此研究最主要目的是利用花粉細胞或花藥建立F1青花菜雙單倍體植株組織培養技術，五種F1青花菜AVBR1101、 B45、KY、AVBR1102和AVBR1103為試驗品種，只有這些植株種植在生長箱設在20/15°C日夜溫16小時光照，將三種不同時期的花粉細胞(單核、雙核及三核)進行花粉細胞培養及兩種不同的大小(2-3 mm 和 3-4 mm)的花苞進行花藥培養， 配子體細胞建立雙單倍體植株主要利用全濃度NLN-13培養基分離花粉細胞再用半濃度NLN-13培養，其中也只有B45和AVBR1102有成功誘導配子體體胚。品種、花苞大小、培養基質(Murashige & Skoog和B5 Gamborg培養基)及賀爾蒙種類(2,4-dichlorophenoxyacetic acid 和 1-naphthaleneacetic acid)的不同處理組合對花藥癒傷組織誘導有明顯的影響，例如AVBR1101、 B45和KY成功從癒傷組織得到植株，而且用2-3 mm花苞和B5培養基用AVBR1101(54%)和KY(62.5%)兩個品種比起其他品種癒傷組織誘導率最高。在培養基加活性碳只對AVBR1101有正面影響癒傷組織誘導率，對其他品種甚至有負面影響。事先花苞的4C冷處理也只對AVBR1101有促進效果。本研究也利用SSR分子標誌、流式細胞儀、計算染色體及氣孔保衛細胞內葉綠體數目及氣孔數目及大小及表觀特徵等技術鑑定雙單倍體植株，目前利用SSR分子標誌及表觀特徵成功鑑定本研究所建立的雙單倍體植株。

The purpose of this research is to compare microspore and anther culture based doubled haploid (DH) development protocols with the aim for routine production of DH F1 broccoli (Brassica oleracea L. var. italica) plants. Five broccoli F1 hybrids AVBR1101, B45, KY, AVBR1102, and AVBR1103 were used to test DH protocols. Hybrid plants were grown in growth chambers with a 16/8 h light/dark cycle at 20/15°C, respectively. Flower buds containing three microspore developmental stages (uninucleate, binucleate and trinucleate) and two flower bud sizes (2-3 mm, 3-4 mm) were used for microspore and anther cultures, respectively. For microspore culture two kinds of growth media, full and half strength NLN-13 medium, were used. In total ten plants were successfully regenerated from B45 and AVBR1102. For anther culture, in addition to variation in bud size and growth media strength, various combinations of the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) were tested. In total seven plants were regenerated from anther culture from the hybrids AVBR1101, B45 and KY. Callus induction was highest when anthers were isolated from buds of 2-3mm and were grown on B5 basal medium. Hybrids AVBR1101 (54%) and KY (62.5%) explants produced callus with highest frequency. Adding active charcoal to the growth medium showed no effect on callus induction in AVBR1101, and had an inhibitory effect in B45 and KY. The effect of precold treatment varied with the genotype. Precold treatment increased the callus induction in AVBR1101, decreased in KY, and had no effect in B45. DNA ploidy determination of regenerated plants was first done by genotyping with SSR markers showing heterozygote genotype in hybrids and homozygote genotype in DH plants. Other methods such as flow cytometry, chromosome counting, and phenotyping by measuring stomata number and size, and seed pods production were also performed for DH validation. SSR marker genotypes indicated that all microspore regenerated plants were haploid, while all plants regenerated from anther culture were in fact diploid. The flow cytometry experiment failed to confirm these results, as the cuticular tissues interfered with the measurements. Stomata and seed pod formation are indications that the ploidy of microspore regenerants duplicated spontaneously.

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