Studies On The Techniques Of Isolated Microspore Culture In Broccoli (Brassica Oleracea L.)

Brassica oleracea L.var.italica Phenck belongs to raphanus of Cruciferae.It is an annual eutrophy herbaceous plant.The study of cauliflower has just started in recent years in our country,so the materials are less.It takes 5～7 years for broccoli breeding.It is needed to raise efficiency of broccoli breeding and accelerate the advancement of broccoli breeding.Isolated microspore culture is of great importance in vegetable genetics and breeding,through which homozygote could be obtained rapidly and it is easy for us to screen out breeding material with resistance in a shorter time.On the other hand,the genotype and phenotype of double haploid (DH) obtained from microspore culture are identical,which is suitable for genetic analysis,genetic map construction,gene direction,trans-gene breeding,chromosome engineering,gene discovery,etc.In the process of microspore culture,determination of the condition suitable for embryoid induction and plant regeneration is very important.In this experiment,directed towards the difficulties in microspore culture,broccoli materials of 30 genotypes were chosen to improve the broccoli microspore culture system.The suitable size of flower bud,pretreatment method,and culture condition of the microspore culture and embryoid regeneration culture were studied.The main results are as follows:1.Screened out 16 embryogenesis material from 30 kinds of broccoli,the induction of the microspore were 53.33%.The relationship between the cytological characteristics of microspore at different development stages and the corresponding morphological characters of flower organ.Through the observation on the morphological characters of flower organ of three broccoli materials,the result showed that the lengths of the flower bud ranged 4.1 to 5.0mm,when the microspore stage was between the late uninucleate-staged to binucleuses-staged.Based on the observation result,it was possible to rapidly and conveniently choose the flower bud at suitable developmental stages.2.The optimal concentration of sucrose for microspore development was 13%.The heat-shock pretreation on the microspore at 32℃,last for 24 hours.After 24 hours,the longer the time was,the lower the embryogenesis would be.It was advantage for microspore survival in the medium with 15% sucrose.To the medium during the culture,the embryo yield could be significantly improved.3.The optimal concentration for adding hormones 6-BA in the Medium was 13%.But hormones NAA was not make an effect for the embryogenesis.4.The concentration for adding the active charcoal at 0.05mg·L-1 had a good effect on embryogenesis and Plant regeneration. Without the active charcoal,the microspore would turn brown.If the concentration at 0.1 mg·L-1,embryogenesis would be stopped.5.The plants regenerated from embryonic were gotten on the suitable medium with 1.2% agar.At the same time the abnormal plants decreased a lot.And these seedlings mostly came from the ripe cotyledon embryos.6.95% regenerated plants were suitable for this rooting medium:1/2MS+0.01% NAA+ 0.02% IBA.The longest root was 16cm.The best medium for regenerated plants was peat and vermiculite with a 2:1 mixture.The rate of the alive plant were 100%.7.The policy level of microspore-derived plants was analyzed by flow cytometry. The same results showed that flow cytomerty could identify haploid,chimera and doubled haploid accurately with the morphology way.At the same time,80% of naturally haploid were observed from the broccoli regenerated plants.