Influence of Preculture Variables on Microspore Embryo Production in Brassica napus ssp. oleifera cv. Duplo

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Abstract
The effect of four preculture variables on microspore embryo induction and growth were examined: (1) the source of the bud selected for culture (apical or axillary inflorescence; (2) the method of harvest (single harvest of whole inflorescence or sequential harvest of individual buds; (3) the length of the bud (2, 3 or 4 mm); and (4) the application of a 4 °C pretreatment to the bud after harvest. Microscopic and macroscopic analysis of every anther used for culture permitted an assessment of the following parameters: (1) the percentage of induced buds; (2) the number of induced anthers per induced bud; (3) the number of productive buds (with macroscopic embryos) as a percentage of the induced buds; (4) the degree of induction per induced anther (an estimate of the number of microspores in which initial embryogenic divisions had commenced); and (5) embryoid survival (the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of induced anthers and all five parameters were components of the final yield - the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67·0 per cent) occurred with 2 mm sequentially harvested non-pretreated buds. Overall, the values decreased with increasing bud length and were lower for pretreated and axillary buds. In contrast, the two other estimates of induction - number of induced anthers per induced bud and degree of induction per induced anther - both had maximum values from 3 mm sequentially harvested, pretreated buds from apical inflorescences. The highest final yield of embryos per cultured bud (44·9) was found with 2 mm non-pretreated buds taken from a single harvest of the apical inflorescence. The study therefore confirmed that the different components of the final embryo yield are differentially affected by the four preculture variables tested. These variables must be controlled if reproducible results are to be achieved.