In 2001, prior to the impoundment of the Three Gorges Reservoir on the Yangtze River, China, Plantago fengdouensis (Plantaginaceae) was discovered along the flood-prone banks of Fengwei Dam in Fengdu County (Zhao et al., Reference Zhao, Wu and Wang2002). Initially classified as a variety of Plantago asiatica, subsequent morphological analyses led to its recognition as a distinct species (Wang et al., Reference Wang, Meng, Li, Wang and Tao2004a). Following the completion of the Three Gorges Dam in 2009, the species’ entire known natural habitat, restricted to three isolated mid-river islets in Chongqing Zhongxian, Fengdu and Banan districts, was permanently submerged (Wang et al., Reference Wang, Li, Wu and Huang2004b). Consequently, P. fengdouensis was declared extinct in the wild as a result of the dam’s construction (Li et al., Reference Li, Wei, Hoggard, Wu, Raven and Hong2011). However, in 2023, a population of c. 200 clusters was discovered on Chenghua Island, Jiang’an County, Sichuan Province, in the Yangtze River, c. 291 km from Fengwei Dam (Fig. 1; Gao & Chen, Reference Gao and Chen2024). This finding significantly extended the species’ known distribution, but the population was vulnerable to summer flooding and extreme temperatures. Plantago fengdouensis is listed as a second-class protected plant in China, but has not yet been assessed on the IUCN Red List of Threatened Species (National Forestry and Grassland Administration of China, 2021).

Fig. 1

Chenghua Island, Jiangan County, Sichuan Province, China, showing the locations of the eight extant Plantago fengdouensis populations. The conservation area indicates where we reintroduced P. fengdouensis from propagated seedlings (see text for details).

Anticipating complete loss of the habitat of P. fengdouensis from the Three Gorges Project, researchers implemented ex situ conservation at Three Gorges Botanical Garden (Wu et al., Reference Wu, Li, Zhang, Ma, Yang, Huang and Zhang2022). Comparative flooding experiments demonstrated that the roots of P. fengdouensis exhibited greater tolerance to waterlogging stress than those of P. asiatica (Yang et al., Reference Yang, Han, Li, Guo and Chan2015). Investigation of flowering characteristics, the reproductive system and pollination adaptations identified heterodichogamy and a dual pollination system (combining anemophily and entomophily), traits that likely enhance survival in environments prone to summer flooding (Wan et al., Reference Wan, Deng, He, Jiang and Liu2018), and the species’ chloroplast genome was sequenced (Wang et al., Reference Wang, Mao, Ding, Li, Fu and Deng2020). The Shenzhou XIII manned spacecraft carried seeds of P. fengdouensis in 2021 for mutation breeding. The aim was to induce favorable mutations in the seeds through microgravity and cosmic radiation. After 183 days, the seeds were returned to Earth, but germination success remained relatively low.

Since 2023, there has been a focus on improving germination and survival rates through cultivation and reintroduction, with preliminary successes (Gao & Chen, Reference Gao and Chen2024). In October 2023, seedling propagation trials used seeds collected from eight populations of P. fengdouensis on Chenghua Island. Eighty mature dry seeds were placed on moist filter paper in Petri dishes and incubated at a constant temperature of 26 °C. After five days, no germination occurred, with some seeds exhibiting decay symptoms. Dissection of ungerminated seeds revealed intact embryos (plumule, radicle and cotyledons), confirming structural integrity, but noted the hardened testae. In germination of Plantago seeds for agricultural purposes, the seeds are rubbed with sand to make the seed coats thinner and they then germinate at 20–30 °C (Yang et al., Reference Yang, Han, Li, Guo and Chan2015). We attempted this for P. fengdouensis, but the seeds failed to germinate. Additional mechanical scarification via scalpel incisions in the testae also failed to induce germination, eliminating physical dormancy as the primary constraint to germination. We subsequently broke dormancy with gibberellic acid solutions of 0.5, 1.0, and 1.5% concentrations with 24 h soaking. By day 3 post-treatment, we observed partial radicle emergence in some seeds, although germination rates remained suboptimal.

In May 2023, to obtain further seeds for germination experiments, we collected five fruiting P. fengdouensis from Chenghua Island. We harvested 273 seeds from these five plants, of which we used 40 immature seeds to test germination viability under previously successful conditions: 24 h soak in gibberellic acid solution followed by Petri dish incubation. By day 3, the radicles of three seeds emerged, with germination observed on days 4 and 5. In total, 29 of the 40 seeds germinated within 1 week. This suggested that persistently low germination may be a result of seed maturity status, with immature seeds potentially exhibiting higher viability. The remaining seeds were subjected to comparative experiments with several gibberellic acid concentrations (0.5, 1.0, 1.5%), temperatures (25, 28 °C), and light intensities (1,000, 1,500, 2,000, 3000 Lux). Optimal germination conditions were soaking in 1.0% gibberellic acid for 24 h at 28 °C and 1,500 Lux, with an average germination rate of 87.5% (Tables 1 & 2). The maximum germination rate was reached on the third day of germination.

Table 1

Per cent seed germination rates of Plantago fengdouensis under various light intensities and gibberellic acid (GA) concentrations, at 25 °C.

Table 2

Per cent seed germination rates of P. fengdouensis in five replications under various temperatures and gibberellic acid (GA) concentrations, at 1,500 Lux.

We then investigated cultivation conditions. A substrate mixture containing 60% river sand was used to simulate native soil conditions. We selected seeds with an embryo root length of 1–2 cm, and transplanted the embryo roots to c. 1 cm below the soil substrate. Seedlings were maintained in a greenhouse at 28 °C. Vigorous growth ensued, with three seedlings emerging by day 3 post-transplantation, successive emergence throughout the following week, and a 98.5% survival rate.

However, the warm environment of the greenhouse enabled the aphid eggs in the soil to hatch and infest the plants, causing the leaves to wrinkle. Imidacloprid solution was applied to eliminate the aphids, but subsequently the seedlings exhibited high susceptibility to powdery mildew, a fungal disease with conidia spread aerially, infecting adjacent plants. Successive applications of two fungicides was ineffective, but triadimefon, a fungicide typically used for wheat, demonstrated efficacy against the powdery mildew affecting P. fengdouensis. This highlights the need for species-specific management protocols in ex situ conservation.

By January 2025, these seedlings had grown 8–9 leaves and their root systems had fully developed. We selected 150 healthy seedlings and transplanted them to Chenghua Island. The transplants exhibited vigorous growth (Plate 1). In June 2025, survival rate was 86%. A timeline from discovery of the species through to this successful reintroduction is illustrated in Fig. 2.

Plate 1

Plantago fengdouensis (a) in cultivation, and (b) growth in situ 12 months after transplantation to Chenghua Island (Fig. 1).

Fig. 2

Timeline of the discovery, putative extinction, rediscovery and subsequent conservation of P. fengdouensis, from its discovery in 2001 through to successful in situ transplantation to Chenghua Island (Fig. 1) in 2025.

We hypothesize that the reasons for the restricted range and small natural population of P. fengdouensis are (1) higher germination rates of immature versus mature seeds combined with poor natural seed abscission, (2) limited dispersal capacity because of dwarf plant stature and large, smooth seeds unsuited for wind or water dispersal, and (3) habitat contraction exacerbated by the formation of the Three Gorges Reservoir.

Following successful reintroduction into the wild, future research needs to focus on monitoring the survival rate of the transplanted seedlings, and optimizing cultivation from seeds, to achieve large-scale reproduction. Although the individual methods we used are not novel, their successful application to P. fengdouensis, a species previously considered extinct in the wild, is a novel contribution to the conservation of floodplain species. The key to successful germination lies in the use of immature seeds, a finding that may inform conservation efforts for other species with similar dormancy traits.