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Various levels (0, 0.01, 0.1, 1.0 μM) of n-nitroso n-methyl urethane (NMU) was treated to the microspores of broccoli (Brassica oleracea L. var. italica) during culture to find out LD 50 dosages at 4 different cultural times (before culture and on the 2nd, 4th, and 7th day after culture). The embryo was almost not induced at 1.0μM, regardless of the treatment time. When the concentration was less than 0.1μM, the lower the dosage and the later the application time was, the more the embryo was induced. The treatments of 0.1 μM on the 4th day after culture and 0.01 μM before culture led to embryo induction of around 50% of the control. Embryos induced from these two treatments and the control were transplanted to the MS2 medium for plant regeneration. Approximately 61, 57 and 77% of the embryos were developed into plants in control, in 4th day treatment, and in preculture treatment, respectively. Regenerated plants were divided into two groups, that is, normal plants consisted of the normal shoots and roots, and abnormal plants had hypertrophic or the vitrificated shoot without root. The ratio of the normal plant regeneration was 29, 26, and 35%, and 57, 43, and 59% of the abnormal plants transplanted to the MSK medium were converted into normal plants. The acclimatization rate of the normal plant was 87, 77, and 91%, and dihaploid plants which the chromosome number was doubled spontaneously during culture were 74, 76, and 85%. In consequence, the plants transplanted to soil after acclimatization were 41, 30, and 55%, and those of the dihaploids were 31, 23, and 47% out of the embryos subcultured first.
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