Isolated Microspore Culture And Plant Regeneration In Broccoli

Eight hybrid varieties(F₁) of broccoli were tested in isolated microspore culture. Several main factors in embryogenesis of microspore culture and plant regeneration were studied.Cytological observations were performed throughout the developmental process of microspore embryogenesis.Meanwhile,ploidy level of regenerants was determined by using flow cytometry(FCM),and the techniques of chromosome doubling and transplantation of plantlets derived from microspore were discussed.The objective of this study is to develop the protocol for haploid embryogenesis induction in broccli.The main results are as following:1) Genotype is one of the key factors affeeting the embryoid induction in broccoli.7Genotypes among 10 get embryoids successfully and 3 get regenerants in the research.Q7 has the highest frequency of embryoid induction with 14.5 embryoids per petri dish,the lowest embryoid yield is XLX with 0.17 embryoids per petri dish.2) Bud size also has an impact on microspore culture.The cytological observation of different bud length for four different genotypes showed that the buds with length ranging from 3.5mm to 4.5 mm and 54.6%-73.7%of the pollen at the late uninucleate stage are optimal for isolated microspore culture of broccoli.3) Low major salts are beneficial to broccoli embryogenesis.Compared with NLN-13 medium,reducing the content of major salts of NLN-13 medium by half can not only increase the the genotypes that can be induced to get embryo but also can remarkably raise the embryoid induction rate.4) Activated charcoal in suitable content(approximately 0.1 mg/mL) can promote embryoid induction and development,especially for recalcitrant genotypes to get embryoids.5) Effects of sucrose concentration on embryogenesis.13%of sucrose in the mediun is appropriate for embryogenesis.However,culturing the microspores in 1/2NLN with 17%sucrose for 2 days and followed by culturing in 1/2 NLN with 13%sucrose can induce embryoids more efficiently.6) For the recalcitrant genotypes,the pre-treatment(32℃) for 48 h is more effective than that for 24 h.7) The cytological observation revealed that two basic routes for microspore embryogenes are asymmetrical division(A) and symmetrical division(B).The symmetrical division is the most predominant one. 8) It is found that plantlet regeneration rate reached highest when cotyledon-type embryos were strong enough at the embryonic age of 30-35 days while the regeneration rate decreased remarkably as long as their age exceeded 40 days.And the suitable medium for embryoid regeneration might be MS(with 1.2%agar) or B5.9) "MS+3%sucrose+1.0%agar" is the optimal medium for subculture and renewal in broccoli,"MS+0.2 mg/L NAA+3%sucrose+0.8%agar" is suitable medium for rooting.10) The analysis of ploidy level revealed that the majority of regenerated plantlets tested were haploids,and also there were a few tetraploids and monoploid-diploid chimeras.The proportion of diploids is about 30%-35%.Those plants that determined as haploids were doubled by 2%colchicine.