Lan, Li, Duan and Gao (2019). Seed Science and Technology, 47, 1, 13-24. https://doi.org/10.15258/sst.2019.47.1.02 Effects of pappus removal and low-temperature short-term storage on interspecific and intraspecific variation in seed germination of Luobuma Yanru Lan1, Tao Li1, TingYu Duan1* and Peng Gao2 1 State Key Laboratory of Grassland Agro-ecosystems; Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs; Collage of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, Gansu, China 2 College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi Province 030801, P.R. China * Author for correspondence (E-mail: [email protected]) (Submitted October 2018; Accepted January 2019; Published online February 2019) Abstract Seed germination rarely occurs in Luobuma (Apocynum venetum, Poacynum pictum and P. hendersonii) under natural conditions. The present study was conducted to assess interspecific and intraspecific variation in seed germination and the effect of pappus removal and low-temperature short-term storage on seed germination of Luobuma. The study identified significant differences in germination percentage, germination index, time to 50% germination, bud length and root length between three Luobuma species Apocynum venetum, Poacynum pictum and P. hendersonii, and between six ecotypes of P. pictum. The highest seed germination percentage was recorded in A. venetum (> 90%) followed by P. pictum (53–66%), while the lowest was recorded in P. hendersonii (26–34%). Among the different ecotypes of P. pictum, Pp-BMX recorded the highest seed germination percentage, while Pp-BMQ recorded the lowest. In addition, the intraspecific variation in germination was considerably more than the interspecific variation under different treatments. Pappus removal increased the germination percentage of Luobuma seeds, which indicates that pappus acts as a mechanical barrier to germination. Low temperature significantly reduced (P < 0.05) the time to 50% germination of A. venetum and P. hendersonii seeds compared with seeds with and without pappus. Keywords: intraspecific, interspecific, low-temperature storage, Luobuma, pappus Introduction Luobuma are perennial herbaceous or half-shrub medicinal plants widely distributed in Central Asia and northwest China. This group of plants include two genera with three species Apocynum venetum L., Poacynum pictum Baill. and P. hendersonii Woodson (Jiang and Li, 1977). These plants are commonly used in tea, medicine, the textile © 2019 Lan 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 13 YANRU LAN, TAO LI, TINGYU DUAN AND PENG GAO industry and eco-friendly construction (Xie et al., 2012). A. venetum leaf extract (AVLE) is particularly well-known for its antihypertensive, cardiotonic, hepatoprotective, free radical scavenging, antidepressant and anxiolytic effects (Zhang, 2004; Xie et al., 2012). They are widely distributed in low temperature, semi-arid regions with saline-alkali soil, riverbanks, fluvial plains and sandy soils (Thevs et al., 2012). Luobuma can grow up to 4 m tall and its horizontal roots can extend up to 6 m (Zhang et al., 2006; Thevs et al., 2012). Apocynum venetum and Poacynum spp. are known as red hemp and white hemp, respectively, based on the colour and shape of their flowers and leaves (Su et al., 1997; Xie et al., 2012; Gao et al., 2015). They also differ in seed morphological characteristics such as size, shape, colour and surface ornamentation (Ma et al., 2000). Seed germination is a critical growth phase that influences population regeneration in many plant species (Khan and Ungar, 1996; Keiffer and Ungar, 1997). In Luobuma, seeds rarely germinate under natural conditions in the field. Instead, Luobuma plants reproduce vegetatively through roots (Gao et al., 2015). This may affect maintenance and expansion of Luobuma populations in the natural ecosystem. However, studies have reported seed germination as high as 84% in Luobuma (Qian et al., 2016). Soaking A. venetum seeds in water at 40 to 50°C improved germination from 55 to 64.3% (Qian et al., 2016). Research has also shown that Luobuma seeds germinate normally under natural light (Zhang et al., 2015) at temperatures ranging from 16.6 to 45.9°C, with an optimum temperature of 25.0 to 30.0°C (Zhang et al., 2011; Rong et al., 2015). However, A. venetum seeds could not germinate and grow normally at temperatures below 15°C and above 40°C (Du, 2018). Temperature is an important factor that affects the final germination percentage and the mean germination time (Bellairs and Bell, 1990; Cony and Trione, 1996; Yamauchi and Yamaguchi, 2004), as well as the level of dormancy acquired during seed maturation (Fenner, 1991). Previous studies have shown that low temperature affects metabolic processes by slowing down enzyme activity and by improving mobilisation of total lipids and protein reserves (Nykiforuk and Johnson-Flanagan, 1993). Studies mainly focused on the effects of low temperature (−4 to +4 °C) on seed germination. Yamauchi and Yamaguchi (2004) reported the activation of gibberellin biosynthesis and response pathways during seed imbibition at low temperature. Li et al. (2007) found that germination in Luobuma is related to the processing temperature before sprouting ; a processing temperature of −16 or 4°C resulted in high germination percentage. Pappus (also referred as pilose) is a hairy structure in the germinal aperture of Luobuma seeds that plays an important role in wind dispersal (Hua, 2017). Reports on the effect of manual removal of pappus on seed germination are limited. Miguel et al. (2017) showed that Centaurea solstitialis L. produces two types of seeds, seeds without pappus and seeds with an intact pappus. However, they did not study the relationship between pappus and seed germination. Our study evaluated the effect of intact pappus and manual removal of pappus on Luobuma seed germination. Although Luobuma rarely reproduces via seeds under natural conditions, the seeds are widely used for cultivation (Gao et al., 2015). High germination percentage and high seedling vigour are important for population growth. It is important to address the effect of low temperature and pappus on Luobuma seed germination, which could form the basis for subsequent studies aiming to enhance expanison of Luobuma populations. 14 PAPPUS REMOVAL AND LOW TEMPERATURE AFFECT SEED GERMINATION OF LUOBUMA Therefore, differences in seed germination between the three species (A. venetum, P. pictum and P. hendersonii) and between the six ecotypes of P. pictum were evaluated in the present study. In addition, we compared the germination potential of the seeds of A. venetum, P. pictum and P. hendersonii, and of the six ecotypes of P. pictum under low temperature and after pappus removal to identify the most suitable species for field cultivation in terms of high final germination percentage and short mean time to maximum germination. We hypothesised that there is more interspecific variation in Luobuma seed germination than intraspecific. We also hypothesised that low-temperature, short-term storage decreases seed germination, while the removal of pappus increases seed germination. Materials and methods Experimental site and seed collection The experimental site was set up at Alakak township, Altay Prefecture, Xinjiang Uyghur Autonomous Region, China (87°33'50" E, 47°42'41" N). This area has a temperate monsoon climate with an average annual rainfall of 126.7 mm and an average annual temperature of 4.5°C. The highest and lowest temperatures recorded are 45 and −41°C, respectively. A nursery comprising three Luobuma species (A. venetum, P. hendersonii and P. pictum), and six ecotypes of one specific species (P. pictum) was established in 2009. Taxonomy and phenotype of the different species/ecotypes are presented in table 1 (Gao et al., 2015). Seeds of Luobuma were sown in the nursery, and seedlings were transplanted from the nursery to the field. A. venetum, P. hendersonii and the six ecotypes of P. pictum were planted in an area of about 0.067 ha with regular manual weeding. The plants were transplanted into the field at a spacing of 1 m within rows and 1 m between rows, and watered by a drip irrigation system every six days. Mature seeds were collected from at least ten individual shrubs in October 2016. Seeds within the fruit pods were allowed to dry naturally and stored in paper bags at room temperature for 12 months. Measurement of seed morphological characteristics Ten fruit pods per species/ecotype were opened by hand; 200 (4 replicates × 50 seeds) seeds were used to determine seed length and seed breadth using Vernier calipers. Seed size was calculated using the formula: Seed size = seed length × seed width × seed depth. Thousand (10 replicates × 100 seeds) seeds were used to measure thousand seed weight (TSW). Germination procedure Seeds of each species/ecotype were removed from the pods, and the seeds with pappus were placed on potato dextrose agar (PDA) in Petri dishes at 23 ± 1°C to test the presence of culturable pathogenic fungi or bacteria. Since no fungi or