DSpace at VNU: Effect of storage time and pretreatment on seed germination of the threatened coniferous species Fokienia hodginsii

bs_bs_banner Plant Species Biology (2014) ••, ••–•• doi: 10.1111/1442-1984.12062 NOTES AND COMMENTS Effect of storage time and pretreatment on seed germination of the threatened coniferous species Fokienia hodginsii DUC QUANG NGUYEN,* THI PHUONG HOA PHAN† and VAN TAN DAO‡ *University of Engineering and Technology, †Institute of Biotechnology, Vietnam National University Hanoi, and ‡Faculty of Biology, Vietnam National University of Education Hanoi, Hanoi, Vietnam Abstract We report the effects of storage time and pretreatment on seed germination of Fokienia hodginsii Lower mean germination was observed in seeds stored for years (6.41 ± 1.23 seeds/replicate) compared with those stored for year (8.52 ± 1.06 seeds/replicate) Seeds collected from a southern location had statistically higher mean germination (9.67 ± 1.28 seeds/replicate) than those collected from a northern location (7.99 ± 1.36 seeds/replicate) Higher mean T50 was observed in seeds stored for years (37.02 ± 4.43 days) compared with those stored for year (30.69 ± 5.06 days) Mean germination of untreated fresh seeds was 9.97 ± 1.34 seeds/replicate and that of treated fresh seeds in 60°C water was 12.95 ± 1.24 seeds/replicate Fresh seeds treated with 50°C and 70°C water had a significantly lower mean germination compared with untreated seeds and seeds treated in 60°C water Mean T50 was lowest in seeds treated with 60°C water Keywords: conservation, ex situ, Fokienia hodginsii, germination, seeds, Vietnam Received 28 February 2014; revision received May 2014; accepted 28 July 2014 Introduction Despite the existence of many other conservation approaches used nowadays, ex situ conservation offers for many threatened plants an important insurance policy for the future In ex situ conservation, species’ populations are protected and managed outside their native or original environment either as seed gene banks or field gene banks (Kozlowski et al 2012; Westengen et al 2013) This approach is preferred in situations where the populations are in real danger of physical destruction or genetic deterioration due to excessive pressures in their natural habitat Ex situ conservation efforts are necessary to minimize the loss of genetic diversity (Fay 1992), reduce the risk of extinction (Bowes 1999; Godefroid et al 2010), and produce propagation material for the expansion or reintroduction of populations (Brusa et al 2007; Godefroid et al 2010; Ren et al 2010) Understanding the seed germination requirements of the species to be reintroduced is paramount to the success of reintroductions Local adapCorrespondence: Duc Quang Nguyen Email: quangdhsp1@gmail.com © 2014 The Society for the Study of Species Biology tation to environmental conditions may result in strict species-specific requirements for seed germination (Rayburn et al 2013) To efficiently and effectively produce cultivated specimens for research and conservation purposes, requirements for seed germination of the species must be known (Meyer & Monsen 1991; Cerabolini et al 2004; Brusa et al 2007) Differential germination responses may be observed among seeds of the same species, if those seeds are stored under different conditions and/or treated differently before being sown (Cerabolini et al 2004) Fokenia hodginsii is a monotypic taxon of the Cupressaceae family The genus Fokienia comprises only one living species and one fossil species (F ravenscragensis) This tree species is ancient and endemic to Laos, Vietnam, and southern China, and it is currently listed as globally threatened Fokenia hodginsii is highly valued, both economically and culturally, throughout its distribution range The tree has become part of Vietnamese culture, much like the Hinoki tree in Japan The timber of this species is durable and highly fragrant and is prized for high-value furniture and craftwork as well as internal D Q NGUYEN ET AL paneling in houses The demand for the timber is the principal driver behind illegal logging, which is the most serious threat to F hodginsii despite it receiving legal protection in China and Vietnam Even in high-profile National Parks such as BiDoup Nui Ba and Chu Yang Sin, mature trees are being felled despite constant monitoring The problem is even worse in more remote areas In Vietnam and Laos subpopulations have been heavily exploited by legal and illegal logging over the last 50 years and are now highly fragmented and reduced in size In China, there has also been a significant decline; lowaltitude forests of F hodginsii have been converted for agriculture or forestry (Luu & Thomas 2000) Despite legal protection, illegal logging continues to reduce the number of mature trees of F hodginsii throughout most of their range Recently, the Vietnamese Government has introduced many laws to protect F hodginsii forests Nevertheless, trade in illegally harvested timber has caused local extinction or extreme scarcity of individuals in many localities At present, several plantation plans have been started in some northern Vietnam areas, however, the planted areas are not significant Failure in producing seedlings was frequently reported in many localities in northern Vietnam One of the difficulties in plantation programs is the limited current knowledge regarding the biology, and particularly ecology, of F hodginsii Research on germination ecology, storage conditions, and pretreatment of the seeds of F hodginsii is crucial in addressing questions of conservation and sustainable harvest of this forest tree To the best of our knowledge, the present paper is the first to address the questions regarding seed germination of F hodginsii under the effects of different storage and pretreatment conditions Materials and method Seed collection Fokienia hodginsii flowers twice a year The first flowering time occurs in spring from April to May and the cone is ripe in October of the same year The spring seeds are not viable The second flowering period is in autumn from September to October, and the cone is ripe in October of the next year The autumn seeds are viable All seeds used in this study were collected during the period from early November to early December 2010 Only seeds of the mature fruits with a dark brown fruit coat were collected The seeds were dry-stored immediately after collection for a period of either or years Seeds of F hodginsii collected from a northernmost Vietnam location (Hagiang Province at 22°02′N) were symbolized as “N-seeds” and from a southernmost Vietnam location (Ninhthuan Province at 11°33′N) were symbolized as “S-seeds.” The © 2014 The Society for the Study of Species Biology N-seeds and S-seeds were classified into upper and lower elevation groups according to the altitudes of the original mother trees from which the seeds had been collected Seeds collected from higher altitudes (1990 m for S-seeds and 1870 m for N-seeds) were called “upper seeds” and seeds from lower altitudes (1610 m for S-seeds and 1500 m for N-seeds) were called “lower seeds” Effect of storage time on seed germination Germination experiments were carried out in Hanoi (Vietnam) during the period from 2011 to 2013 with a total of 16 treatments that represented all combinations of differences in seed storage time (1 year and years), latitudes of seed collection (N-seeds and S-seeds), and altitudes of seed collection (upper and lower seeds for northernmost and southernmost location) A total of 1600 seeds were equally distributed to across 16 treatments with four replicate germination trials per treatment (25 seeds per replicate for a total of 100 seeds per treatment) Effect of hot water pretreatment on seed germination In 2009 when first plantation programs of F hodginsii were initiated, some households of northern Vietnam started to try planting F hodginsii in their own gardens and they reported that the germination rates of fresh seeds were low (approximately 20%) Some people tried pretreating the seeds with hot water (approximately 50°C), and the germination rate was improved They reported that the germination rates were improved especially for the seeds immersed in hot water from to h (pers comm.) In the present research, a second experiment was performed in which seeds were pretreated by immersion in three different temperatures of hot water (50°C, 60°C, and 70°C) for either or h The seeds were then rinsed with KMnO4 0.05% to remove the acidity, and were washed with cool water before being sown All seeds used in this experiment were fresh seeds that had been left to dry naturally within days after collection from Hagiang Province, northern Vietnam A total of 600 seeds were equally distributed to six treatments (3 temperatures × treatment times) with four replicate germination trials per treatment (25 seeds per replicate for a total of 100 seeds per treatment) Untreated seeds were sown at the same time to evaluate the effect of pretreatment on seed germination, and four replicates (25 seeds per replicate) were also set up for the untreated lot The seeds were sown in standard germination boxes on moist filter paper and continuously kept in a dark germination chamber Germination was monitored every days with a green light in order to detect radicle emergence A seed was considered germinated when the radical pierced the coats up to mm Plant Species Biology ••, ••–•• S E E D G E R M I N AT I O N O F F O K I E N I A H O D G I N S I I Data analysis All data in this study are reported as means ± one standard error For the first experiment, a three-way anova for a completely randomized design in program R (Version 2.12.0, R Development Core Team 2008) was used to test for the main effects of storage time, latitudes, and altitudes on mean percentage germination and mean time to 50% germination (T50) as well as two-way and three-way interactions between the predictor variables For the second experiment, a two-way anova was used to test for the main effects of temperature and pretreatment time on mean germination as well as two-way interactions between the predictor variables For both experiments, a Turkey–Kramer adjustment for multiple comparisons using the multcomp package in R (Hothorn et al 2008) was used to compare mean percentage germination and mean T50 between treatments Results Effect of storage time on seed germination First germination was observed after 15 days, independent of treatment (Table 1) The approximate time to 50% germination (T50) across treatments was 34 days Out of 1600 seeds sown, a total of 502 seeds germinated (31.38%) Mean germination was significantly affected by storage time (P < 0.05), with lower mean germination observed in seeds stored for years (6.41 ± 1.23 seeds/replicate, 25.64%) compared with those stored for year (8.52 ± 1.06 seeds/replicate, 34.08%) (Table 1) Independent of storage time and altitudes, seeds collected from the southern location had statistically higher mean germination (9.67 ± 1.28 seeds/replicate, 38.68%) than those collected from the northern location (7.99 ± 1.36 seeds/replicate, 31.96%; P < 0.05; Table 1) Independent of storage time and latitudes, germination of seeds collected from upper and lower altitudes did not differ significantly (7.78 ± 1.21 seeds/replicate, 31.12% and 7.84 ± 1.54, 31.36%, respectively, P > 0.05) (Table 1) There were also significant differences in mean germination between certain treatment combinations (Table 1) For example, the mean germination of 1-year stored seeds collected from southern populations at lower sites (9.78 ± 1.67 seeds/replicate) was significantly higher than those collected from northern populations at both lower and upper sites (8.02 ± 1.33 and 8.44 ± 1.98 seeds/ replicate, respectively) (P < 0.05) (Table 1) Independent of latitudes and altitudes, the mean germination of 1-year stored seeds was significantly higher than that of 2-year stored seeds in all treatment combinations, except for the 2-year stored seeds collected from southern populations at upper sites (7.53 ± 1.91 seeds/replicate) which was significantly higher than those of the other treatment combinations for 2-year stored seeds (P < 0.05) (Table 1) On the other hand, T50 values were significantly different across all treatments (P < 0.05) Independent of latitudes and altitudes, mean T50 was significantly affected by storage time (P < 0.05), with higher mean T50 was observed in seeds stored for years (37.02 ± 4.43 days) compared with those stored for year (30.69 ± 5.06 days) (Table 1) There was no statistically significant effect of Table Mean germination and mean T50for the eight treatments representing pairwise combinations of seed storage time (1 or years), latitudes (northernmost or southernmost), and altitudes (upper or lower) of the original populations from which seeds were collected Storage time (years) Latitude Altitude Approximate days to first germination 1 1 2 2 Mean of 1-year stored seeds Mean of 2-year stored seeds Mean of northern seeds Mean of southern seeds Mean of upper seeds Mean of lower seeds Northern Northern Southern Southern Northern Northern Southern Southern Upper Lower Upper Lower Upper Lower Upper Lower 15 43 15 22 34 33 19 30 Mean germination (seeds/replicate) Mean T50 (days) 8.44 ± 1.98a 8.02 ± 1.33a 9.45 ± 1.24ab 9.78 ± 1.67b 6.03 ± 1.77c 6.11 ± 1.55c 7.53 ± 1.91ab 6.07 ± 1.11c 8.52 ± 1.06 6.41 ± 1.23 7.99 ± 1.36 9.67 ± 1.28 7.78 ± 1.21 7.84 ± 1.54 33.76 ± 4.47a 31.54 ± 3.39a 36.88 ± 5.23b 29.43 ± 4.62a 37.09 ± 5.29b 35.23 ± 5.67ab 37.49 ± 5.55b 37.51 ± 3.69b 30.69 ± 5.06 37.02 ± 4.43 33.11 ± 3.29 33.16 ± 3.37 34.10 ± 4.11 33.95 ± 3.99 Values are means ± one standard error Means in a column with the same superscript letter not differ significantly from one another according to Tukey’s test for multiple comparisons (α = 0.05) Plant Species Biology , â 2014 The Society for the Study of Species Biology D Q NGUYEN ET AL Water temperature (°C) 50 50 60 60 70 70 Untreated seeds Treatment time (h) 4 Approximate days to first germination Mean germination (seeds/replicate) Mean T50 (days) 34 37 15 39 27 32 29 10.36 ± 1.68b 10.44 ± 1.56b 12.95 ± 1.24a 12.89 ± 1.33a 9.78 ± 1.65bc 8.90 ± 1.56c 9.97 ± 1.34bc 28.02 ± 4.89b 29.88 ± 5.44c 25.11 ± 5.13a 25.66 ± 4.22a 26.45 ± 3.14a 26.17 ± 4.01a 28.66 ± 4.55b Table Mean germination and mean T50for the six treatments representing pairwise combinations of seed pretreatment with water temperature (50, 60, and 70°C) and treatment time (3 and h) Values are means ± one standard error Means in a column with the same superscript letter not differ significantly from one another according to Tukey’s test for multiple comparisons (α = 0.05) latitudes and altitudes on mean T50, but there were statistically significant differences in mean T50 values between certain treatment combinations (Table 1) For instance, the mean T50 value for seeds collected from southern population at lower site and stored for year was significantly lower than that for seeds collected from southern population at upper site (29 ± 4.62 and 36 ± 5.23, respectively, P < 0.05) (Table 1) Effect of hot water pretreatment on seed germination Mean germination of fresh seeds of F hodginsii was significantly affected by hot water pretreatment (Table 2) Mean germination of untreated seeds was 9.97 ± 1.34 seeds/replicate (39.88%) (Table 2) Mean germination of treated seeds in 60°C water for h or h was 12.95 ± 1.24 and 12.89 ± 1.33, respectively which were the highest compared with that in 50°C and 70°C ((Table 2) Pretreatment with 70°C water caused a slight decrease in mean germination compared with 50°C and untreated seeds, however the difference was not statistically significant (P > 0.05) It was noted that time of seed treatment in hot water did not significantly affect mean germination as the difference in the mean germination at h and h was not statistically significant (P > 0.05) in all temperatures (Table 2) On the other hand, mean T50 was significantly lower in seeds treated with 60°C water (25.11 ± 5.13 and 25.66 ± 4.22 seeds/replicate for and h, respectively) and 70°C water (26.45 ± 3.14 seeds/replicate and 26.17 ± 4.01 seeds/replicate for and h, respectively) compared with that of seeds treated with 50°C (28.02 ± 4.89 seeds/replicate and 29.88 ± 5.44 seeds/ replicate for h and h, respectively) and untreated seeds (28.66 ± 4.55 seeds/replicate) (P < 0.05) (Table 2) The twoand three-way interactions were not significant Germination was calculated at each time-step over the 360-day duration of the experiment for 60°C-water treated © 2014 The Society for the Study of Species Biology Fig Seed germination of Fokienia hodginsii over 360-day duration of the study At each time step the bars represent the number of newly germinated seeds observed for untreated seeds and for seeds treated in 60°C water for h , 60°C treated; , untreated seeds (in h) and untreated seeds (Fig 1) Histograms of the germination response and timing for treated seeds compared with untreated seeds show that, in general, treated seeds had more pronounced peaks in frequency (approximately 25–30 days) than untreated seeds Germination of treated seeds mostly occurred during the period from 20 to 40 days, whereas germination of untreated seeds occurred during the period from 25 to 40 days (Fig 1) Discussion Our past efforts to germinate cold-stored seeds of F hodginsii were unsuccessful, as seeds stored in 9°C for months lost their germination ability (unpubl data) In the first experiment, we found that dry-stored seeds of F hodginsii were able to germinate after years There has not been a thorough study on the germination ecology of this threatened coniferous species In the second experiPlant Species Biology ••, ••–•• S E E D G E R M I N AT I O N O F F O K I E N I A H O D G I N S I I ment we found that mean germination of fresh seeds of F hodginsii was approximately 40% and that pretreatment of seeds with 60°C water for 3–4 h raised mean germination to approximately 51% Our data add critical preliminary insight into the germination biology of F hodginsii that adds to our understanding of the basic ecology of this species and provides an initial guideline for germination in ex situ conservation The present study revealed that, averaged over altitude and latitude, mean germination of ≤ 2-year dry-stored seeds of F hodginsii was approximately 9% lower than that of fresh seeds Mean germination of 1-year stored seeds was approximately 12% higher than that of 2-year stored seeds In another study, mean germination of 4-month cold-stored seeds of F hodginsii was approximately 20% lower than that of fresh seeds (Canh et al., pers comm.) Consistent with the present study, Canh et al also found that cold-stored seeds of F hodginsii were not able to germinate after 6-month storage Some research has shown that seed viability of coniferous species is significantly reduced following periods of storage greater than 2–3 years (Berdeen et al 2007) Seeds may deteriorate in dry storage, losing vigor and becoming more sensitive to stress during germination (Rajjou & Debeaujon 2008; Probert et al 2009) Temel et al (2011) reported that mean germination of seeds of black pine (Pinus nigra subsp pallasiana) stored for 10 years was approximately 38% lower than that of fresh seeds Liu et al (2011) found that mean germination of seeds of 489 grass species stored in warm dry conditions had decreased by 16%–18% Our results suggest that ≤ years dry storage of F hodginsii seeds for ex situ conservation purposes does not dramatically reduce seed viability Although mean germination was significantly different between some treatments, all treatments had between 24% and 40% germination Future research on the seed storage of F hodginsii should include a more comprehensive germination trial in which the length of dry storage time and temperature are manipulated Such research would help address some of the limitations in our present study and provide useful information for conservation strategy makers and practitioners For example, mean germination may decrease and T50 may be higher for ≥ 2-year dry stored seeds; T50 may be reduced if seeds of F hodginsii are exposed to higher temperatures as part of a germination trial Mean T50 of fresh seeds of F hodginsii was approximately 29 days and that for 1-year stored and 2-year stored were approximately 31 and 37 days, respectively A previous study on the effect of storage time on seed germination in white bark pine also found that seeds stored for shorter periods of time germinated more quickly than those stored for longer periods of time (Berdeen et al 2007) However, Rayburn et al (2013) found a reverse trend in the threatened distylous primrose (Primula Plant Species Biology ••, ••–•• cusickiana var maguirei); that is, seeds stored for years had a higher germination percentage and lower mean T50 compared with seeds stored for year Mean germination of seeds collected from southern Vietnam populations was approximately 7% higher than that of seeds collected from northern Vietnam populations A previous study on black pine (Pinus nigra subsp pallasiana) (Temel et al 2011) found that germination characteristics seemed to associate with population location, especially with humidity of the region from which the seeds were collected While Vietnam lies entirely in the tropics, there is quite a large difference in climate between northern and southern regions Northern Vietnam features a humid subtropical climate and has a full four seasons, with much cooler temperatures than in the south, as well as winters that can get quite cold Southern Vietnam, with its much hotter temperatures, has only two main seasons: a dry season and a rainy season The cold experienced during northern winters is intensified by the humidity Snow can even be found to an extent up in the mountains of the extreme northern regions in places, especially on top of high mountains In southern China, Hou et al (2005) reported that biological and phenological characters of flower and cone of F hodginsii populations are closely linked to geography and climatic factors, including location, altitude, and temperature of the natural distribution area Autumn flowering, fruit, maturity, and seed falling in mountainous areas occurred earlier than in lower mountainous areas, and much earlier than in hilly areas, and in high-altitude areas earlier than in low-altitude areas There are obvious geographic variations in cone diameter, cone height, seed number, 1000seed weight, and germination percentage among populations of F hodginsii in China (Hou et al 2005) Further research needs to be carried to study geographic variation of the seed germination characteristics of F hodginsii populations in Vietnam Our studies (Quang et al 2012; Nguyen & Nguyen 2012) on the genetic variation of F hodginsii across its distribution range in Vietnam found that southern populations had a relatively lower amount of diversity than northern populations Our results revealed that pretreatment of seeds of F hodginsii with 60°C water in h increased mean germination and decreased mean T50 and resulted in uniform germination compared with untreated seeds We observed that after being left in a dry shaded place for 10 days, approximately 90% of the F hodginsii fruit coats opened naturally and seeds were split from the fruit coat However, the seed coat of F hodginsii is relatively hard These seeds will usually germinate in 30–360 days, but even under good conditions germination may be erratic The hard seed coat prevents imbibitions and gaseous exchange that leads to physical dormancy and subsequently the foremost cause in poor and erratic germina© 2014 The Society for the Study of Species Biology D Q NGUYEN ET AL tion Overcoming hard-seededness is an important initial step in the use of seeds of F hodginsii in ex situ conservation and restoration programs The present study showed that pretreatment with 60°C water in h made approximately 48% of the seeds of F hodginsii germinate within 120 days, whereas approximately 40% of untreated seeds germinated within 290 days Acknowledgment Special thanks to The Vietnam National Foundation for Science and Technology Development (NAFOSTED) for the financial support, with project number 106.99– 2010.25 Special thanks to two reviewers with their insightful and constructive comments and suggestions on our manuscript References Berdeen J C., Riley L E & Sniezko R S (2007) Whitebark pine seed storage and germination: a follow-up look at seedlots from Oregon and Washington Proceedings of the Conference Whitebark Pine: A Pacific Coast Perspective, pp 113–121 Bowes B G (1999) A Colour Atlas of Plant Propagation and Conservation Manson Publishing, London Brusa G., Ceriani R & Cerabolini B (2007) Seed germination in a narrow endemic species (Telekia speciosissmia, Asteracear): implications for ex situ conservation Plant Biosystems 141: 56–61 Cerabolini B., De Andeis R., Ceriani R M., Pierce S S & Raimondi B (2004) Seed germination and conservation of endangered species from the Italian Alps: Physoplexis comosa and Primula glaucescens Biological Conservation 117: 351–356 Fay M F (1992) Conservation of rare and endangered plants using in vitro methods In Vitro Cellular Developmental Biology– Plant 28: 1–4 Godefroid S., Piazza C., Rossi G., Buord S., Stevens A., Aguraiuja R., Cowell C., Weekley C W., Wogg C., Iriondo J M., Johnson I., Dixon B., Gordon D., Magnanon S., Valentin B., Bjureke K., Koopman R., Vicens M., Virevaire M & Vanderborght T (2010) How successful are plant species reintroductions? 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