全 文 :弥勒苣苔种子的休眠萌发特性
刘 成ꎬ 秦少发ꎬ 胡枭剑∗
(中国科学院昆明植物研究所中国西南野生生物种质资源库ꎬ 昆明 650201)
摘要: 弥勒苣苔为中国特有的濒危植物ꎬ 一度被认为灭绝后又被重新发现ꎬ 引起了学界及公众的广泛关
注ꎮ 种子库是保护珍稀濒危物种的一种有效手段ꎬ 对物种开展种子保存需要较为深入的了解其种子休眠及
萌发特性ꎻ 但对于弥勒苣苔ꎬ 现有资料十分有限ꎬ 本研究系统地探讨了弥勒苣苔种子的休眠状态及萌发习
性ꎮ 在对其种子进行 X光透视后ꎬ 发现其种子的胚发育完全ꎬ 且基本充满整个种子ꎬ 因此认为该种子不
具有形态休眠ꎮ 在多个变温及恒温条件下对新鲜种子进行萌发测试ꎬ 发现在较高温度 (高于 20 ℃) 下萌
发率很高且萌发迅速ꎬ 赤霉素及低温层积处理能够显著提高种子在较低温度下 (低于 20 ℃) 的萌发率ꎬ
因此可以判定其种子存在浅的生理休眠ꎮ 种子在恒温下的萌发率比相应的变温下萌发率要高ꎬ 可能是其对
所处荫蔽环境的一种适应ꎮ 硝酸钾对种子萌发有显著的促进作用ꎬ 在野外环境下ꎬ 硝酸钾有对生境空窗及
较少竞争的指示作用ꎮ
关键词: 弥勒苣苔ꎻ 种子ꎻ 休眠类型ꎻ 萌发
中图分类号: Q 945 文献标志码: A 文章编号: 2095-0845(2015)03-278-05
Dormancy and Germination of Paraisometrum mileense
and Their Ecological Implications
LIU Chengꎬ QIN Shao ̄faꎬ HU Xiao ̄jian∗
(Germplasm Bank of Wild Species in Southwest Chinaꎬ Kunming Institute of Botanyꎬ Chinese Academy of Sciencesꎬ Kunming 650201ꎬ China)
Abstract: Paraisometrum mileense is endemic to China and was considered to be extincꎬ after its rediscovery it re ̄
ceived much attention from the botanists as well as the public. Preserving its seeds in a seed bank gives a reliable
way for long term conservationꎻ however its seed dormancy status and germination requirements are scarcely studied.
In this study we checked its embryo size by X ̄ray imagingꎬ the presence of fully developed embryo ruled out the pos ̄
sibility of morphological dormancy. Freshly matured seeds germinated readily at relatively high temperatures (over
20 ℃) and gibberellic acid as well as moist chilling promoted germination at lower temperatures (below 20 ℃) sig ̄
nificantlyꎬ this indicates the seeds are non ̄deep physiologically dormant. Seeds germinated better at constant temper ̄
aturesꎬ this is probably an adaption to shaded environment. The germination also responded significantly to KNO3ꎬ
and KON3 is considered to be an indication of gap and less competition in the field.
Key words: Paraisometrum mileenseꎻ Seedꎻ Dormancy statusꎻ Germination
Paraisometrum mileense W T. Wangꎬ belonging
to the monotypic genus Paraisometrum W T. Wang
(Gesneriaceae)ꎬ is endemic to Southwest China (Wei ̄
tzmann et al.ꎬ 1997). It was first discovered in Yun ̄
nan by a French missionary in 1906 ( Tan et al.ꎬ
2011)ꎬ and was considered to be extinct before 2007.
In 2007ꎬ Shui Yumin from Kunming Institute of Bot ̄
any reported the rediscovery of this species in Shilin
County of Yunnan Province ( Shuiꎬ 2007)ꎬ since
then it received much attention from the botanists as
植 物 分 类 与 资 源 学 报 2015ꎬ 37 (3): 278~282
Plant Diversity and Resources DOI: 10.7677 / ynzwyj201514117
∗ Author for correspondenceꎻ E ̄mail: huxiaojian@mail kib ac cn
Received date: 2014-08-26ꎬ Accepted date: 2014-11-27
作者简介: 刘成 (1986-) 男ꎬ 硕士ꎬ 工程师ꎬ 主要从事植物分类学、 野外植物种子采集和保存工作ꎮ E ̄mail: liucheng@mailkibaccn
well as the public. Though more populations was
found in Guizhou (Gao et al.ꎬ 2011) and Guangxi
(Xu et al.ꎬ 2009)ꎬ it is still considered to be highly
endangered (Guo and Zhengꎬ 2013).
Seed bank provides efficient long term conser ̄
vation for endangered plants which produce orthodox
seeds (Li and Pritchardꎬ 2009). The banked seeds
can be used for the reintroduction and restoration in
the future. For the successful conservation of the target
speciesꎬ information about seed biology such as dor ̄
mancy status and germination requirements is essential.
However this information for P mileense is scarce.
As proposed by Baskin and Baskin ( 2004)ꎬ
there are five classes of dormancy: physiological dor ̄
mancy (PD)ꎬ physical dormancy ( PY)ꎬ morpho ̄
logical dormancy (MD)ꎬ morphophysiological dor ̄
mancy (MPD) and combinational dormancy (PY+
PD). As Gesneriaceae is not in the families that
known to have PY ( Baskin and Baskinꎬ 1998)ꎬ
thus in this study only MD and PD elements were
checked. PD as the most common type of seed dor ̄
mancyꎬ have 3 levelsꎬ non ̄deepꎬ intermediate and
deepꎬ for the first 2 levelsꎬ seed germination usually
response positively to gibberellic acid (GA) ( some
species with intermediate dormancy don’ t response
to GA). For no ̄deep PDꎬ according to the response
to temperature during dormancy breakꎬ five types
are recognized (Baskin and Baskinꎬ 2004).
Besides GAꎬ other chemical compound such as
KNO3 can also promote germination for seeds have
non ̄deep PD (Finch ̄Savage and Leubner ̄Metzgerꎬ
2006)ꎬ and was proved to be effective for seeds of
Begonia guishanensis which also grows in the same
habitat of P mileense (Hu et al.ꎬ 2012). Non ̄deep
and intermediate PD also response to warm or cold
stratification for a relatively short time (few weeks to
few months) ( Baskin and Baskinꎬ 1998ꎻ Baskin
and Baskinꎬ 2004). The aims of this research are
(1) to determine whether seeds of P mileense are dor ̄
mant or notꎻ (2) if the seeds are dormantꎬ to identify
what class (levelꎬ type) of dormancy is presentꎻ (3)
methods to break the dormancy ( if the seeds have
any) and enhance the germination.
1 Materials and methods
1 1 Seed collection
Ripe capsules of P mileense were collected from
Shilin county of Yunnan Province on 22ndꎬ October
2013. Non ̄seed material was removed by hand in the
laboratoryꎬ and only roundꎬ full seeds were used in
the experiments. The experiments were commenced
immediately after seed collection.
1 2 Detection of morphological dormancy
In order to determine if the seeds have MDꎬ
both seed and embryo length and width of randomly
chosen 5 seeds were measured by a Faxitron MX ̄20 ̄
DC12 X ̄ray image system (Faxitron X ̄ray Corpora ̄
tionꎬ Lincolnshireꎬ ILꎬ USA).
1 3 Detection of physiological dormancy
To determine if the seeds have PDꎬ we tested
the germination of the freshly matured seeds as well
as seeds received dormancy release treatments under
various temperatures. The treatments include GA3ꎬ
moist chilling (MC) and KNO3 .
For GA3 and KON3 treatmentꎬ seeds were placed
on 1% agar in distilled water with either 200 mgL-1
GA3 or 90 mgL
-1 KNO3 during the germination test.
For MC treatmentꎬ seeds were placed on 1%
agar in distilled water at 5 ℃ (photoperiod was 12 h
light with 22 2 μmol / m2 / s illumination by cool white
fluorescent light and 12 h dark) for 4 or 8 weeksꎬ af ̄
ter the MC treatmentꎬ seeds were transferred to the
germination test.
Three replicates with 20 seeds were used for
each treatment (GA3ꎬ KNO3 and MC) and the con ̄
trol (freshly matured seeds).
Germination test was carried out in 90 mm Petri
dishes at four constant and four diurnal tempera ̄
turesꎬ these were 10ꎬ 15ꎻ 20ꎬ 25ꎬ 5 / 15ꎬ 10 / 20ꎬ
15 / 25 and 20 / 30 ℃ (night / day)ꎬ the photoperiod
was 12 h light with 22 2 μmol / m2 / s illumination by
cool white fluorescent light and 12 h dark. The Petri
dishes were put in to transparent plastic bags to pre ̄
vent desiccation.
9723期 LIU Cheng et al.: Dormancy and Germination of Paraisometrum mileense and Their Ecological Implications
Germination were checked every 7 daysꎬ seed
with a radical exceeds 1 / 3 the total seed length was
considered to be germinated. The Petri dishes were
rearranged randomly on checking days. The germina ̄
tion test ran for a minimum of 4 weeks after which if
no more seed germinated for continuous 2 weeks the
test was terminated. At the point of termination we
cut the un ̄germinated seedsꎬ empty and rotted seeds
were counted and excluded from the total number of
seeds tested.
1 4 Statistical analysis
Mean±standard deviation of both seed and em ̄
bryo length and width were calculated.
Germination percentage (GP) and mean germi ̄
nation time (MGT) was calculated as:
GP =(∑ni / N)
MGT(days)= ∑(ti∗ni) / ∑ni
Where ti is the number of days from experiment
startingꎬ ni is the number of seeds germinated at each
checking day and N is the total number of seeds tested.
We analyzed the germination percentages and
mean germination time using a univariate General
Linear Model to test the effect of temperature and the
three treatments (GA3ꎬ KNO3 and MC)ꎬ a Student ̄
Newman ̄Keuls ( S ̄N ̄K) post ̄hoc test was applied
for multiple comparisons between different tempera ̄
tures as well as between different treatments. The
percentage data were arcsine transformed before anal ̄
ysis. Differences obtained at a level of P < 0 05 were
considered to be significant. All statistical analysis
was carried out by SPSS 16 0 (Chicagoꎬ ILꎬ USA).
2 Results
The seed length and width are 0 59 ± 0 06 mm
and 0 28 ± 0 02 mm respectivelyꎬ the length and
width of the embryo are 0 52 ± 0 04 mm and 0 21 ±
0 02 mm respectively. Freshly matured seeds germi ̄
nated readily at 20ꎬ 25ꎬ 15 / 25 and 20 / 30 ℃ꎬ the
treatments of GA3ꎬ KNO3 and MC did not show either
positive nor negative effect on germination percentage
significantly (Table 1)ꎬ however MGT was reduced
by all the treatments at those temperatures (Table 2).
At 15 ℃ freshly matured seeds germinated to
77 89% with a MGT of 53 57 daysꎬ GA3 and MC
both promoted GP and reduced MGT significantly while
KNO3 only reduced MGT (Table 1 and Table 2).
Freshly matured seeds germinated poorly at 10
and 10/ 20 ℃ with a germination of 3 42% and 15 0%
respectivelyꎻ all there treatments promoted GP sig ̄
nificantly at both temperatures but only GA3 and MC
reduced MGT significantly at 10 / 20 ℃ (Table 1 and
Table 2).
No germination occurred at 5 / 15 ℃ for freshly
matured seedsꎬ GA3ꎬ KNO3 and 4 weeks of MC
didn’t promote the germinationꎬ only 8 weeks of MC
promoted the germination significantly to 48 33%ꎬ
and as there was no germination occurred for freshly
matured seeds and seeds treated with GA3 and
KNO3ꎬ MGT of these treatment are not available
(labeled as NA) (Table 1 and Table 2).
Table 1 Effects of different treatments on germination percentage (%) of seeds of Paraisometrum mileense at different temperatures
Temperature
/ ℃
Treatment
Control
GA3 KNO3 4MC 8MC
Total by
temperature
10 3 42±2 97a 95 00±5 00b 39 39±20 78c 91 67±2 87b 91 67±5 78b 64 23±39 02
15 77 89±15 43a 100 00±0 00b 75 37±12 60a 100 00±0 00b 100 00±0 00b 90 65±14 06
20 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00
25 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00
5 / 15 0 00±0 00a 0 00±0 00a 0 00±0 00a 1 67±2 89a 48 33±10 40b 10 00±20 26
10 / 20 15 00±15 00a 100 00±0 00b 55 53±17 97c 95 00±5 00b 98 33±2 89b 72 77±35 59
15 / 25 96 67±2 89a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 99 33±1 76
20 / 30 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00a 100 00±0 00
Total by treatment 61 62±45 09 86 88±33 61 71 29±36 60 86 04±32 77 92 29±17 57
Data are means ± standard deviation. Different letters represent significant different treatment means (within the same temperature) by S ̄N ̄K test at
5% level of significance
082 植 物 分 类 与 资 源 学 报 第 37卷
Table 2 Effects of different treatments on mean germination time (days) of seeds of Paraisometrum mileense at different temperatures
Temperature
/ ℃
Treatment
Control
GA3 KNO3 4MC 8MC
Total by
temperature
10 35 00±9 90a 30 20±0 25a 46 29±13 82a 36 36±3 87a 37 84±3 33a 37 29±8 50
15 53 57±2 63a 17 62±0 88b 28 65±2 32c 18 08±1 73b 17 03±0 73b 26 99±14 54
20 13 30±0 35a 8 98±0 53b 9 80±0 35b 12 25±0 70c 7 71±0 02d 10 41±2 17
25 8 52±0 73a 7 00±0 00b 7 37±0 64b 7 36±0 37b 7 25±0 43b 7 50±0 69
5 / 15 NA NA NA 35 00 68 87±5 96 61 15±18 10
10 / 20 44 33±16 50a 19 68±3 76b 35 02±7 31ab 22 18±2 66 b 23 19±1 01b 27 78±10 68
15 / 25 16 64±1 33a 10 15±0 00b 12 83±1 13c 11 99±0 70c 13 42±0 53c 13 01±2 32
20 / 30 13 51±0 85a 8 05±0 35b 11 18±0 95c 10 79±0 97c 11 29±1 38c 10 97±1 98
Total by treatment 25 02±17 67 14 53±8 13 21 59±15 17 17 82±10 02d 23 45±20 42
Data are means ± standard deviation. Different letters represent significant different treatment means (within the same temperature) by S ̄N ̄K test at
5% level of significance. NA means the data is not available
3 Discussion
The X ̄ray image showed the inner part of the
seeds were almost fully filled by the embryoꎬ as
seeds of Gesneriaceae was reported to have little
(one or several layers of cell) endosperm ( Imaichi
et al.ꎬ 2000)ꎬ so we considered the embryo is fully
developed thus the seeds are not morphologically
dormant (Baskin and Baskinꎬ 2004).
Freshly matured seeds germinated well at higher
temperatures (20ꎬ 25ꎬ 15 / 25 and 20 / 30 ℃) and
after the dormancy alleviation treatmentsꎬ seeds gra ̄
dually gained higher germinations at lower tempera ̄
tures (10ꎬ 15ꎬ 10 / 20 and 5 / 15℃)ꎬ according to the
classification system of Baskin and Baskin (2004)ꎬ
seed dormancy of P mileense belongs to the type 2 of
non ̄deep PD.
The habitat of P mileense in Yunnan is influ ̄
enced strongly by the annual monsoonꎻ almost 90% of
the total precipitation of the year occurred during the
wet season (May to October) (Kunming Bureau of
Meteorologyꎬ 2006). Capsules of P mileense matured
and dispersed during late October to early Novemberꎬ
during this transitional periodꎬ a superficially favora ̄
ble condition for germination may occurꎬ thus seeds
of P. mileense may need dormancy to avoid germina ̄
tion under such conditionꎬ otherwise the seedlings
can not survive the following dry season. The crucial
function of dormancy is considered to be preventing
germination when conditions are suitable for germina ̄
tion but the probability of survival and growth of the
seedling is low (Fenner and Thompsonꎬ 2005).
Howeverꎬ the dormancy of P mileense seeds is
only non ̄deep PDꎬ and even less detectable than
other species which also have seeds with non ̄deep
PD from the same region ( e g. seeds of Begonia
guishanensis and B lithophila) (Hu et al.ꎬ 2012)ꎬ
if we only checked the germination at 20ꎬ 25ꎬ 25 / 15
and 30 / 20 ℃ꎬ we would infer seeds of P mileense
have no dormancy. The shallow dormancy of these
seeds indicates the danger of germination under su ̄
perficially favorable conditions after seeds dispersal
is probably not imminent. This is consistent with the
late dispersal of the seedsꎬ the seeds usually dispers ̄
ed during Novemberꎬ at this time in the mountainous
area of this region both precipitation and temperature
are not suitable for germinationꎬ so the seeds do not
need to impose deeper dormancy to avoid germination.
Seeds of P mileense germinated better at constant
temperatures than at the corresponding diurnal temper ̄
atures (which has the same accumulated temperatureꎬ
e g. 10 ℃ and 5/ 15 ℃ꎬ 15 ℃ and 10/ 20 ℃). The
seed of P mileense is smallꎬ and usually small seeds
germinated better at fluctuated temperatures (Fenner
and Thompsonꎬ 2005). Fluctuating temperatures can
act as a sensing mechanism for both soil depth and
vegetation cover (Koutsovoulou et al.ꎬ 2014)ꎬ and
it is unfavorable for small seeds to germinate under
deep soil for their seedlings may fail to penetrate the
soil above (Jankowska ̄Blaszczuk and Dawsꎬ 2007).
1823期 LIU Cheng et al.: Dormancy and Germination of Paraisometrum mileense and Their Ecological Implications
Howeverꎬ even smaller seeds of B guishanensis from
the same habitat also germinate better at constant
temperatures ( Hu et al.ꎬ 2012). Both of the two
plants grow in shaded environment and shade re ̄
duces the amplitude of the diurnal temperature. This
is consistent with some of the new results which sug ̄
gested for very small seedsꎬ light instead of tempera ̄
ture fluctuation is used for detecting the burial depth
(Koutsovoulou et al.ꎬ 2014). Moreover their habitat
is a typical karst plateau mountain areaꎬ both of
them mainly grow on limestone with a thick layer of
mossesꎬ and mosses is consider to be able to provide
a micro ̄environment with stable water and tempera ̄
ture status (Belnap et al.ꎬ 2001).
The germination responded to KNO3 significant ̄
lyꎬ KNO3 is considered to be either a germination
cue which promote germination after dormancy alle ̄
viation (Fenner and Thompsonꎬ 2005) or a dorman ̄
cy release factor as it widened the environmental
range for germination ( Finch ̄Savage and Leubner ̄
Metzgerꎬ 2006). Though the mechanism of KNO3 on
promoting seed germination is still not clearꎬ ecolog ̄
ically it is considered to be an indication of gap pres ̄
ence and soil disturbance ( Fenner and Thompsonꎬ
2005) of the habitat. In this researchꎬ seeds of
P mileense responded positively to both constant
temperature and KNO3ꎬ similar results were also ob ̄
tained for B guishanensis ( Hu et al.ꎬ 2012) and
Stellaria nemorumꎬ this is considered to be a mecha ̄
nism for detecting small ̄scale disturbances under a
closed forest canopy (Vandelook et al.ꎬ 2008).
4 Conclusions
Seeds of P mileense have a shallow type 2 non ̄
deep physiological dormancy. Freshly matured seeds
germinated readily at 20ꎬ 25 and 20 / 30 ℃ꎬ and o ̄
ver all germinated better at constant temperatures
than the corresponding diurnal temperatures. Dorman ̄
cy release treatments such as GA3ꎬ MC and KNO3
can promote the germination significantly at lower
temperatures. For viability test of the seeds in the
labꎬ we recommend 25 ℃ without any pretreatment.
Reference:
Baskin CCꎬ Baskin JMꎬ 1998. Seeds: Ecologyꎬ Biogeographyꎬ and Evolu ̄
tion of Dormancy and Germination [M]. San Diego: Academic Press
Baskin JMꎬ Baskin CCꎬ 2004. A classification system for seed dor ̄
mancy [J] . Seed Science Researchꎬ 14 (1): 1—16
Belnap Jꎬ Prasse Rꎬ Harper GTꎬ 2001. Influence of biological soil
crusts on soil environments and vascular plants [A] / / Belnap Jꎬ
Lange OL ed.ꎬ Biological Soil Crusts: Structureꎬ Functionꎬ and
Management [M]. Berlin: Springer
Fenner Mꎬ Thompson Kꎬ 2005. The Ecology of Seeds [M]. London:
Cambridge University Press
Finch ̄Savage WEꎬ Leubner ̄Metzger Gꎬ 2006. Seed dormancy and the
control of germination [J]. New Phytologistꎬ 171 (3): 501—523
Gao Q (高乞)ꎬ Xu WB (许为斌)ꎬ 2011. Paraisometrum W T.
Wangꎬ a newly recorded genus of Gesneriaceae from Guizhouꎬ
China [J] . Acta Botanica Boreali ̄Occidentalia Sinica (西北植
物学报)ꎬ 31 (4): 858—860
Guo Z (国政)ꎬ Zang RG (臧润国)ꎬ 2013. Evaluation Index system of
endangered levels of the wild plants with tiny population in China
[J]. Scientia Silvae Sinicae (林业科学)ꎬ 49 (6): 10—17
Hu XJꎬ Yang LHꎬ Guan KYꎬ 2012. Physiological dormancy in seeds of two
endemic species of Begonia from Yunnan Provinceꎬ China: diagnosis
and classification [J]. Plant Species Biologyꎬ 27 (3): 201—209
Imaichi Rꎬ Nagumo Sꎬ Kato Mꎬ 2000. Ontogenetic anatomy of Strep ̄
tocarpus grandis (Gesneriaceae) with implications for evolution of
monophylly [J] . Annals of Botanyꎬ 86 (1): 37—46
Jankowska ̄Blaszczuk Mꎬ Daws MIꎬ 2007. Impact of red: far red ratios
on germination of temperate forest herbs in relation to shade toler ̄
anceꎬ seed mass and persistence in the soil [ J] . Functional
Ecologyꎬ 21 (6): 1055—1062
Koutsovoulou Kꎬ Daws MIꎬ Thanos CAꎬ 2014. Campanulaceae: a
family with small seeds that require light for germination [ J] .
Annals of Botanyꎬ 113 (1): 135—143
Li DZꎬ Pritchard HWꎬ 2009. The science and economics of ex situ plant
conservation [J]. Trends in Plant Scienceꎬ 14 (11): 614—621
Shui YMꎬ 2007. ‘100 ̄years ̄lost’ plant re ̄emerges in Yunnanꎬ China
[J] . Samaraꎬ 12: 3
Tan Y (谭英)ꎬ Wang Z (王智)ꎬ Sui XY (隋学艺) et al.ꎬ 2011. The
systematic placement of the monotypic genus Paraisometrum (Gesne ̄
riaceae) based on molecular and cytological data [J]. Plant Diversi ̄
ty and Resources (植物分类与资源学报)ꎬ 33 (5): 465—476
Vandelook Fꎬ Van de Moer Dꎬ Van Assche JAꎬ 2008. Environmentalsig ̄
nals for seed germination reflect habitat adaptationsin four temperate
Caryophyllaceae [J]. Functional Ecologyꎬ 22 (3): 470—478
Weitzmann ALꎬ Skog LEꎬ Wang WT et al.ꎬ 1997. New taxaꎬ new combi ̄
nationsꎬ and notes on Chinese Gesneriaceae [J]. Novonꎬ 7 (4):
423—435
Xu WB (许为斌)ꎬ Pang B (盘波)ꎬ Huang YS (黄俞松) et al.ꎬ 2009.
Paraisometrum W T. Wangꎬ a newly recorded genus of Gesneriace ̄
ae from China [J]. Guihaia (广西植物)ꎬ 29 (5): 581—583
282 植 物 分 类 与 资 源 学 报 第 37卷