全 文 ：植物遗传资源的种子基因库保存?
唐安军1 , 2 , 宋松泉3 , 龙春林1
??
( 1 中国科学院昆明植物研究所 , 云南 昆明 650204; 2 中国科学院研究生院 , 北京 100039;
3 中国科学院西双版纳热带植物园 , 云南 勐腊 666303 )
摘要 : 一个物种的灭绝是与其受生物因子和非生物因子的威胁程度相关的。随着物种的加速绝灭 , 保护生
物多样性受到广泛地关注。保护生物多样性的最有效的生物技术之一是建立种子基因库 , 进行迁地保护。
种子库理想的贮藏条件主要取决于种子含水量、贮藏环境 (如温度和湿度 ) 和贮存种子的容器。进行种子
贮藏 , 了解种子生命力和活力的影响因子的作用机理是十分重要和必要的。除了种子自身的生理特征外 ,
种子的贮藏寿命与种子成熟度、收获技术、加工处理方法也是息息相关的。即使在最适的库存条件下 , 种
子也会随时间发生劣变。因此 , 必须根据种子特定的贮藏行为 , 加以考虑影响种子存活的 3 个主要方面
(贮藏环境、贮藏期和植物种类 ) 而选择有效的贮藏方案。本文试图讨论种子贮藏生理的几个重要方面及
其需解决的技术问题 , 以便更好地通过种子基因库 , 长期有效地保存植物种质资源。
关键词 : 生物多样性 ; 种子库 ; 植物种质 ; 种子贮藏行为 ; 种子生命力与活力 ; 贮藏寿命
中图分类号 : Q 948 文献标识码 : A 文章编号 : 0253 - 2700 (2007) 01 - 043 - 08
Ex Situ Conservation of Plant Genetic Resources
through Seed-Gene Bank
TANG An-Jun1 , 2 , SONG Song-Quan3 , LONG Chun-Lin1 **
(1 Kunming Instituteof Botany, Chinese Academy of Sciences, Kunming 650204 , China; 2 Graduate School of
ChineseAcademy of Sciences, Beijing 100039 , China; 3 Xishuangbanna Tropical Botanic Garden,
Chinese Academy of Sciences, Mengla 666303 , China)
Abstract: With the acceleration of extinction of species, biodiversity conservation is extensively concerned . The extinc-
tion of species is concerned with the degree of threat bybiotic and abiotic factors . So, taking action to preserve plant spe-
cies is verynecessary and paramount beforetheir extinction . Oneof themost effectivebiological techniques to conservethe
biodiversity is the establishment of genebanks, i. e . ex situ conservation . The elucidation of various factors that regulate
seedviability and vigor in storage isessential . An ideal condition toprolongthe longevity ismainlydepended on seedwater
content, temperature, humidity and types of containers used during storage . The optimum stage of seed maturity, har-
vesting techniques and processing, inaddition to physiological features such as degree of dormancy, also play key roles in
seed storage . Certainly, desiccated seeds deteriorate with time even under extremely good genebanking conditions . Ac-
cording to seed storage behavior, it is necessary to consider three principal factors: storage environment, storageduration
and plant specieswhich will affect seed survival under good genebanking conditions . The present review is an attempt to
discuss the importance of the aforementioned aspects of seeds in detail in order to conserve plant germplasms ( especially
wild rareand endangered plants) for ex situ conservation through seed-gene bank .
云 南 植 物 研 究 2007 , 29 (1 ) : 43～50
Acta Botanica Yunnanica

?
?? ?Author for correspondence; E-mail : long@mail .kib. ac. cn
Received date: 2006 - 03 - 10 , Accepted date: 2006 - 04 - 30
作者简介 : 唐安军 (1976 - ) 男 , 博士研究生 , 主要从事种子生物学研究工作。E-mail: tanganjun@ mail .kib. ac. cn ?
基金项目 : 中国科学院知识创新工程重要方向性项目 ( KSCX2-SW-117) 、科技部国家科技基础条件平台工作项目 ( 2004DKA30430)
和 (2005DKA21006)
Key words: Biodiversity; Seed bank; Plant germplasm; Viability and vigor; Storage behavior; Storage longevity
That biodiversity is sharply decreasing is beyond
dispute . In many regions, especially, tropical re-
gions, complex and species-rich ecosystems, are be-
ing rapidly destroyedor changed, and somefragile en-
vironments, such as wetlands, arid and semi-arid re-
gions, are threatened by the increasing stress fromhu-
man disturbance and fluctuating climates . At the cur-
rent rate of deforestation, extinction of speciesmust be
imminent . Moreover, a considerable amount of genet-
ic diversity within plants that cansurvive is likely to be
lost . Fragmentation of habitats is bound to affect the
fate of plants, especially, rare and valuable plants .
If someplants in fragmented habitatsmay bereduced to
such low numbers that they cannot constitute viable
populations . In such populations, genetic drift and
inbreeding may result in inbreeding depression . Thus,
a combination of demographic and genetic factors may
hasten the extinction of some plants in small , isolated
fragments . Generally, extinction is a consequence of
themutually accelerating demographic and genetic de-
cline of a population . Appropriate scientific informa-
tion is required to design an effective conservation plan
for any threatened species . There has been a general
recognition in recent years that thegenetic variation pr-
esent in akindof species is avery valuable resource for
genetic engineering ( Lu, 1998 ) . The loss of genetic
diversity represents a type of partial extinction that of-
ten presages its total extinction . Conservation and evo-
lutional inferences about any impacts of environmental
changes on plant populations should consider not only
both demographic and genetic processes, but also the
complicated interactions of these processes ( Reed et
al . 2002) . So it is necessary to understand the cer-
tain biological features of species to find out the caus-
ative factors which lead to reproductive and regenera-
tion failure . These days, there is a general awareness
that is necessary and crucial to conserve natural plant
resources worldwide . Studies have shown that many
plant species are in danger of extinction, while some
have already become extinct . On a global basis, the
global red list, 2000 IUCN Red List of Threatened
Species (SSC, 2000) , includes5611 threatenedplant
species . In compiling the red list, only approximately
2% of an estimated vascular plant flora of 270 000
species worldwide was assessed (SSC , 2000) . Thus,
the proportion of species listed as threatened is more
than 20% of species that were assessed (SSC , 2000) .
Especially, there are many plants distributed only in
single country or a special region . Thus, in order to
build a relatively small amount of relevant work on rare
and threatened ( plants) species priorities shouldbede-
termined so as to make conservation strategies . ( ht-
tp:??www. wri .org?biodiv?in-situ.html ) .
Facing the conservation biodiversity of a problem,
conservation biologists are scramblingto devisemethods
and techniques for plant species protection and preser-
vation . One of most goals of many preservation pro-
grammes, in addition to habitat preservation, is main-
taining the existinglevel of genetic variation in species .
Presently, there are two strategies commonly used in
conservinggenetic resources . One is in situ conserva-
tion, and the other is ex situ conservation ( Cheng,
2005) . In reality, we think, both in situ conserva-
tion and ex situ conservation are complementary and
should not view as alternatives . On the contrary, this
complementary conservation should be described as in-
ter situ conservation .
In agriculture, most crop species are conserved
by ex situ accesses, such as seed-gene banks, field-
seedling banks, tissue culture and cryopreservation
gene-bank . However, in forestry, becauseof thelong
regeneration time required by trees, the perfect con-
servation approach is likely to incorporate in situ con-
servation principles into sustainable using and manage-
ment . But, if in situ conservation is difficult for some
tropical ecosystems, exsitu conservation through seed-
gene bank will becomean effective approach . To fulfill
thegoal , we needbasic knowledgeonseedbiologyand
technology such as seed mature index, seed harvest-
ing, processing, germination, dormancy, viability,
44 云 南 植 物 研 究 29 卷
vigor and storage physiology for various seeds . Though
much knowledgeof different seeds has been document-
ed, it is still quite inadequate . This article focuseson
all the aboveaspects of seedsof temperate, subtropical
and tropical plant species .
1 Categories of Seed Storage Behavior
Seeds are the most convenient forms by which to
store and distribute plant germplasm . In other words,
seed storage plays a complementary role in germplasm
conservation in sustainable development of social econ-
omy . To fulfill the conservation roles, seed-storage
lifemust be maintained in a viable condition from the
time of collection until the time of thenext generation .
Successful long-termstorage is depended on continuous
viability monitoring with re-collection or regeneration,
whenever the viability drops to a limited minimum lev-
el . The longevity of seeds varies fromspecies to spe-
cies, though they are provided identical storage condi-
tions . Practically longevity of seeds in storage depends
on their sensitivity to temperatures and tolerance to
desiccation .
In the 1970s, Roberts (1973) divided seeds into
two major groups, i . e . orthodox and recalcitrant seed
depending on their inherent nature . In other words,
“orthodox” and“ recalcitrant” are used to describe
storage behaviors of different seeds . Orthodox seeds
can be dried without damage to low moisture contents
and, over a wide rangeof environments, their longev-
ity increases with decrease in seed storage moisture
content and temperature in a quantifiable and predict-
able way ( Roberts, 1973; Ellis, 1988; Dickie et
al . 1990) . The latter is defined by the seed viability
equation:
V = Ki - p?10KE - Cw log10 m - CH t - CQ t2
WhereV is probit percentage viability after p days in
storage at m percent moisture content (w.b .) , t℃ ,
Ki is a constant specific to the seed lot, and KE , CH
and CQ are species viability constants ( Ellis and Rob-
erts, 1980; Cromarty et al . 1982 ) . Seeing fromthe
above equation, many researchers have found that the
longevity of orthodox seeds varies with different seed
water content and temperature .
Generally, orthodox seeds acquire desiccation tol-
erance during development and may be stored in the dry
state for predictable periods under defined conditions .
Unless debilitated by zero-tolerant storage fungi , ortho-
dox seeds should maintain high vigor and viability from
harvest until the next growing season . Over the years,
the techniques, such as ultra-dry storage, for conserv-
ing orthodox seeds have been developed ( Cheng,
2005) . These involve drying seed to lowmoisture cont-
ent (MC) (3 - 7% fresh weight, dependingon thespe-
cies) and storing them, in hermetically-sealed contain-
ers, at low temperature, preferably at - 18℃ or cooler
( FAO?IPGRI , 1994 ) . These procedures have been
widely adopted by seed-banks worldwide .
Many species of tropical , subtropical or temperate
origin have seeds that are sensitive to drying and chill-
ing and cannot be stored in conventional genebanks .
Seedsof this type are termed recalcitrant seeds ( Rob-
erts, 1973; Berjak and Pammenter, 2001 ) . Recal-
citrant seeds aren′t equally sensitive, and the variable
degrees of dehydration are tolerated depending on the
plant species . This implies that the processes and
mechanisms that course desiccation tolerance are vari-
ably developed or expressed in the nonorthodox condi-
tions . Differential desiccation sensitivities among re-
calcitrant seeds of various species are clearly shown by
their different responses when subjected to the same
drying regime ( Farrant et al . 1989; Berjak and Pa-
menter, 2001 ) . Thereby, the recalcitrance of seeds
can be also classified into three types ( although be fur-
ther subdivided) : minimal , mediumand high recalci-
trancemakeup acontinuumof seed recalcitrance (Far-
rant et al . 1988 ) . As is known, the response to de-
hydration depends upon the metabolic activity of the
seed and the rate of drying . Thereby, this makes it
difficult to measure desiccation tolerance—there is no
such thing as“critical water content”that is character-
istic of a species ( Pammenter and Berjak, 2000 ) .
Based on it, Song et al .(2003) suggested anewwork-
ing approach be to quantify the degree of seed recalci-
trance that does not depend on an absolute specific wa-
541 期 TANG An-Jun et al .: Ex Situ Conservation of Plant Genetic Resources Through Seed-Gene Bank
ter content related to desiccation damage but depends
on storage lifespan of seeds . If seeds of this type are
stored under conditions by means of traditional meth-
ods, their life spans are limited to a fewweeks, occa-
sionally months (Berjak and Pammenter, 2001) .
In the 1990s, seeds of woody plants were again
classified into four categories based on the length of
their viability and tolerance to freezing temperature,
i . e . true orthodox, sub-orthodox, temperate recalci-
trant and tropical recalcitrant ( Bonner, 1990) . Tem-
perate recalcitrant seeds of plants from genera such as
Quercus cannot bedried at all , but they can be stored
for several years at near-freezingtemperatures . By ma-
intaininghigh water content and necessary gas, seeds
of Quercus species can be stored for 3 - 5 years (Bon-
ner, 1996 ) . But, given the same environment,
tropical recalcitrant seeds such as Dipterocarpus (Yap,
1986; Tompsett, 1987 ) and Jackfruit ( Chin and
Roberts, 1980) cannot survive .
Besides, a third seed storage behavior had been
identified as intermediate seed ( Ellis et al . 1990 ,
1991) . Seedsof many plants showintermediatestorage
behavior, surviving desiccation to fairly low moisture
content, but suffering injury due to low temperature .
In comparison with truly recalcitrant seeds, partial
drying can prolong thestorage lifeof these intermediate
seeds, but it remains impossible to achieve the long-
termconservation, which has been realized for ortho-
dox seeds . Sothe long-termmaintenanceof viability of
intermediate seeds resembling recalcitrant seeds is a
vexed problem, provided the storage environment is
defined well and controlled . Therefore, if an acces-
sion of seeds of aparticular plant is to be conserved, it
is necessary to determine whether these seeds show or-
thodox, recalcitrant or intermediate storage behavior .
At present, however, the information of seed storage
behavior is fairly inadequate . Remarkably, the same
plant species of somegenera that aredistributed in dif-
ferent regions have many individuals impacted by dif-
ferent ecological factors for many years . Thus, they
can produce different seeds with different features, in
particular, their storage behaviors . In other words,
seeds of the same species from different provenances
may show diverse storage behavior ( Hong and Ellis,
1998) .
To summarize, before being stored in seed
banks, storage behaviors of seeds collected have to be
identified according to complex physiological proper-
ties . Only after seed storage behavior is confirmed,
can suitablemethods and technologies be selected . For
storage techniques, besides ultradry storage and low-
temperature storage, cryopreservation is thoughtof as a
perfect approach, but how to carry it out successfully
for recalcitrant seeds is still a vexed problem (Walters
et al . 2004; Panis and Lambardi , 2005) .
2 Seed maturity , viability and vigor test
High quality seed is a prerequisite for higher and
reliable yield of crops and establishments of healthy
seedlings for forestry . Don′t forget, seed maturity is
close related to seed viability and vigor . The mature
stage of seeds has a critical effect on seed vigor . Gen-
erally speaking, vigor of mature seeds is higher than
that of half-mature and immature seeds . So collection
period for conservation purposes should be ideally se-
lected . Only in this way, can seed vigor and viability
tested reflect truly the quality of seed lots .
Under certain environments, it is impossible to
estimate the viability of seeds by a standard laboratory
germination test . Seed researchers have been interest-
ed in indirectmethodsof assessingtheviability of seeds
without the necessity of a routine germination test,
particularlywhen dealing with the deep dormant seeds
or seeds requiring a rather long period for the comple-
tion of germination . Indirect tests can be performed
within a few hours and are thus a great favor in cases
where results of the tests are required as soon as possi-
ble . The triphenyl tetrazolium choride ( TTC) , elec-
trical conductivity of seed leachates, excised embryo
test, X-ray cutting test, electrical impedance spec-
troscopy ( EIS) and fluorescein diacetate ( FDA ) are
some of the indirect, reliable, routine viability tests
( ISTA , 1993) . In thepast, the resultsof storage re-
search were evaluated primarily in terms of germination
64 云 南 植 物 研 究 29 卷
and?or viability percentage . Now, all well-planned
storage works incorporate some type of vigor test as an
integral part of the evaluation . It is worth pointingout
the value of seed leachate conductivity . Loss of via-
bility and increase in seed leachate conductivity indi-
cate that the changes in thermodynamic properties of
seed water reflecting the seed deterioration during stor-
age under accelerated ageing conditions ( Krishnan et
al . 2004) .
The importance of vigor as an important aspect in
seed quality is clearly indicated by the trends in recent
seed storage research . Loss in vigor can be thought of
as an intermediate stage of seeds, occurring between
theonset and terminationof ability of germination . Pr-
esently, no general accepted and satisfactory method
has been found to measure the vigor of a particular
plant, but some vigor test methods havebeen used for
different purposes . These methods include germination
value, accelerated aging test, cool temperature test,
germination rate, meangermination time, excised em-
bryo test, andgermination index (Song et al . 2005) .
Except for methods mentioned, liquid nitrogen quick
test is also a good tool ( Becwar et al . 1983 ) . In
short, all themethods play key roles in testing physio-
logical quality of seeds, especially, in testing their vi-
ability during long-termstorage .
3 Seed germination and pretreatment
The standard for judging seed quality is always a
germination test under suitable conditions . Moisture
content, temperature, media and light are the critical
factors affecting seed germination . Optimum tempera-
turevaries with ecotype; seeds are biochemically ac-
tive at this temperature above and below which any
fluctuation retards the rate of biochemical activity,
which in turn results in inhibition or slowingof theger-
mination rate ( Kebreab and Murdoch, 1999 ) . Simi-
larly, the light and media requirement for optimum
germination percentage varies with plant species . Cor-
binean and C?me (1982 ) found that the intensity and
duration of light at various temperatures had profound
effects on the germination of Oldenlandia corybosa L .
seeds . During the past years, considerable progresses
havebeen madeon thequantificationof germination re-
sponses to temperature and themodel of thermal timeof
seedgermination has developed (Moot et al . 2000 ) .
Seed germination is to calculate the thermal time (Ke-
breab and Murdoch, 1999; Alvarado and Bradford,
2005) . Besides, several researchers showed that the
cardinal temperature and thermal time for the rate of
germination depend on species and may vary signifi-
cantly amonggenotypes (Mohamed et al . 1988) . De-
termination of the cardinal temperature and thermal
time for seed germination rate will facilitate conserva-
tionists or seed-gene bank managers to select a suitable
sowing season and agro-climatic zone for introductionof
plants species in field for regeneration and as in situ
conservation stand .
In some mature seeds of woody trees or crops,
seeds fail to germinate promptly even under the opti-
mumgermination conditions . The absence of germina-
tionof an intact viableseed under favorablegermination
conditions within a specified time lapse is termed as
dormancy (Bewley and Black, 1984) . Seed dormancy
was divided into three types depending on how each of
them arises: viz ., as innate, enforced and induced
(Harper, 1977) . Baskin and Baskin ( 2004 ) brought
forwards a current classification system, including
physiological dormancy, morphological dormancy,
morphophysiological dormancy, physical dormancy,
and combinational dormancy . Of course, the dormant
conditions varyevenwithin aspecies, dependingon the
differences between individuals, location, climatic
conditions, time of collection, as well as nature and
duration of seed storage after collection . Depending on
thedormancy type and its degree, the pretreatment is
different from species to species . The most common
requirements are exposure to periods of warmth and?or
cold, soaking of in hot and cold water . Generally,
there are three types of stratifications, which include
warm-moist stratification, cool-moist stratification and
warm-moist-cool-warm stratification ( Steadman, 2003;
Baskin and Baskin, 1991 ) . In other cases of hard
seeds, soaking in concentrated sulfuric acid and scarifi-
741 期 TANG An-Jun et al .: Ex Situ Conservation of Plant Genetic Resources Through Seed-Gene Bank
cation are also effective methods for breaking dormancy
of hard seeds (Nasreen et al . 2002; Morris et al .
2000) . In addition, a biochemical change controlled
by the interaction between the inhibitor and growth pro-
moter does have a major role in actual breaking of
dormancy (Khan, 1977; Duan et al . 2004; Rinaldi ,
2000; Jacobsen et al . 2002) . Currently, it is recog-
nized that smoke-stimulatedgermination is not limited to
species from fire-prone habitats, and a variety of spe-
cies fromfire-free habitats also respondpositively (Light
and van Staden, 2004 ) . As are aforementioned as-
pects, knowledge of optimum germination and pre-
treatment conditions is essential prior to routineviabili-
ty test during seed storage, even before being sowing .
4 Conclusion
While scientific and sociopolitical communities
around the world are awareof thenatural and economic
importance of biodiversity, we are facing with an ever-
increasingnumber of plant species under threat of ex-
tinction . Conservation is thus a vital part of the plant
scientist′s work, in the field, in the botanic gardens,
in institutes and in universities .
The diversegenetic informationof plants is scien-
tific material in the era of genetic engineering, as well
as material base of mankind . But, large-scale de-
stroyingof forests and wetlands are accelerating species
extinction, so conserving them through in situ conser-
vation and ex situ conservation is highly necessary for
social economic sustainable development . Moreover,
we feel strongly that it is high time to explore the stor-
age physiological knowledge for their cost-effective
long-term conservation . Here, we recommended that
efforts should be made to develop post-harvest technol-
ogy for proper handling of various seeds . At the same
time, we must understand the relationship between
seed vigor and desiccation tolerance . Studies of the
premature harvest on seed vigor and viability suggest
that maximumdesiccation tolerance is achieved step by
step (Galau et al . 1991) . Indeed, desiccation toler-
ance is acquired continuously during seed maturation,
many orthodox seeds acquire maximum vigor and via-
bility after maximumdrymatter accumulation, at time,
in dry storage (Demir and Ellis, 1992; Welbaumand
Bradford, 1989) . Recalcitrance appears to be a prod-
uct of either postvascular separation ( PVS) stageor an
early termination of development ( Finch-Savage,
1992) .
According to their storage behaviors, seeds col-
lected are classified into a certain category . Once
seeds of aparticular species are classified, it is essen-
tial to develop complementary strategies for their con-
servation according to their storage physiology . For in-
stance, ultradry of orthodox seeds and cryopreservation
of various seeds canoffer effective and economically vi-
able alternatives for long-term ex situ germplasm con-
servation through seed-genebank . Although the techn-
ology of seed ultradry storage is promising in plant
germplasmconservation, its application in seed banks
has still many challenges such as various safe water
contents and methods of their obtaining . In addition,
plant cryopreservation technologies have been evolving
rapidly, opening the door to the possibility of long-
term storageof valuablegenetic resourcesof many plant
species, but there are two important aspects (freezing
safe water content and procedures) to be solved in aca-
demic studies . Perhaps additionally, the damaging
consequences could be realizedonceseeds are removed
fromeither cold- or cryo-storage . As observed by Ben-
son and Bremner (2004) , Levitt (1962) originally hy-
pothesized that four potentially injurious phases exist in
freezing injury: the moment of freezing; in the frozen
state; the moment of thawing; and during the post-
thaw period . Of course, to facilitate the development
of even more efficient cryopreservation protocols, a
better knowledge of the physio-chemical background of
cryopreservation is needed . This canonly beunraveled
through fundamental studies that involve both thermal
analysis and athorough examinationof thedifferent pa-
rameters that can influence the cryo-behavior, like en-
dogenous sugars, membrane composition, oxidative
stress and cryoprotective proteins . So it is essential for
seed biologists to investigate these parameters for differ-
ent seeds, particularly recalcitrant seeds .
84 云 南 植 物 研 究 29 卷
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