Polyploid breeding is an effective way to improve ornamental traits of horticultural plants. Colchicine, an anti-mitotic agent, is widely used to induce 2n gametes in horticultural plants, allowing for the production of polyploid plants through sexual hybridization. Based on an understanding of flower bud development and meiosis in pollen mother cells (PMCs) of Paeonia lactiflora cv. ‘Fen Yu Nu’, 2n pollen were obtained by injecting different concentrations of colchicine (0.1, 0.2, 0.3, 0.4 or 0.5%) into flower buds multiple times (once, twice or three times with a 24-h interval in between). External morphology, viability and possible hybridization of colchicine-induced 2n pollen were observed. Finally, P. lactiflora cv. ‘Lian Tai’ was pollinated with colchicine-induced pollen from ‘Fen Yu Nu’. Polyploids were identified among offspring by flow cytometry, chromosome counting, and molecular assessment using simple sequence repeats (SSR). Flower bud diameter of herbaceous peony was related with meiosis in PMCs. The most suitable period to induce 2n pollen was when most PMCs were in prophase I of meiosis, and when flower buds were about 11-12 mm in diameter. The highest induced incidence (%) of 2n pollen was 47.39% following treatment with 0.4% colchicine solution injected twice. The average diameter of 2n pollen was 46.01 μm, which was 1.53-fold wider than haploid pollen. SEM results indicated that 2n pollen were considerably different from haploid pollen in terms of external shape and surface decoration, and 2n pollen were mostly spherical or with an irregular shape. Induced 2n pollen could germinate in vitro and on the stigma of the female parent, but abnormalities such as pollen tube bifurcation or thickening were observed in both germinated states. The percentage of germinated 2n pollen in vitro was 12.78%. In addition, two tetraploids (2n = 4x = 20) were obtained by 2n pollen hybridization. SSR analysis indicated that tetraploids were produced by the spontaneous fusion of 2n female gametes with colchicine-induced 2n male gametes. This indicates that colchicine did not entirely inactivate 2n pollen. The production of few polyploids may be due to the abnormal germination of 2n pollen and obstacles in ploidy hybridization. Our findings fortify the foundation for Paeonia polyploid breeding.

多倍体育种是提高园艺植物观赏性状的有效途径。秋水仙碱是一种抗有丝分裂剂，广泛用于在园艺植物中诱导 2n 配子，从而通过有性杂交产生多倍体植物。基于对芍药花粉母细胞 (PMC) 花芽发育和减数分裂的了解。'粉玉女'，2n花粉是通过将不同浓度的秋水仙碱（0.1、0.2、0.3、0.4或0.5%）多次（一次、两次或三次，间隔24小时）注入花蕾中获得的。观察秋水仙碱诱导的2n花粉的外部形态、活力和可能的杂交。最后，P. lactiflora简历。'莲台'用'粉玉女'的秋水仙碱诱导花粉授粉。通过流式细胞术、染色体计数和使用简单序列重复 (SSR) 的分子评估在后代中鉴定出多倍体。芍药花芽直径与PMCs减数分裂有关。诱导2n花粉最适宜的时期是大多数PMC处于减数分裂前期，花芽直径约11-12毫米时。用0.4%秋水仙碱溶液注射两次后，2n花粉的最高诱导发生率（%）为47.39%。2n花粉的平均直径为46.01 μm，比单倍体花粉宽1.53倍。SEM结果表明，2n花粉在外形和表面装饰方面与单倍体花粉有很大不同，2n花粉多为球形或不规则形状。诱导2n花粉可以发芽在体外和母本的柱头上，但在两种萌发状态都观察到花粉管分叉或增厚等异常。2n花粉体外发芽率为12.78%。此外， 通过2n花粉杂交获得了两个四倍体（2 n  = 4 x = 20）。SSR分析表明，2n雌配子与秋水仙碱诱导的2n雄配子自发融合产生四倍体。这表明秋水仙碱并未完全灭活 2n 花粉。多倍体的产生可能是由于2n花粉的异常萌发和倍性杂交的障碍。我们的研究结果为芍药多倍体育种奠定了基础。