Luo, Zhang, Ren, Ma and Liu (2019). Seed Science and Technology, 47, 3, 343-349. https://doi.org/10.15258/sst.2019.47.3.09
Research Note
Dormancy and germination of Firmiana danxiaensis, an endangered tree endemic to South China
Xiaoying Luo1, Qianmei Zhang 2*, Hai Ren 2*, Guohua M a 2 and Hong L i u 3, 4
1 College of Tourism and Geography, Shaoguan University, Shaoguan 512005, China 2 Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China 3 Department of Earth and Environment and International Center for Tropical Botany, Florida International University, Miami, FL 33199, USA 4 Forestry College, Guangxi University, Nanning 530004, China *Authors for correspondence (E-mail: [email protected]; [email protected])
(Submitted July 2019; Accepted October 2019; Published online November 2019)
Abstract
Firmiana danxiaensis (Malvaceae) is a rare and endemic tree of South China. It has a very narrow distribution with only two extant populations in the wild. The aim of this study was to understand the nature of its seed dormancy, how to break the dormancy and the environmental requirements for the natural germination of its seeds. To identify whether seeds of F. danxiaensis are dormant, germination experiments with 12 treatments
(three light levels × four temperatures) were conducted. To determine the optimal way to break dormancy, we conducted the dormancy-breaking experiment with five treatments, including physical and chemical methods. The results showed that the dormancy of F. danxiaensis seeds was associated with a water- impermeable seed coat, i.e., the seeds have physical dormancy. Soaking the seeds in 98% concentrated sul phuric acid for one hour overcame the physical dormancy and enabled imbibition followed by germination (up to 73%). Once dormancy was broken, F. danxiaensis seeds could germinate over a wide range of temperatures in both light and darkness. The results suggest that ex situ conservation and reintroduction of F. danxiaensis might be achieved by sowing acid-treated seeds at restoration sites at the appropriate time of year.
Keywords: acid scarification, endangered plant, Firmiana danxiaensis, germination, physical dormancy, South China
Experimental and discussion
The perennial deciduous tree Firmiana danxiaensis H.H. Hsue & H.S. Kiu (Malvaceae) was first reported in 1987 (Hsue et al., 1987). It was listed as a Second Class Key Protected Wild Plant of China in 1999, a Critically Endangered (CR) species in the China
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343 XIAOYING LUO, Q IANMEI ZHANG, HAI REN, GUOHUA M A AND HONG L I U
Species Red List in 2004, and a Plant with Extremely Small Populations (PESP) in China in 2012 (Ren et al., 2016). Firmiana danxiaensis has an extremely narrow distribution with only two known extant populations, which are located in the Danxia Landform in northern Guangdong Province in South China (Flora of China Editorial Committee, 2007). Research on F. danxiaensis has included taxonomy (Hsue et al., 1987; Qiu, 1994), population genetics (Fan et al., 2013; Chen et al., 2014, 2015), floristic characteristics (Luo et al., 2015) and reintroduction trials (Zhang et al., 2014). Germination studies, however, have not been reported for F. danxiaensis, and little is known about the seed biology of this rare species. Seed ecology is important for the conservation of endangered plants (Herranz et al., 2010). Insufficient understanding of F. danxiaensis seed ecology hampers efforts to increase the population size of this plant through seed propagation. The current research aimed to identify reliable germination protocols to make ex situ propagation possible, which in turn would contribute to the species recovery program, including reintroduction, ex situ conservation and sustainable utilisation. Of the two extant populations of F. danxiaensis, one is located in the Danxiashan
National Nature Reserve (113°36'25' - 113°47'53''E, 24°51'48'' - 25°04'12"N) (DX) and the other i s located in Cangshizai County Na ture Reserve (114°2'45"E, 25°08'19"N) (NX). Both locations are in Shaoguan City, northern Guangdong Province, South China. Mature seeds of F. danxiaensi s were manually collected from dehisced fruits at DX and NX on 20 August 2016. The seeds were then transferred to the laboratory, where they were stored in sealed plastic bags at ambient room temperature (25-30°C) and relative humidity (50-60%) for about one month until they were used in experiments. Seed viability was determined by using the 1% tetrazolium chloride (TTC) test on three replicates of 15 decoated seeds at 30°C for 12 hours in darkness. Seeds with more than two-thirds of the embryo stained red were considered viable (Wu et al., 2016). The results showed that the seed viability was 58.3% for NX seeds and 81.8% for DX seeds. Water imbibition was assessed for (i) scarified seeds (a small hole was pierced in the seed coat at the chalazal end with a dissecting needle); (ii) decoated seed s (the entire seed coat was removed); and (iii) intact seeds. Three replicates of 15 seeds were used for each treatment and population. The amount of water taken up was determined as the percentage increase in seed weight (Baskin et al., 2004): WI (%) = [(Wi – Wd) / Wd] × 100, where
WI is percentage of increase in seed weights, Wi and Wd are the masses of imbibed and dry seeds, respectively. The imbibition curves of DX and NX seeds were very similar (figure 1). Intact seeds had only small increases (about 20%) in mass at 96 hours. When seeds were scarified, water absorption significantly increased (P < 0.01), i.e., the mass increased by 100% for both seed samples at 24 hours and increased to 120% for DX seeds and to 140% for NX seeds at 96 hours. In addition, the coat of some scarified seeds began to crack at 48 hours. The mass of decoated seeds increased to 130% for DX seeds and to 170% for NX seeds at 48 hours. To determine whether seeds of F. danxiaensis are dormant, we conducted a germina- tion experiment. The experiment included 12 treatments, i.e., three light levels × four temperatures. The three light levels were continuous light, continuous darkness and 12 hours light / 12 hours dark. The four temperature treatments were a constant 25°C and three 12 hours light / 12 hours dark daily alternating temperature regimes of 35/25, 25/15
344 DORMANCY AND GERMINATION OF FIRMIANA DANXIAENSIS
180 DX 160
140 a b 120 100 (%) I 80 W 60 40 c 20 0 0 102030405060708090100 Time (hours)
intact scarified decoated
180 NX a 160 140 b 120 100 (%) I 80 W 60 40 c 20 0 0 102030405060708090100 Time (hours)
intact scarified decoated Figure 1. Percentage increase in weight of intact, scarified and decoated seeds of Firmiana danxiaensis in the imbibition experiment. Different letters indicate significant differences between treatments based on maximum mass increase (P < 0.01). DX = seeds collected from Danxiashan National Nature Reserve; NX = seeds collected from Cangshizai County Nature Reserve. and 20/10°C. Three replicates of 15 seeds were used for each treatment. The experiment was terminated after four weeks. The final germination percentage (GP), germination index (GI) and germination rate index (GRI) were calculated. GI = ∑(Gt / Dt), where Gt -1 is the number of germinated seeds after t days (Dt) (Shen et al., 2016); GRI (% d ) =
∑[( Gi - Gi-1) / i], where i is the germination count-day, Gi is the percentage of seeds that germinated at time i and Gi-1 is the percentage of seeds that germinated the previous count-day (Cook et al., 2008). The germination of fresh DX and NX seeds ranged from 0.0 to 4.4% at three incubation temperatures (35/25, 25, 25/15°C). No seeds germinated at 20/10°C. The germination percentage was significantly lower than the seed viability percentage, which was 58.3%
345 XIAOYING LUO, Q IANMEI ZHANG, HAI REN, GUOHUA M A AND HONG L I U for NX seeds and 81.8% for DX seeds. Illumination did not affect germination. Freshly matured, viable seeds may be defined as dormant if no seeds or only a few seeds have germinated at a range of temperature regimes in both light/dark and continuous darkness after about four weeks (Baskin and Baskin, 2014). Based on this criterion, we conclude that freshly matured seeds of F. danxiaensis are dormant, i.e., the germination of fresh seeds was < 5% at 15-35°C with or without light. Embryo type was determined by excising and examining embryos from fully imbibed seeds. The determination of dormancy class for F. danxiaensis seeds was facilitated by the use of a dichotomous seed dormancy key (Baskin and Baskin, 2014). We conclude that F. danxiaensis seeds have physical dormancy (PY) based on the following results: 1) embryos extracted from imbibed seeds had differentiated organs and were fully developed; 2) imbibition by intact seeds was low, indicating the presence of a water-impermeable seed coat; and 3) scarified seeds became fully imbibed in 24 hours and germinated within four weeks. PY has been reported for F. simplex (L.) W. Wight (Wang, 1989), Apeiba tibourbou Aubl. (Daws et al., 2006) and Kosteletzkya virginica (L.) C. Presl ex A. Gray (Poljakoff-Mayber et al., 1994), which are the other members of the Malvaceae; the latter is one of 18 angiosperm families reported to have seeds with PY (Gama-Arachchige et al., 2013; Mahadevan and Jayasuriya, 2013). The seeds of F. danxiaensis did not imbibe water or germinate until the integrity of the seed coat, which contains an outer layer of palisade cells (Baskin et al., 2006), was disrupted. To identify the optimal way to break dormancy, we conducted an experiment with five treatments. Acid treatment: seeds were soaked in 98% H2SO4 (concentrated sulphuric acid) for on e hour, rinsed with running water and then soaked in distilled water for 48 hours at room temperature. Boiling water treatment: seeds were placed in boiling water and allowed to naturally cool to room temperature for 48 hours. 90/0°C cycles treatment: seeds were placed in 90°C water for five minutes and then immediately transferred to 0°C water (a beaker filled with ice and water) for five minutes; this hot-cold cycle was repeated three times and the seeds were then soaked in distilled water for 48 hours at room temperature. 50°C water treatment: seeds were soaked in distilled water at 50°C for 48 hours. CK: seeds were soake d i n distilled wat er for 48 hours at room temperature. Three replicates of 30 seeds were used for each treatment. Seeds were incubated in 12/12 hours light/dark with an alternating (12/12 hours) temperature of 35/25°C for four weeks. After four weeks of incubation, seeds treated wi t h concentrated sulphuric acid had germination percentages of 73% for DX and 50% for NX (figure 2). The results of TTC test and germination data of acid treatment were subjected to ANOVA, which showed that there is no significant difference between these two sets of data (for DX, P = 0.27 > 0.05; for NX, P = 0.65 > 0.05). Therefore, soaking in concentrated sulphuric acid for one hour will not kill the seeds. Seeds that are not germinated should be non-viable seeds before subjected to the treatment. The germination percentages in the remaining treatments were significantly lower (P < 0.01) than in acid treatment and ranged from 1 to 8% for DX and from 1 to 4% for NX (figure 2). Most germination occurred during the first two weeks in acid treatment, with minimal additional germination by the end of the fourth week. Seeds treated with concentrated sulphuric acid began to germinate during the fi rst week of incubation, whereas seeds in the other treatments did not begin to germinate until the
346 DORMANCY AND GERMINATION OF FIRMIANA DANXIAENSIS
80 DX a 70
60 T1 50 T2 40 T3 30 T4 20
Germination (%) Germination T5 10 b 0 0 5 10 15 20 25 30 Time (days)
80 NX 70
60 T1 50 a T2 40 T3 30 T4 20 Germination (%) Germination T5 10 b 0 0 5 10 15 20 25 30 Time (days)
Figure 2. Germination of fresh seeds of Firmiana danxiaensis in 12/12 hours light / dark at 35/25°C as affected by five treatments: T1, soaked in 98% H2SO4 one hour; T2, placed in boiling water and allowed to cool for 48 hours; T3, hot (90°C) and cold (0°C) cycles; T4, soaked in water at 50°C for 48 hours; and T5, soaked in water at room temperature 48 hours. Different letters indicate significant differences in final germination at four weeks (P < 0.01). DX = seeds collected from Danxiashan National Nature Reserve; NX = seeds collected from Cangshizai County Nature Reserve. second to fourth week of incubation. GI and GRI values were signifi cantly higher for seeds in acid treatment than in the other treatments (P < 0.01), which indicated that seeds in acid treatment germinated more rapidly. This study demonstrated that the physical dormancy of F. danxiaensis seeds can be removed through acid scarification. Acid scarification results in the substantial eros ion of the seed coat and is regarded as an effective way to break PY. The effect of the approach has also been demonstrated for other members of the M alvaceae, including Adansonia gregorii F. Muell. (Turner and Dixon, 2009), Ambroma augusta L.f. (Dutta et al., 2002) and Corchorus fa scicularis Guill. & Perr. and C. pseudo-olitorious Isla m & Zaid (Kak et al., 2009). Although many other methods have been developed to break PY, such as treatment with hot water, low temperature, dry heat or dry storage, the success of these other methods varies with the species and treatment intensity and duration (Baskin and
347 XIAOYING LUO, Q IANMEI ZHANG, HAI REN, GUOHUA M A AND HONG L I U
Baskin, 2014). PY is broken in the seeds of many species by either hot water treatments or wet heat/ice water cycles. For example, PY is broken for Corchorus olitor ius L. by 80°C for 5-15 minutes (Velempini et al., 2003), for Abutilon theophrasti Medik by 70°C for one hour (Horowitz and Taylorson, 1984), for Guazuma ulmifolia Lam. by 60°C for 16 minutes (Sobrinho et al., 2012) and for Sophora moorcroftiana Benth. by 90°C for 30 seconds and ice water for two minutes (Baskin et al., 2007). This was not, however, true for F. danxiaensis seeds. None of the wet heat treatments broke the dormancy of F. danxiaensis seeds. In the present study, the effect of wet heat treatments was time and temperature dependent. The germination percentage was low with boiling water or with 90/0°C cycl es probably due to heat injury. On the other hand, 50°C water might not be hot enough to disrupt the seed coat and thereby enable seeds to absorb water. Because the PY o f F. danxiaensis seeds can be easily broken by acid scarifi cation, seeds can be pre conditioned i n the laboratory and then sown at restoration sites at appropriate time s, i.e., during the wet s eason and when temperatures are between 15 and 35°C. The ability to sow preconditioned seeds and achieve a high rate of seedling establishment would facilitate reintroduction and would require less labour and material than transplanting young plants (Baskin et al., 2007).
Acknowledgements
This study was s upported by the Guangdong Na tural Science Foundation (grant no. 2016A030307046), the Program for Excellent Young Teachers in Guangdong Colleges and Universities (grant no. Yq2014157) and Shaoguan Science and Technology Plan Project (grant no. 2019sn061) to Xiaoying Luo, and by the National Natural Science Foundation of China (grant no. 31360146) to Hon g Liu. We would like to thank Zaixiong Chen and Pingsheng Zhong for assistance in seed collection. The first author dedicates this article to Professor Xueying Zhuang (1961-2016), who devoted her life to the forestry and conservation of endangered plants in South China.
References
Baskin, C.C. and Baskin, J.M. (2014). Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germina- tion, 2nd Edition, San Diego, California, Academic Press. Baskin, J.M., Davis, B.H., Baskin, C.C., Gleason, S.M. and Cordell, S. (2004). Physical dormancy in seeds of Dodonaea viscosa (Sapindales, Sapindaceae) from Hawaii. Seed Science Research, 14, 81-90. Baskin, J.M., Baskin, C.C. and Dixon, K.W. (2006). Physical dormancy in the endemic Australian genus Stylobasium, a first report for the Family Surianaceae (Fabales). Seed Science Research, 16, 229-232. Baskin, C.C., Wurts, J., Liu, Z.M. and Baskin, J.M. (2007). A method for breaking physical dormancy in seeds of the endemic Tibetan Plateau shrub Sophora moorcroftiana var. moorcroftiana (Fabaceae) and implications for restoration. Natural Areas Journal, 27, 118-123. Chen, S.F., Li, M.W., Hou, R.F., Liao, W.B., Zhou, R.C. and Fan, Q. (2014). Low genetic diversity and weak population differentiation in Firmiana danxiaensis, a tree species endemic to Danxia Landform in northern Guangdong, China. Biochemical Systematics and Ecology, 55, 66-72. Chen, S.F., Li, M.W., Jing, H.J., Zhou, R.C., Yang, G.L., Wu, W., Fan, Q. and Liao, W.B. (2015). De novo transcriptome assembly in Firmiana danxiaensis, a tree species endemic to the Danxia Landform. Plos One, 10, e0139373.
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Cook, A., Turner, S.R., Baskin, J.M., Baskin, C.C., Steadman, K.J. and Dixon, K.W. (2008). Occurrence of physical dormancy in seeds of Australian Sapindaceae: a survey of 14 species in nine genera. Annals of Botany, 101, 1349-1362. Daws, M.I., Orr, D., Burslem, D.F.R.P. and Mullins, C.E. (2006). Effect of high temperature on chalazal plug removal and germination in Apeiba tibourbou Aubl. Seed Science and Technology, 34, 221-225.
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