Effects of Pappus Removal and Low-Temperature Short-Term Storage on Interspecific and Intraspecific Variation in Seed Germination of Luobuma

Home , Apocynum venetum, Pappus (botany)

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 bacteria were isolated, seeds used in subsequent tests were not surface-sterilised. The pods were not opened until the start of the germination test to avoid contamination. Filter papers and Petri dishes were autoclaved at 121°C for 20 minutes. For each species/ecotype, three types of seeds were

15 YANRU LAN, TAO LI, TINGYU DUAN AND PENG GAO

Table 1. Morphological characteristics of three Luobuma species and six of ecotypes of Poacynum pictum population. Flower Flower Plant Leaf Leaf Stem Taxonomic status Code size colour type type colour colour

Apocynum - Small Pink Upright Elliptical Dark green Green- venetum and ovate fuchsia

Poacynum pictum Pp-BMQ Medium Fuchsia Upright Ovate and Dark green Turquoise elliptical

P. pictum Pp-BMH Medium Fuchsia Upright Ovate Green Fuchsia

P. pictum Pp-BMZ Medium Fuchsia Lodging Ovate Dark green Purple spots

P. pictum Pp-BMB Medium White Upright Ovate Bright green Turquoise

P. pictum Pp-BMK Medium Pink Upright Ovate and Green Green oblong

P. pictum Pp-BMX Medium Fuchsia Upright Ovate Green Green

P. hendersonii - Large White Lodging Ovate and Bright green Turquoise elliptical sown: intact seeds, seeds with pappus removed by hand and seeds stored at −20°C for seven days. Twenty-five seeds were sown in six Petri dishes with two layers of sterilised filter paper moistened with 5 ml of sterile water and incubated at room temperature. One mL of sterile distilled water was added to each dish every three days to keep the filter paper moist. Total seed germination was recorded daily. The germination percentage, germination index and time to 50% final germination (T50) of each species/ecotype were calculated using the following equations (Farooq et al., 2005; Feng et al., 2018):

Germination index = Σ (Gt / Dt) where, Gt represents the number of germinated seeds on t day and Dt represents the germination days: T50 = ti + [(N/2ni)(tj−ti) / (nj−ni)] where, N is the final number of germinated seeds, and ni and nj are the cumulative number of seeds germinated by adjacent counts at times ti and tj, respectively, when ni < N/2 < nj. Bud length and root length were measured on the last day (the tenth day) of the experiment.

Seeds were considered germinated once the radical was 2 mm long.

Statistical analysis The experiment involved a completely randomised design with six replicates. Germination percentage was arcsine-transformed to stabilise the variance. All results are presented as mean values (six replicates ± standard error of the mean, SE). Two-way ANOVA was used to determine the effect of species or ecotypes and treatments on germination parameters. Tukey’s honestly significant difference (HSD) procedure was used to compare and determine the significant differences between treatment means at P < 0.05. Statistical analysis was performed using the software SPSS v.17.0 (USA).

16 PAPPUS REMOVAL AND LOW TEMPERATURE AFFECT SEED GERMINATION OF LUOBUMA

Results

Measurement of seed morphological characteristics Species/ecotypes of Luobuma differed significantly in seed length, width and size, and

in TSW (P < 0.05; table 2). Seed length, width and size, and TSW ranged from 2.4 to

4.0 mm, 0.4 to 0.8 mm, 0.3 to 3.0 mm, and 358.4 to 1382.6 mg, respectively. A. venetum seeds recorded the lowest and P. hendersonii seeds recorded the highest values of these parameters. Among the six ecotypes of P. pictum, Pp-BMQ recorded the lowest seed length, seed width and TSW.

Table 2. Basic seed characteristics of three Luobuma species and six ecotypes of Poacynum pictum. Seed length Seed width Seed size Thousand seed weight Taxon Code (mm) (mm) index (TSW) (mg)

Apocynum venetum - 2.37 ± 0.07 c 0.35 ± 0.07 b 0.29 ± 0.01 b 358.4 ± 0.00 c

Poacynum pictum* - 3.64 ± 0.11 b 0.63 ± 0.03 ab 1.42 ± 0.02 ab 676.5 ± 0.00 b

Poacynum hendersonii - 3.97 ± 0.07 a 0.82 ± 0.14 a 2.99 ± 0.10 a 1382.6 ± 0.02 a

P. pictum Pp-BMX 3.82 ± 0.11 a 0.60 ± 0.01 ab 1.37 ± 0.02 a 659.1 ± 0.00 c

P. pictum Pp-BMK 3.64 ± 0.16 a 0.72 ± 0.06 ab 1.90 ± 0.04 a 690.7 ± 0.00 c

P. pictum Pp-BMH 3.96 ± 0.04 a 0.88 ± 0.18 a 3.04 ± 0.04 a 806.6 ± 0.00 a

P. pictum Pp-BMQ 3.11 ± 0.18 b 0.54 ± 0.02 b 0.89 ± 0.17 a 483.8 ± 0.00 d

P. pictum Pp-BMB 3.65 ± 0.05 a 0.62 ± 0.02 ab 1.39 ± 0.01 a 666.0 ± 0.00 c

P. pictum Pp-BMZ 4.02 ± 0.03 a 0.58 ± 0.04 ab 1.36 ± 0.03 a 751.4 ± 0.00 b * mean of the six ecotypes.

Same lowercase letters mean data do not differ significantly for the different species/ecotypes at P  0.05 by Tukey’s HSD.

Germination of different Luobuma species The three species of Luobuma differed significantly in seed germination percentage, germination index, time to 50% germination, bud length and root length (P < 0.05). Germination percentage and germination index of the seeds were highest in A. venetum

(> 90%; 24.2–39.8, respectively) and lowest in P. hendersonii (26–34%; 5.6–7.8, respectively) over all the treatments (P < 0.05). Seed germination in P. pictum ranged from 53.0 to 66.0% (figure 1A, B). Germination percentage, bud length and root length were not significantly affected by the different treatments in any of the species studied. However, the removal of pappus significantly (P < 0.05) affected the germination index of A. venetum (12.8% increase) and P. pictum (10.1% increase) seeds (figure 1B). The time taken to 50% germination

(T50) ranged from 1.7 to 5.0 days in the three species over all the treatments (figure 1C).

T50 of P. hendersonii seeds was significantly more than that of A. venetum and P. pictum seeds with or without pappus (P < 0.05). Low temperature significantly reduced (P < 0.05) the T50 of A. venetum and P. hendersonii seeds. T50 of P. hendersonii seeds with pappus

17 YANRU LAN, TAO LI, TINGYU DUAN AND PENG GAO

140 (A) Apocynum venetum Poacynum pictum P. hendersonii 120 a a 100 a

80 b b 60 b c 40 c c Germination (%) 20

0 Non-pappus Pappus -20°C + Non-pappus

50 (B) 45 a 40 a 35 30 b b 25 bc 20 15 cd d Germination index 10 d d 5 0 Non-pappus Pappus -20°C + Non-pappus

7 (C) 6 a 5

b 4 bc bcd cd

(days) 3 d cd e 2 e

1 Time to 50% germination

0 Non-pappus Pappus -20°C + Non-pappus

Treatment

Figure 1. Germination percentage (A), germination index (B), and time to 50% germination (C) of three Luobuma species under different treatments. Different lowercase letters indicate significant differences within or across the same treatments at P < 0.05 by Tukey’s HSD.

18 PAPPUS REMOVAL AND LOW TEMPERATURE AFFECT SEED GERMINATION OF LUOBUMA

was significantly high (P < 0.05) compared with seeds without pappus. Bud length ranged from 8.0 to 23.1 mm and root length ranged from 2.5 to 6.9 mm in the three species. Bud length and root length of P. hendersonii was shorter compared with the other two species (P < 0.05); A. venetum and P. pictum had similar bud and root lengths (figure 2).

35 (A) Apocynum venetum Poacynum pictum P. hendersonii

a 25 a a a a b 20

15 c 10 b b Bud length (mm)

0 Non-pappus Pappus -20°C + Non-pappus

10 9 (B) 8 a 7 a a 6 a 5 ab b 4 b 3 b b

Root length (mm) 2 1 0 Non-pappus Pappus -20°C + Non-pappus Treatment

Figure 2. Bud length (A) and root length (B) of three Luobuma species under different treatments. Different lowercase letters indicate significant differences within the same treatments at P < 0.05 by Tukey’s HSD.

Germination of six ecotypes of Poacynum pictum Germination percentage, germination index, bud length, and root length of the six ecotypes of P. pictum differed significantly (P < 0.05). Germination percentage ranged from 21.3 to 96.7% and germination index ranged from 7.1 to 33.2. Germination percentage and germination index were the highest for Pp-BMX (84.0–96.7%; 16.9–33.2) and the lowest for Pp-BMQ (21.3–40.0%; 7.1–9.9) in each treatment (figure 3A, B). Bud length ranged from 16.7 to 26.2 mm and root length ranged from 2.8 to 9.7 mm (figure 4). Seed germination percentage of the ecotypes was not significantly influenced by the treatments, except for Pp-BMH ecotype. Pappus removal significantly influenced the germination percentage of Pp-BMH ecotype (P < 0.05). Germination percentage of

19 YANRU LAN, TAO LI, TINGYU DUAN AND PENG GAO

140 (A) Pp-BMX Pp-BMK Pp-BMH 120 Pp-BMQ Pp-BMB Pp-BMZ a 100 ab abc abcd 80 abcd bcdef bcdef bcdef bcdef cdefg 60 efgh defgh cdefg fgh fgh fgh 40

Germination (%) h gh 20

0 Non-pappus Pappus -20°C + Non-pappus

50 45 (B) 40 35 a ab 30 abc bcd bcd 25 bcde cde cdef defg defg 20 efgh efgh gh 15 fg gh fgh gh Germination index 10 h 5 0 Non-pappus Pappus -20°C + Non-pappus

7 (C) 6

5 a a 4 ab bc de cd cde cde 3 de de

(days) de de cde e de de de e 2

1 Time to 50% germination 0 Non-pappus Pappus -20°C + Non-pappus Treatment Figure 3. Germination percentage (A), germination index (B), and time to 50% germination (C) of six ecotypes of Poacynum pictum under different treatments. Different lowercase letters indicate significant differences across the treatments at P < 0.05 by Tukey’s HSD.

20 PAPPUS REMOVAL AND LOW TEMPERATURE AFFECT SEED GERMINATION OF LUOBUMA

40 Pp-BMX Pp-BMK Pp-BMH 35 (A) Pp-BMQ Pp-BMB Pp-BMZ 30 a a a a a ab a 25 a a ab bc a b a b b b 20 b 15

Bud length (mm) 10

0 Non-pappus Pappus -20°C + Non-pappus

14 (B) 12 a a 10 ab ab abc abc abcd abcd 8 abcd abcd bcde 6 cde cde e de e 4 e e

Root length (mm) 2

0 Non-pappus Pappus -20°C + Non-pappus Treatment Figure 4. Bud length (A) and root length (B) of six ecotypes of Poacynum pictum under different treatments. Different lowercase letters indicate significant differences across the treatments at P < 0.05 by Tukey’s HSD.

Pp-BMH ecotype seeds with pappus was 46.7% and with low-temperature treatment was 42.0%. The seeds without pappus recorded 77.3% germination, which was 30.7 and 35.3% higher compared with the seeds with pappus and low-temperature treatment, respectively (figure 3A). However, the seed germination index of the ecotypes were significantly different, except for Pp-BMQ ecotype. Removal of pappus significantly increased the germination index of Pp-BMX (16.3), Pp-BMK (9.5), Pp-BMH (16.8) and Pp-BMZ ecotypes (12.2) compared with the seeds with pappus. In addition, the germination index of the seeds of Pp-BMX, Pp-BMB, and Pp-BMZ ecotypes given low-temperature treatment was significantly higher (P < 0.05) compared with the seeds with pappus (figure 3B). The time taken to 50% germination for the six ecotypes ranged from 2.4 to 4.1 days

(figure 3C). Pappus removal reduced the T50 of Pp-BMX, Pp-BMH and Pp-BMB ecotypes by 0.7 to 1.5 days compared with seeds with pappus (P < 0.05); however, pappus removal had no impact on Pp-BMK and Pp-BMQ ecotypes. Additionally, the T50 of seeds given low-temperature treatment was less compared with seeds with pappus in Pp-BMX, Pp- BMH, Pp-BMB and Pp-BMZ ecotypes.

21 YANRU LAN, TAO LI, TINGYU DUAN AND PENG GAO

Discussion

This study identified significant differences in germination percentage, germination index,

T50, bud length and root length between the three different species of Luobuma and between the six ecotypes of one specific Luobuma species (Poacynum pictum). A. venetum seeds were the best in terms of germination percentage and germination index, followed by P. pictum and P. hendersonii. There was no significant difference in germination percentage between the ecotypes of P. pictum, except for Pp-BMX and Pp-BMQ which recorded the highest and lowest germination percentage, respectively. The findings of this study prove that the interspecific variation in seeds is high compared with the intraspecific variation. Seed size and mass can strongly inﬂuence germination requirements and timing, especially with regard to light (Bewley and Lack, 2013). Interestingly, length, width and size of A. venetum seeds were the lowest but had the highest germination percentage, while P. hendersonii seeds were the largest but showed the lowest germination percentage. This indicates the inverse relationship between seed characteristics and germination ability, which is consistent with the conclusion that there is a significantly negative correlation between germination and seed size in mesad plants (Wang et al., 2007). The effect of low-temperature and pappus removal on Luobuma seed germination was more pronounced within species than between species, which is consistent with an earlier report (Loha et al., 2009). Pappus removal significantly improved germination percentage and germination index, and reduced time to 50% germination of Luobuma. Under natural conditions, Luobuma seeds retain pappus after being released from the broken pod, which may act as a mechanical barrier during germination. Earlier studies have proven that a multilayered perisperm-endosperm envelope in developing seeds delays germination by acting as a mechanical barrier to radicle emergence (Sreenivasulu and Amritphale, 1999) and the micropylar endosperm in Podophyllum hexandrum Royle presents a physical barrier to the protrusion of the radicle (Sreenivasulu et al., 2009). For Luobuma under natural conditions, seed germination may be affected if the pappus does not fall off quickly from the mature seeds resulting in rotting of seeds in the soil and leading to poor renewal of population from the seeds. Moreover, pappus might act as a mechanical barrier at the germinal aperture of seeds and limit oxygen absorption, thereby decreasing seed germination. Temperature is an important factor that influences seed germination percentage. Xie et al. (2012) demonstrated an improvement in germination percentage on soaking A. venetum seeds in warm water at 40–50°C. Our study suggested that even a short period of low- temperature treatment may lead to low germination of Luobuma seeds in the field, which may indirectly lead to decline in Luobuma populations under natural conditions. Our hypothesis that the removal of pappus and short-term low-temperature storage affect the germination of seeds was supported. However, further research is necessary to understand the detailed mechanism of temperature regulation in Luobuma seeds. The present study is the first to analyse the effect of low-temperature short-term storage and pappus removal on seed germination in different ecotypes of Luobuma, and the findings help improve our understanding on the role of pappus in Luobuma seed germination.

22 PAPPUS REMOVAL AND LOW TEMPERATURE AFFECT SEED GERMINATION OF LUOBUMA

In summary, the present study elucidated the interspecific and intraspecific variation in seed germination of Luobuma. The research also demonstrated that low temperature and pappus may be the possible reasons for lower germination percentage in Luobuma seeds under natural conditions, and this indirectly may lead to Luobuma population decline.

Acknowledgements

We acknowledge the financial support from Key Project of Science and Technology Department of Xinjiang Autonomous Region, China (2016E02015, 2016A03006).

References

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