Isolated Microspore Culture And Plant Regeneration In Brassica Juncea Czern. Et Coss

Brassica juncea Czern. et Coss. belongs to raphanus of Cruciferae.It is an annual eutrophy herbaceous plant.It takes5-7years for breeding an inbred.It is needed to raise efficiency.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.Their ploidy level usually were identified by observing their morphological characteristics during budding and flowering. However,this chromosome doubling method is labour intensive, time consuming,and isn’t suitable for treating large number of plants.In the process of microspore culture,determination of the condition suitable for bryoid induction and plant regeneration is very important. Based on previous studies, the optimization on the microspore culture technology of Brassica juncea Czern. et Coss.under field condition was studied systematically in the paper.1.Genotype is one of the key factors affeeting the embryoid induetion in broecoli.2Genotypes among3get embryoids successfully and2get regenerants in the research. Zi has the highest frequency of embryoid induction with16embryoids per petri dish, the lowest embryoid yield is Liuchuan with0embryoid per petri dish.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 brassica juncea czern.et coss. materials,the result showed that the lengths of theflower bud ranged2.5to3.5mm,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.Pretreatment in4℃low-temperature couldn’t increase the embryoid yield,but it could keep material fresh for period of time.While keeping for5-6days in4℃,the embryoid yidld brought down obviously,the result was that most of pollens losed activity through microscope.3.The optimal concentration of sucrose for microspore development was13%. Theheat-shock pretreation on the microspore at33℃,last for48hours.After72hours,the longer the time was,the lower the embryogenesis would be.4.Adding hormones6-BA and hormones NAA in the Medium was13%was not make an effect for the embryogenesis,and when6-BA and NAA with high concentration had opposite effect.5.With the suitable medium for shooting was B5+TDZ0.02mg·L-1+NAA0.02mg-L-1+KT0.05mg-L-1+Active Carbonl g-L-1+3%sugar+0.8%agarose.,the shoot induction rate come up to88%.6.100%regenerated plants of Zi were suitable for this rooting medium MS+NAA0.2mg-L-1. The best medium for regenerated plants was peat and vermiculite with a2:1mixture.The rate of the alive plant were100%.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,100%of naturally haploid were observed from the Brassica juncea Czern. et Coss. regenerated plants.