Li, Song, Guan and Li (2020). Seed Science and Technology, 48, 2, 303-314. https://doi.org/10.15258/sst.2020.48.2.17 Seed dormancy and germination in Cardiocrinum giganteum var. yunnanense, a perennial herb in China with post-dispersal embryo growth Ye-Fang Li1, Jie Song2, Wen-Ling Guan1* and Feng-Rong Li1 1 Faculty of Horticulture and Landscape, Yunnan Agricultural University, Kunming 650201, PR China 2 Flowers Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650204, PR China *Author for correspondent (E-mail: [email protected]) (Submitted February 2020; Accepted May 2020; Published online June 2020) Abstract Seeds of Cardiocrinum giganteum var. yunnanense, which is native to China, has underdeveloped embryos when dispersed from parent plants that did not grow until the second autumn and winter after exposure to summer temperatures. Radicles and cotyledons emerged in late winter and spring. Thus, a 15–16 month period was required from dispersal to seed germination. Under laboratory conditions, this period could be shortened to 5–6 months in a 25°C / 15°C (60 days) → 15°C / 5°C (60 days) → 5°C (60 days) temperature sequence. Based on dormancy-breaking requirements, the seeds have deep simple morphophysiological dormancy (MPD). This is practical knowledge for propagation of the species from seeds. Keywords: Cardiocrinum giganteum var. yunnanense, embryo development, morphophysiological dormancy, seed germination, temperature requirement Introduction Dormancy is a mechanism whereby seeds do not germinate during periods that may be favourable for germination but unfavourable for subsequent seedling establishment (Vleeshouwers et al., 1995). Differing climatic conditions are often reflected in the dormancy breaking requirements of seeds (Skordilis and Thanos, 1995). Some species have an underdeveloped embryo at the moment of dispersal, meaning that it has to grow within the seed before germination. These seeds are described as morphologically dormant (MD) or morphophysiologically dormant (MPD), with an additional physiological block preventing germination (Nikolaeva, 1977). Species with underdeveloped embryos are especially common in Liliales and certain Asparagales families (Kondo et al., 2006). Although dormancy has been studied in some monocotyledon taxa, data on embryo growth requirements are particularly scarce. © 2020 Li et al. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/licenses/by-nc/4.0 303 YE-FANG LI, JIE SONG, WEN-LING GUAN AND FENG-RONG LI The genus Cardiocrinum (Liliaceae s. str.) grows in the Sino-Himalaya, and consists of three species: C. giganteum, C. cathayanumm and C. cordatum. Cardiocrinum giganteum is distributed in Nepal, Sikkim, Bhutan into north-west Burma and south-west China. The variety found in Yunnan, C. giganteum var. yunnanense has bronze-coloured stems and greener flowers. Cardiocrinum cathayanum occurs from central to east China; and C. cordatum from Sakhalin Island to Japan (Wu and Peter, 2000). This group of species are bulbous plants in the Liliaceae, distinguished from the genus Lilium by their heart-shaped leaves and hapaxanthic life history (bulbs die after flowering, but produce new bulbous offsets, which root and grow independently). In China, C. giganteum var. yunnanense grows in forests (altitudes 1200-3600 m a.s.l.) in Gansu, Guangdong, Guangxi, Guizhou, Henan, Hubei, Hunan, Shaanxi, Sichuan and Yunnan (Wu, 1993; Wu and Peter, 2000). The sweetly scented flowers are enormous and appear in May-July. In addition to being an introduced ornamental in Yunnan, the species has edible and medicinal value (Liu, 1984; Pei and Long, 1998; Shou et al., 2018). Unfortunately, this lily resource has been declining rapidly due to changing environmental conditions over past decades and excessive harvesting of the wild plants from native fields in Yunnan (own unpublished survey data). To mitigate this undesirable situation, it is necessary not only to prevent further loss of the native plants, but also to reproduce them horticulturally. Cardiocrinum giganteum var. yunnanense can propagate by seeds or through division, although the asexual propagation rate by plant division is much lower than from seedlings. The main disadvantage to seed propagation is that the breeding of commercial plants from seedlings takes 5–7 years. Many growers have long awaited the development of a method to shorten the seedling production time. Seeds with an underdeveloped embryo at the time of dispersal and requiring specific temperature conditions over a prolonged period of time for embryo growth and for radicle or cotyledon emergence are described as having morphophysiological dormancy (MPD) (Nikolaeva, 1977; Baskin and Baskin, 1998, 2004). Seeds of Cardiocrinum var. glehnii have an underdeveloped embryo at dispersal and require a sequence of warm and cold temperatures to break dormancy and to germinate (Kondo et al., 2006), so seeds of this taxon have deep simple morphophysiological dormancy (MPD). Phartyal et al. (2012) reported that the seeds of the Himalayan Cardiocrinum giganteum var. giganteum from India has similar dormancy and germination requirements as C. cordatum var. glehnii from Japan. However, so far, there are no reports on the germination of C. giganteum var. yunnanense seeds. The primary objective of the study was to clarify whether the Cardiocrinum from China has the same characteristics for dormancy break and germination as the other Cardiocrinum species. In order to achieve these goals, two experimental studies have been carried out on seeds of C. giganteum var. yunnanense: (1) phenology of embryo growth and of seedling emergence outdoors in Kunming; (2) testing temperature requirements for dormancy break and for seedling emergence in laboratory conditions. The results were compared with those of previous reports on seed germination of C. cordatum var. glehnii from Japan by Kondo et al. (2006) and C. giganteum var. giganteum from India by Phartyal et al. (2012). 304 SEED DORMANCY IN CARDIOCRINUM GIGANTEUM VAR. YUNNANENSE Materials and methods Seed collection Eighty mature capsules were collected from ten plants in a population of Cardiocrinum giganteum var. yunnanense growing naturally in a moist subtropical evergreen broad- leaved forest woodland at an altitude of 2400 m a.s.l. in the Ailaoshan Mountain of Yunnan Province, China (101°01'E, 24°32'N), on 10 November 2015. Mean air temperatures in January and July at the nearest weather station (Ailaoshan National Ecosystem Observation Research Station Network, CERN, Yunnan; 2450 m a.s.l., 101°01'E, 24°32'N), were 5 and 15.3°C, respectively (China FLUX). The area is located in the southwest monsoon climate zone and is a subtropical mountain climate. Thus, November-April is the dry season, and May–October is the peak wet season in the natural habitat of the study species. The capsules harvested from different plants were combined and were put into non- woven fabric envelopes. The capsules arrived at Yunnan Agricultural University, Kunming, Yunnan, China on 11 November 2015 and were allowed to dry naturally in a laboratory (approximately 20°C) for three days, during which time the capsules dehisced. Then, seeds were collected by hand from the opened capsules and were spread onto porcelain dishes and allowed to dry naturally again at ambient room temperature (approximately 20°C) for three days. Undeveloped seeds were discarded. Only visibly well-developed dry seeds were put into plastic envelopes and stored in a drying basin with silica gel at 4°C until used in germination studies. Phenology of embryo growth and of seed germination When a seed of C. giganteum var. yunnanense germinates, the radicle emerges first and then the cotyledon emerges. In this study, the criteria for radicle and cotyledon emergence were when the radicle tip had emerged 1 mm or more from the seed coat (figure 1A) and when the cotyledon tip had completely emerged above ground (figure 1B). (A) (B) radicle Figure 1. Seedling emergence in Cardiocrinum giganteum var. yunnanense. (A) growing radicle inside seed; (B) cotyledon emergence complete. 305 YE-FANG LI, JIE SONG, WEN-LING GUAN AND FENG-RONG LI The phenology of embryo growth and of radicle and cotyledon emergence was scored in a non-temperature-controlled shade shed located outdoors on the campus of Yunnan Agricultural University. In order to simulate conditions in a native forest habitat, the shade shed was covered with shade cloth throughout the year, so that illuminance inside this structure was about 30% of that in the open (measured with an illuminance meter). Pots and trays containing the seeds (see later in this section) were placed in the shade shed. Soil (1:1 v/v mixture of pearl rock and peat) in the tray was kept moist throughout the experiment. Monthly mean, maximum and minimum temperatures were collected from the nearest weather station (Jindian Reservoir, Kunming). The study continued from 20 November 2015 through 10 April 2017. Embryo development On 20 November 2015, about 30 seeds were placed in each of 20 fine-mesh polyester bags and buried at a soil depth of 30 mm in a tray in the shade shed. On the same day, ten seeds were used to observe embryo development and the initial embryo length of each seed was measured with a micrometer after scraping the seed coat with a scalpel under an anatomical microscope. Subsequently, one bag was removed at random from the tray at about 30-day intervals until 18 March 2017 and embryo length for each of ten seeds was measured using a dissecting microscope equipped with a micrometer. After 20 November 2016, when embryos began to curve (figure 2D), embryos were cut into 3–4 straight sections and their lengths measured and summed to get total embryo length.