75-1.0 was the most suitable for embryogenesis in isolated microspore culture.4.NLN-13 was the best medium for radish microspore culture.The concentration of macro-elements in the medium did not affect the embryoid yield.5.The supplement of 0.75g/L activated charcoal(AC) to NLN-13 could increase embryoid yield and quality.6.Pretreatment in 4℃low-temperature couldn't increase the embryoid yield,but it could keep material fresh for period of time.While keeping for 5～6 days in 4℃,the embryoid yield brought down obviously,the result was that most of pollens losed activity through microscope.7.The results showed that 6-BA with low concentration can promote embryoid yield.It stimulated cell division.While 6-BA with high concentration had opposite effect.8.The survival embryoids subcultured on solid B₅ were significantly higher than those on MS medium.The embryoids from L-4,L-9 and L-11 did not germinate either on MS or B₅ medium.Further observation indicated that these embryoids were abnormal and the embryoids which survived in the germination medium were all cotyledonary embryoids. 9.The chromosome number of regenerant was analyzed.It was confirmed that the regenerated plantlets consisted of haploid,diploid and tetraploid plants as compared with the diploid donor plant(2n=2x=18).Ploidy level of regenerated plants was also determined by flow cytomatic analysis and the results were in accordance with those from chromosome analysis,which could identify ploidy of the regenerated plantlets quickly and exactly.10.The chromosome number and karyotype of three different coloured coat and two different zones species were studied.The karyotype formulae were as follows: NAU-SJMSH:2n=2x=18=18m,NAU-WYQ:2n=2x=18=2M+16m,NAU-YZBLB:2n=2x=18 =18m,みのZaoshengdagen:2n=2x=18=18m(2SAT).The karyotypes of the four species were uniform and belonged to Stebbins's 1A type.According to the karyotype analysis data, the relationship among them was primarily discussed.And we found that the karyotype of NAU-WYQ was more original than those of the other two colour genotypes.It can be inferred that Japan may be one of the radish origin centers in the world.