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

NOTES AND COMMENTSEffect 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

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 ofFokienia

hodginsii Lower mean germination was observed in seeds stored for 2 years (6.41± 1.23

seeds/replicate) compared with those stored for 1 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 meanT50 was observed in seeds stored for 2 years (37.02 ± 4.43 days) compared

with those stored for 1 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

signifi-cantly lower mean germination compared with untreated seeds and seeds treated in 60°C

water MeanT50 was lowest in seeds treated with 60°C water.

Keywords: conservation, ex situ, Fokienia hodginsii, germination, seeds, Vietnam.

Received 28 February 2014; revision received 4 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

dete-rioration due to excessive pressures in their natural

habitat Ex situ conservation efforts are necessary to

mini-mize 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

rein-troduction of populations (Brusa et al 2007; Godefroid

et al 2010; Ren et al 2010) Understanding the seed

germi-nation requirements of the species to be reintroduced is

paramount to the success of reintroductions Local

adap-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 pur-poses, 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

eco-nomically 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

Correspondence: Duc Quang Nguyen

Email: quangdhsp1@gmail.com

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

pro-tection 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;

low-altitude 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

extinc-tion 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

germina-tion ecology, storage condigermina-tions, 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

pre-treatment 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 1 or 2 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

Prov-ince at 11°33′N) were symbolized as “S-seeds.” The

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 dif-ferences in seed storage time (1 year and 2 years), lati-tudes 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 pre-treating 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 2 to 4 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 3 or 4 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 7 days after collection from Hagiang Province, northern Vietnam

A total of 600 seeds were equally distributed to six treat-ments (3 temperatures× 2 treatment times) with four rep-licate 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 germi-nation chamber Germigermi-nation was monitored every 5 days with a green light in order to detect radicle emergence A seed was considered germinated when the radical pierced the coats up to 2 mm

Data analysis

All data in this study are reported as means± one

stan-dard 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

alti-tudes 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

T50between treatments

Results

Effect of storage time on seed germination

First germination was observed after 15 days,

indepen-dent 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 2 years (6.41± 1.23 seeds/replicate,

25.64%) compared with those stored for 1 year (8.52± 1.06

seeds/replicate, 34.08%) (Table 1) Independent of storage

time and altitudes, seeds collected from the southern loca-tion had statistically higher mean germinaloca-tion (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 lati-tudes, 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%,

respec-tively, P> 0.05) (Table 1)

There were also significant differences in mean germi-nation between certain treatment combigermi-nations (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 sig-nificantly higher than those of the other treatment

combi-nations for 2-year stored seeds (P< 0.05) (Table 1)

On the other hand, T50values were significantly

differ-ent across all treatmdiffer-ents (P< 0.05) Independent of

lati-tudes and altilati-tudes, mean T50was significantly affected by

storage time (P< 0.05), with higher mean T50 was observed in seeds stored for 2 years (37.02± 4.43 days) compared with those stored for 1 year (30.69± 5.06 days) (Table 1) There was no statistically significant effect of

Table 1 Mean germination and mean T50 for the eight treatments representing pairwise combinations of seed storage time (1 or 2 years), latitudes (northernmost or southernmost), and altitudes (upper or lower) of the original populations from which seeds were collected

Approximate days to first germination

Mean germination

Values are means ± one standard error Means in a column with the same superscript letter do 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

statis-tically significant differences in mean T50values between

certain treatment combinations (Table 1) For instance, the

mean T50value for seeds collected from southern

popula-tion at lower site and stored for 1 year was significantly

lower than that for seeds collected from southern

popu-lation 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

sig-nificantly 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 3 h or 4 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)

Pretreat-ment 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 3 h and 4 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 3 and 4 h, respectively)

and 70°C water (26.45± 3.14 seeds/replicate and

26.17± 4.01 seeds/replicate for 3 and 4 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 3 h and 4 h, respectively) and untreated seeds

(28.66± 4.55 seeds/replicate) (P < 0.05) (Table 2) The

two-and 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

seeds (in 3 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 Germi-nation 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 9

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 2 years There has

not been a thorough study on the germination ecology of this threatened coniferous species In the second

experi-Table 2 Mean germination and mean

T50 for the six treatments representing pairwise combinations of seed pretreat-ment with water temperature (50, 60, and 70°C) and treatment time (3 and 4 h)

Water temperature

(°C)

Treatment time (h)

Approximate days to first germination

Mean germination (seeds/replicate) Mean T50 (days)

Values are means ± one standard error Means in a column with the same superscript

letter do not differ significantly from one another according to Tukey’s test for multiple

comparisons (α = 0.05).

Fig 1 Seed germination of Fokienia hodginsii over 360-day

dura-tion 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 3 h , 60°C treated; , untreated.

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

germina-tion to approximately 51% Our data add critical

prelimi-nary 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

ger-minate 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 ≤ 2 years dry storage of F hodginsii

seeds for ex situ conservation purposes does not

dramati-cally 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

practi-tioners For example, mean germination may decrease

and T50may 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

approxi-mately 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

ger-mination 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

cusickiana var maguirei); that is, seeds stored for 2 years

had a higher germination percentage and lower mean T50

compared with seeds stored for 1 year

Mean germination of seeds collected from southern Vietnam populations was approximately 7% higher than that of seeds collected from northern Vietnam

popula-tions A previous study on black pine (Pinus nigra subsp.

pallasiana) (Temel et al 2011) found that germination

char-acteristics 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 fea-tures 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, matu-rity, 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 varia-tions in cone diameter, cone height, seed number, 1000-seed 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

varia-tion of F hodginsii across its distribuvaria-tion 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 3 h increased mean

germi-nation and decreased mean T50and 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 subse-quently the foremost cause in poor and erratic

germina-tion Overcoming hard-seededness is an important initial

step in the use of seeds of F hodginsii in ex situ

conserva-tion and restoraconserva-tion programs The present study showed

that pretreatment with 60°C water in 3 h made

approxi-mately 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

Conser-vation 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?

Bio-logical Conservation 144: 672–682.

Hothorn T., Bretz F & Westfall P (2008) Simultaneous inference in

general parametric models Biometrical Journal 50: 346–363.

Hou B., Lin F G., Yu Z., Cheng X Z & Tao S (2005) Study on law

of flower and cone of Fokienia hodginsii provenance Journal of

Plant Genetic Resources 6: 163–167.

Kozlowski G., Gibbs D., Huan F., Frey D & Gratzfeld J (2012)

Conservation of threatened relict trees through living ex situ

collections: lessons from the global survey of the genus

Zelkova (Ulmaceae) Biodiversity and Conservation 21: 671–685.

Liu K., Baskin J M., Carol C B., Haivan B., Liu M., Liu W & Du

G (2011) Effect of storage conditions on germination of seeds

of 489 species from high elevation grasslands of the eastern Tibet Plateau and some implications for climate change.

American Journal of Botany 98 (1): 12–19.

Luu N D T & Thomas P I (2000) Conifers of Vietnam Foreign

languages pub Hou Hanoi.

Meyer S E & Monsen S B (1991) Habitat-correlated variation in

mountain big sagebrush (Artemisia tridentata ssp vaseyana)

seed germination patterns Ecology 72: 739–742.

Nguyen M T & Nguyen I P T (2012) Molecular identification of Cupressaceae (Coniferales) in Vietnam based on 18S-rRNA

sequence African Journal of Biotechnology 11 (18): 4158–4162.

Probert R J., Daws M L & Hay F R (2009) Ecological correlates

of ex situ seed longevity: a comparative study on 195 species.

Annals of Botany 104: 57–69.

Quang N D., Hoa P T P., Tan D V & Thang N V (2012) Nucleo-tide variation at the methionine synthase locus in an

endan-gered tree species, Fokienia hodginsii (Cupressaceae) in

Vietnam African Journal of Biotechnology 11: 15398–15404.

Rajjou L & Debeaujon I (2008) Seed longevity: survival and

maintenance of high germination ability of dry seeds Comptes

Rendus Biologies 331: 796–805.

Rayburn A P., Davidson J B & Schupp E W (2013) Effect of storage time, site and floral morph on seed germination of the

threatened distylous primrose Primula cusickiana var maguirei.

Plant Species Biology 28: 101–108.

Ren H., Ma G., Zhang Q., Guo Q., Wang J & Wang Z (2010) Moss

is a key nurse plant for reintroduction of the endangered

herb, Primulina tabacum Plant Ecology 209: 313–320.

Temel F., Gulclcu S., Olmez Z & Gokturk A (2011) Germination

of Anatolian black pine (Pinus nigra subsp pallasiana) seeds

from the lakes region of Turkey: geographic variation and

effect of storage Notulae Botanicae Horti Agrobotanici

Cluj-Napoca 39 (1): 267–274.

Westengen O., Jeppson S & Guarino L (2013) Global ex-situ crop

diversity conservation and the Svalbard global seed vault:

assessing the current status Plos ONE 8 (5): e64146.

doi:10.1371/journal.pone.0064146.