全 文 ：青阳参种子的萌发
?
王定康1 , 2 , 孙桂芳3 , 翟书华2 , 岑晓江2 , 赵 剑4 , 萧凤回1
??
( 1 云南农业大学中药材研究所 云南省中药材规范化种植技术指导中心 , 云南 昆明 650201 ;
2 昆明学院生物系 , 云南 昆明 650031 ; 3 昆明医学院技能中心 , 云南 昆明 650031;
4 昆明市第十四中学 , 云南 昆明 650106)
摘要 : 青阳参 ( Cynanchumotophyllum) 种子在 11 月成熟时有休眠习性。收获后将其种子种植在自然温室
内 , 到第二年的春天种子才会萌发 , 且大多数种子在 3 月 28 至 4 月 4 日间萌发 , 这期间的日平均最高和最
低温度分别为 19 .0℃和 9 .9℃。层积能有效地打破青阳参种子的休眠 , 休眠种子通过大约 1 周的层积便能
萌发。种子在有光的条件下层积 1 周后转移到 25?15℃的黑暗条件下萌发率可达到 75 .4%。青阳参种子不
论在有光的条件下还是在黑暗环境中层积 2～3 周后转入 30?20 和 25?15℃进行变温处理 , 其萌发率最低能
达到 66 .4% , 而转入 20?10℃变温处理其萌发率最多只能达到 20 .1% , 但若层积 6 周 , 即便在 20?10℃变温
处理的情况下其萌发率也可以达到 65 .3%以上。
关键词 : 青阳参 ; 萌发 ; 种子 ; 层积 ; 温度
中图分类号 : Q 945 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 02 - 173 - 05
Seeds Germination of Cynanchumotophyllum
(Asclepiadaceae ) Endemic to China
WANG Ding-Kang
1 , 2
, SUN Gui-Fang
3
, ZHAI Shu-Hua
2
, CEN Xiao-J iang
2
,
ZHAO Jian4 , XIAO Feng-Hui1 * *
(1 Instituteof Chinese Medicinal Materials of Yunnan Agricultural University, Yunnan Provincial
Center for Chinese Medicinal Material GAP Technology, Kunming 650201 , China;
2 Biology Department, Kunming University, Kunming 650031 , China;
3 Skill Center, Kunming Medical College, Kunming 650032 , China;
4 Kunming No . 14 Middle School , Kunming 650106 , China)
Abstract: Intact seeds of Cynanchumotophyllumwere innately dormant at maturity in November . Seeds planted in anon-
heated greenhouse in November germinated the following spring and most of thegermination occurred between 28 March -
4 April , when the meanmaximumandminimum daily temperatures were19 .0 and 9 .9℃ , respectively . Stratificationwas
effective in overcomingdormancy, andnondormantseeds germinated in both light and darkness . About oneweek of stratifi-
cation was effective in overcomingdormancy in some of the seeds; seedsstratified in light for 1 week andthen incubated in
darkness at 25?15℃ germinated to 75.4% . Seeds stratified in light or darkness for 2 and 3 weeks and then incubated in
light or darkness germinatedto66 .4% or moreat thermoperiods of 30?20 and 25?15℃ but germinated to20 .1% or less at
20?10℃ . Seeds stratified for 6 weeks also germinated to high percentages (65 .3% or more) at 20?10℃ .
云 南 植 物 研 究 2009 , 31 (2) : 173～177
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.07330
?
?? ?Author for correspondence; E-mail : fenghuixiao@ 163 . com
Received date: 2008 - 08 - 11 , Accepted date: 2009 - 01 - 05
作者简介 : 王定康 (1966 - ) 男 , 博士 , 教授 , 主要从事药用植物繁殖生物学研究。 E-mail : wdk117@163 . com ?
Foundation items: ?教育部科学技术研究重点项目 (209115) ; 云南省应用基础研究计划项目 (2007C262M) ; 云南省教育厅科研基
金重点项目 (07Z10351)
Key words: Cynanchumotophyllum; Germination; Seeds; Stratification; Temperature
Germination is a crucial phase in the life cycleof
a plant, greatly affecting its fitness (Narbona et al. ,
2007) . In order for the seeds of a species to germi-
nate, they must be in suitable environmental condi-
tions, which usually favour the growth and establish-
ment of the seedlings ( Harper, 1977 ) . However, via-
ble seeds of somespecies do not germinate even though
conditions are favourable . Five classes of dormancy are
known to occur in seeds: morphological (MD) , physi-
ological ( PD) , morphophysiological (MPD) , physical
(PY ) , and combinational ( PY + PD) ( Baskin and
Baskin, 2004) .
Cynanchum is a large genus of some 400 de-
scribed species with a tropical and subtropical distribu-
tion in Africa, Madagascar, Australia, North and
South America, and parts of Asia ( Liede, 1997 ) .
Seeds of most of the studied species of this genus have
physiological dormancy (Sheeley, 1992; Zhou et al. ,
2003; Wang et al. , 2007 ) . Seeds of C . rossicum
stored at 4 .5℃ for 5 weeks in the dark had relatively
high germination percentages under either light (46% )
or dark (36% ) conditions in a controlled environment
chamber when subjected for 7 weeks to fluctuating tem-
peratures of 13 - 21℃ (Sheeley, 1992 ) . In contrast,
seeds of C. komaroviil stored 2 - 4℃ for 40 d, and
then incubatedunder an alternating temperature (25℃?
5℃ ; 12 h light?dark) , the percentage of germination
was 3 .33% (Zhou et al. , 2003) .
CynanchumotophyllumSchneid . is a native Chi-
nese perennial vine found on open forest of upland and
shrubs of hillside, ranging from Xizang to Sichuan,
Guangxi, Hunan and Yunnan . The roots are used as
medicine for rheumatism, epilepsy, hydrophobia, and
detoxification of viper bites ( Li et al. , 1995 ) . Some
information on the biology of C. otophyllum has been
summarized (Li et al. , 1995 ) . Little is known about
the characteristics of C. otophyllumseeds .
Onepurposeof this studywas to determinewheth-
er the freshly matured C . otophyllum seeds are
dormant . The second purpose was to observe what the
time seeds germinate when they are exposed to natural
temperature regimes during autumn-winter and spring
and what the temperatures are when germination oc-
curs . The thirdpurposewas to investigate thegermina-
tion responses of seeds stratified in light and darkness
for various periods of time and then incubated in light
and darkness at four alternating temperature regimes
which represent thoseoccur in the field fromearly spri-
ng to summer .
1 Materials and methods
1 .1 Seed collection site
The studied site of Cynanchumotophyllumwas located on a
hill withyellow soil near the town of Longchang, Xuanwei Coun-
ty, Yunnan Province, P . R . China ( 104°09′E , 26°11′N ) .
Theclimate is subtropical with an annual average temperature of
14 .1℃ . The C. otophyllumplants were growing in a dry area,
coveringabout 3 000 m2 and usually thrive in patches . The sur-
rounding landscape was amixture of small woodlots, andagricul-
tural crops . There were about 500 plantswithin the whole area .
1 . 2 General
We collected mature seeds from a population of C . o-
tophyllumplants on8 November 2006 . The seedswere separated
from thetufts of silky hairs . Thecollected seedswereallowed dry
for 1 week in the laboratory, andthe experimentswere started on
15 November 2006 . Seedswere considered to be 0 weeks old on
that date . In all experiments radicle protruded 1 - 2 mmwas the
criterion germination .
1 . 3 Germination at natural temperatures
Each 250 seeds in two copies were planted on greenhouse
potting soil in large flats and placed in a non-heated greenhouse
in Kunming University′s Botany Experimental Field . The win-
dows in this greenhouse were open during the entire experiment
and temperatureswere similar to thoseout-of-doors . Meanweekly
maximumand minimum daily temperatures were calculated from
continuous thermograph recordsmadein thegreenhouse . The soil
in the flats waswatered daily to keep it similar to field capacity .
Theflatswereexamined at about 1-week intervals, and eachtime
thegerminated seedswere counted and then removed .
1 . 4 Germination responses of stratified seeds
Seeds of C. otophyllum were placed on moist, quartz sand
in Petri dishes (50 seeds in each of 240 dishes) and stratified at
4±2℃ for 1 , 2 , 3 , 6 weeks . One half of the seedswas strati-
fied inconstant darkness, andtheother half was stratified in light
(12 h of 28 .8μmol m- 2 s- 1 of cool white fluorescent light each
day) . All Petri dishes were wrapped with wrap to retard loss of
water, and with aluminum foil to provide constant darkness . At
471 云 南 植 物 研 究 31 卷
the end of thevariousstratification periodsseedsweretransferred
to four alternating temperature regimes ( 15?6 , 20?10 , 25?15 ,
and 30?20℃ ) . These temperature regimes approximate the aver-
age daily thermoperiods that occur near the soil surface in spring
and summer in Kunming . At each temperature, seeds stratified
in light and darkness were incubated in light and darkness, and
seeds stratified in darkness were incubated in light and darkness
too . Non-stratified seeds (0 weeks of stratification) were placed
in light and darkness at each temperature regime on15 November
2006 at thebeginningof theexperiment . Seeds incubated in light
received 12 h of 2 000 lux of cool white fluorescent light each
day . The high and low temperatures were maintained for 12 h
each day . Three replications ( dishes) of seeds were used for
each treatment . Germination percentages were determined for all
seeds 2 weeks after they were placed at the alternating tempera-
ture regimes .
1 . 5 Statistical analysis
Mean germination percentageand standard error were statis-
tically tested with Student′s t-test (Zar, 1984 ) .
2 Results
2 .1 Germination at natural temperatures
Germination of the seeds sown after dried for 1
week was practically null . It began in 17 weeks about
the date between 14 March and 21 March 2006 (Fig .
1) , and during this time themeanmaximumandmini-
mum daily temperatures were 18 .7 and 10 .5℃ (Fig .
1) , respectively . Mostof theseeds germinatedbetween
28 March and 4 April . During this interval the mean
weekly maximum daily temperatures ranged from 18 .6
to 19 .5℃ , and the mean weekly minimum daily tem-
peratures ranged from9 .8 to 10 .0℃ . The mean maxi-
mum and minimum daily temperatures from 21 March
and 4 April were 19.0 and 9 .9℃ , respectively . The
mean maximum of seeds germination percentage was
66 .8±2 .21% at natural temperatures .
2 . 2 Germination responses of stratified seeds
Freshly matured, non-stratified seeds ( 0 weeks)
germinated to low percentages in both light ( 5 .2 ±
1.2% , 8 .2±1.58% ) and darkness (15.3±3.42% ,
8.1±2.12% ) at 25?15 and 30?20℃ , but there was no
germination at 20?10 and 15?6℃ ( Table 1 ) . After 1
week of stratification, germination at 25?15 and 30?20℃
wasgreatly improved, especially for seeds incubated in
darkness, but therewasno germination at 20?10 and 15?
6℃ . Regardlessof whether seeds were in light or dark-
ness during stratification or during incubation at the al-
ternating temperatures, seeds receiving2 weeksof strati-
fication germinated to 66.4±3 .78% or more at 25?15
and 30?20℃ , and to 9 .2±2 .45% or less at 20?10;
no seeds germinated at l5?6℃ . Seeds that received
longer stratification periods (3 and 6 weeks) showed an
Fig . 1 Germination of C. otophyllum seeds planted on soil in a non-heated greenhouse in Kunming, Yunnan
5712 期 WANG Ding-Kang et al. : Seeds Germination of Cynanchumotophyllum (Asclepiadaceae) Endemic . . .
Table 1 Germination percentages of C . otophyllum seeds after 0 , 1 , 2 , 3 and 6 weeks of stratification . Seeds stratified in light were
incubated in light and darkness, and seeds stratified in darkness were incubated in light and darkness ( mean±s . d)
Stratification
treatment
Germination temperature
change ( ℃ )
Stratification time (wks)
0 ?1 ?2 3 46
Light-Light 15 C?6 0 ?0 ?0 0 40
20 0?10 0 ?2 j. 80±1 P. 21 4 ?. 1±0 .78 20 .1±1 p. 32 76 . 5±1 .32
25 0?15 5 .2±1 ?. 2 34 .2±3 P. 65 85 ?. 2±6 .54 90 .3±5 p. 78 94 . 2±3 .55
30 0?20 8 ?. 2±1 .58 20 .0±1 P. 65 75 ?. 3±4 .64 89 .2±3 p. 96 94 . 1±1 .34
Dark-Light 15 C?6 0 ?0 ?0 0 40
20 0?10 0 ?0 ?4 .8±1 .21 7 .5±1 ]. 87 78 . 5±0 .77
25 0?15 0 ?19 .1±2 P. 54 66 ?. 5±3 .33 91 .0±6 p. 21 95 . 2±2 .56
30 0?20 0 ?21 .4±2 P. 45 68 ?. 7±5 .23 85 .1±5 p. 54 95 . 3±2 .23
Dark-Dark 15 C?6 0 ?0 ?0 0 40
20 0?10 0 ?0 ?4 .0±0 .65 18 .3±1 p. 22 65 . 3±3 .12
25 0?15 15 .3±3 ?. 42 65 .2±5 P. 75 75 ?. 5±6 .58 90 .1±4 p. 66 95 . 2±3 .28
30 0?20 8 ?. 1±2 .12 30 .3±3 P. 37 66 ?. 4±3 .78 84 .5±5 p. 64 96 . 1±2 .79
Light-Dark 15 C?6 0 ?0 ?0 0 40
20 0?10 0 ?0 ?9 .2±2 .45 16 .2±1 p. 36 75 . 2±2 .12
25 0?15 0 ?75 .4±3 P. 45 90 ?. 1±6 .23 92 .5±5 p. 21 97 . 1±1 .63
30 0?20 0 ?66 .0±3 P. 69 80 ?. 4±4 .21 91 .5±4 p. 98 86 . 2±5 .31
increase in ability to germinate at 20?10 , 25?15 , and
30?20℃ . After 6 weeks of stratification, regardless of
whether seeds were in light or darkness during and?or
after stratification, seeds germinated to 94 .1±1 .34%
or more at 30?20 and 25?15℃ but no seeds germinated
at15?6℃ . Seedsgerminated to 55 .3±3 .12% or more
incubated in darkness at 20?10℃ .
3 Discussion
Seeds of Cynanchumotophyllumdried for 1 week
at natural temperaturedid not germinate . However, the
fact that the seeds sown under the same conditions did
germinate after 17 weeks indicates that they had physi-
ological dormancy . Physiological dormancy is common .
It has been reported in common milkweed ( Asclepias
syriaca L .) ( Evetts and Burnside, 1972; Jeffery and
Robison, 1971; Oegma and Fletcher, 1972) and but-
terflymilkweed ( Asclepias tuberoseL .) ( Bir, 1986) in
the family of Asclepiadaceae . Dormancy can bebroken
by short period of moist, low-temperature treatment
(stratification) ( Evetts and Burnside, 1972; Jeffery
and Robison, 1971; Oegma and Fletcher, 1972; Yang
and Liu, 2005) , removing the seed coat (Oegma and
Fletcher, 1972; Ren and Abbott, 1992 ) , dry labora-
tory storage ( Evetts andBurnside, 1972) and treatment
withgibberellic acid or kinetin ( Evetts and Burnside,
1972; Oegma and Fletcher, 1972 ) . Physiological
dormancy also has been found in species of the genus
Cynanchum, in which stratified at 2 - 5℃ for 5 - 7
weeks was effective in overcoming dormancy (Sheeley,
1992; Zhou et al. , 2003; Wang et al. , 2007) .
When seeds of C . otophyllum are dispersed in
November, although a small percentage of the seeds is
non-dormant, there is no germination . These non-
dormant seeds have a high temperature requirement for
germination (30?20 and 25?15℃ ) ( Table 1 ) and can
not germinate in natural conditions because tempera-
tures are too low then inKunming . When the seedsbe-
gin to after-ripen during winter, they obtain the ability
to germinate to high percentages at temperatures repre-
sentative of late April and early May ( 25?15 and 30?
20℃ ) , but germination is prevented because tempera-
tures of the environment are too low in the experiment
site . With further after-ripening theseeds become capa-
ble of germinating if subjected to lower temperatures
(20?10℃ ) , but temperatures of the environment are
still below those required for germination . Germination
is delayeduntil springwhentemperaturesof the environ-
ment increaseand correspond tothose required for germ-
ination . Seeds subjected to natural , low winter temper-
atures in the non-heated greenhouse germinated in
April whenmeanmaximumandminimumdaily temper-
ature were about 20 and 10℃ , respectively (Fig . 1) .
In the early stages of after-ripening (after 1 week
671 云 南 植 物 研 究 31 卷
of stratification) seeds germinated better when incubat-
ed in darkness than in light (Table 1 ) . As the seeds
becamemore fully after-ripened, however, they germi-
nated equally well in light or darkness, regardless of
whether or not they received light during stratification .
Thus, seeds that overwinter in the field can germinate
in darkness in spring if they become buried in either
autumn or spring . Jeffery and Robison ( 1971 ) found
that seeds germinated better when planted 1 or 2 cm
deep thanwhen plantedon thesoil surface . One possi-
ble reason for low seed germination in their study was
the fact that they used open pots, causing high surface
evaporation and low water availability to seeds .
To sum up, Cynanchum otophyllum seeds have
non-deep physiological dormancy, which is broken in
late spring and early summer, thus allowing seeds to
germinate in theoptimal wet season . The physiological
dormancy of seeds can be broken by stratifying at a low
temperature . Seeds cangerminateunder thesituationof
light and darkness .
Acknowledgements: We thank Prof . C . F . Voegelin and Dr .
J . H . Jackson for their comments and valuable suggestions on
themanuscript .
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7712 期 WANG Ding-Kang et al. : Seeds Germination of Cynanchumotophyllum (Asclepiadaceae) Endemic . . .