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The effect of low temperature pretreatment of buds or inflorescence on microspore culture for the production of haploids of Brassica rapa (syn. B.campestris) was examined. Incubation of the buds or the inflorescence at 4°C for 3 or 10 days before the culture of microspores induced efficient microspore embryogenesis. Pretreatment of flower buds was more effective than that of the inflorescence. Prolonged pretreatment up to 20 days promoted embryo induction. Microspores in the buds were examined for developmental stage before and after the pretreatment. Buds with a petal length to anther length ratio of about 0.5-0.7, which had microspores were at the late unicellular stage, were collected and were used. All microspores were at the late unicellular stage before the pretreatment. The percentage of the microspores at the stage of late unicellular decreased during the pretreatment while the percentage of bicellular stage microspores with two unequal size nuclei increased. At the same time, a small but notable number of bicellular stage microspores with equal size nuclei, which is the first step of microspore embryogenesis, was observed after the pretreatment.

Shoot-Forming Cultures. The shoot-forming cafli of D. purpurea L., which had been established in the previous study (5), were transferred into the liquid basal medium, supplemented with 1 mg/L BA and 1 mg/L IAA, and cultured in the light. Thus, green, shoot-forming cultures without root were established and subcultured every 3 weeks. White, shoot-forming cultures were obtained by sub-culturing a portion of the green, shoot-forming cultures in the dark.Culture Condition. Murashige and Skoog medium (9)-with 1 mg/L thiamin-HCl and without agar, edamin, IAA, and kinetinwas used as the basal medium. Approximately 1.5 g fresh weight of cells were inoculated into a 500-ml Erlenmeyer flask containing 100 ml of liquid medium and cultured in continuous light (fluorescent lamp: about 4 x 105 erg/s.cm2) or in the dark at 28°C on a reciprocal shaker (100 strokes/min; 2.0 cm in length).Measurement of Growth. Fresh weight was measured after removing culture medium by suction filtration. The harvested fresh culture was lyophilized, and its dry weight was determined.Assay for Digitoxin. Lyophilized cells (0.1-2 g) were homogenized with 50 ml ethanol in a glass homogenizer. The homogenate was heated at 74°C for 4 h and filtered. The filtrate was dried in vacuo, and the residue was taken up in 2 ml ethanol and diluted with 18 ml H20. When necessary, the extract was further diluted with H20 to a desirable level. Determination of digitoxin concentration of the extract was done by radioimmunoassays, as described (5).Assay for Chi. Chl content was determined spectrophotometric-

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