Induction of 2n pollen by colchicine during microsporogenesis to produce polyploids in herbaceous peony (Paeonia lactiflora Pall.)

• The optimal treatment for inducing 2n pollen was 0.4% colchicine solution injected twice in herbaceous peony. • Colchicine increased the diameter of pollen in herbaceous peony. • Induced 2n pollen could germinate in vitro and on the stigma in herbaceous peony. • It is feasible to use colchicine-induced 2n pollen to produce polyploids in Paeonia. 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 (2 n = 4 x = 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.