Applied stress is often used to induce embryogenesis in microspore culture. Cold pretreatment has been used in cereal crops, Brassica napus and Brassica rapa microspore cultures, but seldom attempted in Brassica oleracea microspore culture. In this study, the effect of cold pretreatment (4°C) on embryogenesis in microspore culture and ploidy of regenerants resulting from those cultures was investigated in three broccoli cultivars ÔGreen EliteÕ, ÔGreen StarÕ and ÔTI-111Õ. Cold pretreatment for 1, 2 or 3 days was effective when applied alone using these three cultivars, but the frequency of embryos derived from microspores was low. The combination of cold pretreatment (4°C) for 1 or 2 days and heat shock (32.5°C) for 1 day significantly enhanced microspore embryogenesis efficiency, especially with ÔTI-111Õ for which it was increased by 63-72-fold. In addition, the combined treatment resulted in higher diploid frequency of the regenerated population compared to traditional microspore culture protocol which typical only uses heat shock at 32.5°C for 1 day in broccoli.

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