全 文 ：文章编号: 1007-0435( 2002) 03-0198-05
羊草幼穗离体培养诱导植株再生的研究
刘公社, 汪恩华, 刘　杰* , 齐冬梅, 李芳芳
(中国科学院植物研究所, 北京 100093)
摘要: 采用离体培养方法首次获得羊草体细胞再生植株。其方法要点是:取羊草幼穗为外植体, 经 0. 1% HgCl2溶
液表面消毒后, 接种到含 2mg / L 2, 4-D 的M S 培养基上, 置于恒温 25℃条件下诱导愈伤组织。在加有 1 mg/ L 2, 4-
D MS 培养基上继代 2次后,转移到含 1. 0mg / L KT 和 0. 5mg / L NAA 的M S 培养基上分化培养得到再生芽。在无
激素的基本培养基上获得了生根的试管苗, 试管苗移栽到温室后生长正常。研究结果还表明, 尽管从羊草叶片、幼
穗和成熟胚在同样培养条件下均能诱导出愈伤组织,但只有幼穗愈伤组织能够继续分化出再生植株。羊草试管苗
的分化因基因型和外源激素的不同而异。
关键词: 羊草( L eymus chinensis) ; 幼穗; 外殖体; 再生植株
中图分类号: Q813. 1　　　文献标识码: A
Plant Regeneration of Leymus chinensis Via in Vitro Culture
LIU Gong-she, WANG En-hua , LIU Jie
* , QI Dong-mei, LI Fang-fang
( Ins t itu te of Botany, Th e Chinese Academy of Sciences , Beijing 100093, Ch ina)
Abstract: T he regenerated plants were obtained from L eymus chinensis for the f ir st t ime through t issue
culture. Immatur e-base explants w ere cultured on M urashige and Skoog s basal medium ( MS) supplement-
ed w ith 2 mg / L 2, 4-D af ter surface sterilized with 0. 1% HgCl2 to induce callus at 25℃ constant tempera-
ture. Af ter moved to M S medium containing 1mg / L 2, 4-D for tw o times, the compact calluses w ere select-
ed and transfer red to M S basal medium containing 1. 0mg / L Kt and 0. 5mg / L NAA, which dif ferentiated
into shoot-buds. T he shoo t-buds appeared and regenerated new roo ts, f inally developed plant lets on 2, 4-D-
fr ee basal medium . Regenerated plants g row th w ell w hen transferred into g reenhouse. A compar at ive
study of differ ent explants, in this study, revealed that only could the callus f rom immature stachy s dif fer-
ent iate into regenerated plants. The dif ferent iate potent ial of regenerated plants has close related to the
genotype and external hormone.
Key words : L eymus chinensis; Immatur e stachys; Explant ; Plant regenerat ion
　　L eymus chinensis ( T rin. ) T zv el. is a v ery im-
por tant fo rage species o f gr amineae and w idely dis-
tr ibuted in loess plateau, Songnen plain and Inner
M ongolia plateau in China. It was no t only the king
of g ramineae forage due to high product i-
v ity , high protein content , adaptability to surr oun-
ding s and palatablity to cat t le, but also a major
candidate in art if icial grassland constr uction and
improving ecotope as w ell
[ 1]
. In recent years, how-
ever, ow ing to the over load on g rassland and
收稿日期: 2002-01-31; 修回日期: 2002-03-28
基金项目: Fou ndat ion item : Su pported by Key Project of the Ch ines e Academ y of S cience ( KSCX1-08-03) and State Key Project of the
Minis try of S cience and Techn ology (2001BA707B02)
* Au th or for correspondence.
作者简介: 刘公社( 1958-) ,男,陕西人, 1986年在法国获生物学博士学位。1986—1988年回国从事博士后研究工作,现为中国科学院植物
研究所首席研究员,研究方向为生物工程及其在持续农业上的应用。发表学术论文 72篇,编著 3部
第 10卷　第 3期
　Vo l. 10　　No. 3
草　地　学　报
ACT A AGRESTIA SIN ICA
　2002 年　9月
Sept . 　2002
the exacerbated environment pr oblems, the popula-
tion of L . chinensis has become mo re and more de-
generated
[ 2]
. To alleviate or prevent it f rom further
gr ow ing w or se, some exploring researches have
been done such as morphological descript ion, m i-
cro spore development , chromosome diversity, phys-
iolog ical explanat ion to the reason o f flow er , dor -
mancy and fruct if ication in L . chinensis, genet ic
variance analysis w ith isozyme and molecular
marker and so on
[ 3～8] . Although creat ion o f novel
germplasm and genet ic impr ovement through tech-
niques of t issue culture ho lds g reat potent ial, there
are few repo rts on t issue cul ture of L . chinensis and
very lit t le pro gress has been made in regenerat ion
of plant via in vit ro cul ture up to now .
[ 9, 10] . The
pr esent study is the f irst account o f plant r eg enera-
tion f rom immature spikes on M S medium and es-
tablishment of a reliable regener at ion system via
t issue culture for L . chinensis.
1　Materials and methods
1. 1　Plant material
Eleven L . chinensis genotypes w ere collected
fr om ( Chines A cademy o f Science) Gr assland Ex-
perimental Stat ion of Inst itute Botany in Inner
M ongolia, Heilongjiang and Jilin pro vince ( Table
1) . Seeds w ere sow n in the g reenhouse o f CAS vi-
varium in Beijing. The leaf-base explants ( 0. 5～
1cm ) ( 12 days after g row th) w ere sterilized w ith
HgCl2 ( 0. 1%) fo r 12 m in and cultured on prepar ed
nutrient media ( see below ) af ter w ashed three
t imes at least w ith sterile dist illed w ater . Immature
spikes of boot ing stage w ere sterilized w ith ethanol
( 70% ) for 1 min and fo llow ed by HgCl 2 ( 0. 1%)
fo r 12～15 m in, then w ashing them w ith sterile dis-
tilled w ater 3～5 times. These immature spikes-
base explants ( 1 cm ) w er e excised asept ically and
cultured on nutrient medium . As fo r mature em-
br yos, surface-sterilized seeds were soaked
overnight in sterile dist il led w ater and from slight-
ly sw ollen seeds, mature embr yos w ere excised
asept ically and cultured on nutrient medium .
Table 1　Experimental materials and their origin
Number genotypes Resource and orig in
1 SL Shuan gliao, Jil in, China
2 GLT Gaolintun, Inn er Mongolia, China
3 YHT -w Yihuta, Inner Mongolia, C hina
4 CL -w Changl ing, J ilin, Chin a
5 NM -1 Ximeng , Inner M ongolia, Ch ina
6 HU IG Changl ing, J ilin, Chin a
7 CHC-01 Changchun, Jilin, Ch ina
8 J IS-1 Changchun, Jilin, Ch ina
9 J IS-4 Changchun, Jilin, Ch ina
10 ZY-01 Zhaoyuan, Helon gjiang, Ch ina
11 WZM QY Hailaer, Inner Mong ol ia, Chin a
1. 2　Culture medium
T hree kinds of dif ferent explants ( immature
spikes, mature embryos and the leaf-base
segment ) w ere cultured on MS [ 11] basal medium
supplemented w ith various concentrat ions of 2, 4-D
( 0, 0. 5, 1, 1. 5, 2, 2. 5mg / L ) to induce callus. After
subculture ( on M S medium w ith 1mg/ L 2, 4-D)
tw o t imes ( 4 weeks/ t ime) , the callus w ere t rans-
fer red to 2, 4-D-fr ee basal medium with different
concentrat ion components of hormone ( Kinet in
0. 5, 1, 1. 5, 2mg / L and NAA 0. 5, 1, 1. 5, 2mg / L )
to test the ability of org anogenesis. Root regenera-
t ion medium was 2, 4-D-free M S. T her e w as 3%
sucrose in all the medium gelled w ith 0. 8% agar,
pH o f the medium was adjusted to 5. 8 befo re auto-
claving at 120℃ for 20 m in. Cultur es w ere main-
tained under illum inat ion of w hite f luorescent light
( 15molõm - 2õs- 1) and w ith a photoperiod of 12h/
24h at 25℃. Values show n in Figure 1 and Table 2
w ere the means o f three independent experiments
perfo rmed w ithin 30 day s, w ith 30 repl ications per
t reatment w ith standard error of the mean varying
f rom 0. 05 to 0. 08.
2　Results
All three kinds of explants coming fr om eleven
genotypes of L . chinensis show ed morphogenic re-
sponse on M S medium containing 0. 5, 1. 0, 1. 5,
2. 0, 2. 5mg / L 2, 4-D, respectiv ely , cell pro liferat ion
199第 3期 刘公社等:羊草幼穗离体培养诱导植株再生的研究
was possible in 2 w eeks of induction. Whereas, f re-
quency of response depended on explants and the
concentrat ion of 2, 4-D in the basal medium and an
increase o r decrease in level of 2, 4-D pr oved in-
hibito ry( F ig. 1) .
Fig. 1　Frequency of cultures showing formation of
callus f rom three kinds of explants on MS
media with diff erent 2, 4-D concentration
Leaf sect ions could produce callus on the
medium containing 2, 4-D. Expansion and sw ell ing
at the cut end w as visible w ithin a w eek. At low er
concentrat ions ( 0. 5, 1. 0, 1. 5mg/ L ) of 2, 4-D, the
callus w as w hite, very loo se and mucilaginous,
how ever , at higher concentrat ions ( 2. 5mg/ L ) , the
callus showed relat ively less f riable and sem i-com-
pact after 2 w eeks. A n optimal concentrat ion o f 2,
4-D was 2mg / L under which 50%～60% cultures
developed callusing. In order to achieve different ia-
tion, all these kinds of calli w ere moved to the
medium with dif ferent components o f g row th regu-
lato r ( KT and NAA ) , show ed profuse shoot ing
over the surface and failed to dif ferentiate into ei-
ther embryos or shoot-buds after 8 w eeks gr ow th.
The explants f rom mature embryos cultured on the
2, 4-D medium resulted in cal lusing as well . A
max imum o f 35%～45% cul tures fo rmed friable
and slight-yellow callus at 2mg/ L 2, 4-D level, and
the callus orig inated from peripheral cells of scutel-
lum . Similar to the leaf-base cultur e, no different ia-
tion w as found in mature embryo cultures.
T he highest frequency ( 88%) of callus from
immature spikes-base explants occurred in the M S
medium w ith 2. 0mg / L 2, 4-D in 30 day s ( F ig. 2a) .
Af ter moved to MS medium containing 1mg / L 2, 4-
D for tw o t imes ( 4 weeks ) , the color of callus
turned to pale-yellow and nodule-l ike st ructures
w ere visible. Only the compact cal luses w ere se-
lected and cultured on MS basal medium w ith dif-
fer ent combinat ion of hormone( KT+ NAA) .
Within 3-w eeks after tr ansfer, g reenish spo ts
appeared on these calli ( Fig . 2b) . The components
and levels of ho rmone inf luenced the different iation
f requency o f eleven genotypes ( Table 2) . These
mer istematic spo ts different iated into shoot-buds
and final ly to young shoots ( Fig . 2c) . Calli devel-
oped on M S medium at 1mg/ L KT and 0. 5mg/ L
NA A fo rmed shoot-buds in 38% of culture. T he
shoot-buds formed from immatur e stachys-base re-
generated new roo ts and then developed plant lets
on 2, 4-D-free basal medium ( Fig . 2d) . P lant lets
w ere f inally t ransferred to soil through a g radual
acclimat izat ion( Fig . 2e) .
Fig. 2a　The callus f rom immature spikes-base
explants of Leymus chinensis (NM-1) occurred in the
MS medium with 2. 0mg/L 2, 4-D
3　Discussion
T he repo rted studies on t issue cultur e of L .
chinensis have been rest ricted to callus induc-
t ion [ 9, 10] . T here is no informat ion of reg ener ation
200 草　地　学　报 第 10卷
via o rganogenesis so far. Similar w ork on r egenera-
tion from immature spikes-based explants has been
described in the for ag e like L olium multif lorum, E-
lymus giganteus and Bothriochloa ischaemum
[ 12～14]
.
Fig. 2b　The green shoot-buds formatted in callus
f rom genotype Leymus chinensis(NM-1)
Fig. 2c　The young shoots developed from green shoot-
buds calli of Leymus chinensis(NM-1)
Fig. 2d　The regenerative plantlets of Leymus
chinensis (NM-1) via in vitro
Fig. 2e　The plantlets of Leymus chinensis
(NM-1) via in vitro are transf erred to soil
　　The geno type of L . chinensis is a v ery impor-
tant factor inf luencing o rgan dif ferent iat ion in t is-
sue cultur e. All elev en geno types in our experi-
ment , displayed some potent ial of org anogenesis on
M S medium w ith KT ( 1. 0mg / L ) and NAA
( 0. 5mg / L ) . The percentag e o f g reen shoo t-bud
formation ranged fr om 3% ( WZLQY ) ～ 38%
(NM-1) and signif icant dif ference w as obv ious a-
mong eleven geno types. T he opt imal concentr ation
o f 2, 4-D ( 2m l/ L ) , may lead to produce lo ose and
friable calli. How ever , some compact cull i and
mer istematic centers of ten drive fr om a low 2, 4-D
level ( 1mg/ L ) in combination with NAA ( 0. 5mg /
L ) induces nodular t issue which can further pro-
duce shoo t-bud.
Differ ent kinds of explants responsed to cul-
ture init iat ion dif ferent ly . Wang L et al . ( 1995) re-
ported that the regenerated plant could obtain from
mature embryo of E lymus giganteus in vitr o[ 13] .
Luos paper in 1990 indicated the rapid pr opagation
o f Puccinel lia chinampoensis fr om leaf-based ex-
plants
[ 15]
. Chen et al . ( 1995, 1996) had successful ly
induced the calli and developed a variant HR20-8
r esistant to hydroxyproline f rom immature spikes
o f L . chinensi s, unfo rtunately , failed to the regener-
at ion plant . A comparat ive study o f differ ent ex-
plants, pr esent experiment , r ev ealed that
o ganogenic cal li may be induced only f rom imma-
ture spikes-based explants o f L . chinensis.
201第 3期 刘公社等:羊草幼穗离体培养诱导植株再生的研究
Table 2　Frequency organogenic callus formation with shoot-buds in diff erent levels of hormone components
Genotypes
Frequency of organogen ic callus ( % )
KT
( 0. 5mg/ L)
KT ( 0. 5m g/ L) +
NAA( 0. 5mg/ L)
KT ( 1. 0mg/ L ) +
NAA( 0. 5mg /L )
KT ( 1. 0mg/ L) +
NAA( 1. 0mg/ L)
KT( 1. 0mg / L) +
NAA ( 1. 5m g/ L)
NAA
( 0. 5mg/ L )
SL 2 2 11 9 9 3
GLT 4 6 21 19 18 2
YHT -w 0 2 20 14 16 1
C L-w 0 2 6 5 8 0
NM -1 5 8 38 25 20 6
HU IG 0 0 8 8 7 3
CHC-01 0 1 10 8 7 4
J IS-1 2 2 4 6 5 3
J IS-4 0 1 12 10 9 2
WZLQY 0 1 3 3 2 0
　　 In conclusion, a cultur e sy stem in v itro for
L eymus chinensi s fr om immature stachys-based ex-
plants has been acheiv ed. T he r egeneration pro to-
col is expected to be helpful not only to the genet ic
improvement of L eymus chinensis via recombinant
DNA technolog y, but also to rapid and economical
pr opagation o f superexcellent g ermplasm .
References:
1　Jia S-X(贾慎修) . Forage f lora in people republic of s inica[ M ] .
Beijing: Chin a Agricu ltural Pr ess , 1987, 19～35( in C hines e)
2　Liu G-S (刘公社) , Wang Z-Y(王志远) . S ymposia of cont inu al
developm ent res earch in north ag ricul tu re an d animal husban dry
dis t rict [ M ] . Beij ing: China Science an d T echnolog y Press , 1999,
136～137( in Chinese)
3　Ren W-W(任文伟) , Qian J (钱吉) , Wu T (吴霆) , Zh eng S-Z
( 郑师章) . C om parat ive S tudy on Morph ological An atomic St ruc-
ture of L eymu s Chinensis in Different Geograph ic Populat ions
[ J ] . J ournal of Fudan U inv. ( Natural Science) (复旦学报) (自然
科学版) . 1999, 38( 5) : 561～564
4　Zhao Q-L(赵巧丽) , Gu an L-X(关力学) , Zhu Y(朱宇) . Obser-
vat ion on microsporogenes is of ult ramicros copic st ructure for
L eymus chinensis Trin . T zvel . [ J ] . J Shanxi Med U niv. ( 山西医
科大学学报) 1997, 28(S uppl . ) : 87～89( in Chin es e)
5　Li C-H( 李春红) . S tu dies on th e ch romosome karyotype an d G-
banded chromosomes of L eymus chinensi s and Roegner ia p end uli-
na[ J] . A cta P rataculturae S inica(草业学报) , 1990, 1( 1) : 55～62
( in Chin es e with En glish abst rract )
6　Wang M -L( 王梦龙) , A Study on Seed Produ ct ion of Aneurol e-
p id ium Chinensis[ J] . Grass lan d of China(中国草地) , 1998, ( 1) :
18～20( in Chin ese)
7　Lu P(卢萍) , Yu D-N (余诞年) , Yang X-L( 杨锡麟) , Yang S-C
(杨锁成) . T he analysis of peroxidas e is ozyme of leymus chinensi s
[ J] . J Inn er Mongolia Norm al Un iv ( Natural Science Ed ition )
(内蒙古师大学报) (自然科学汉文版) , 1997, 2: 55～58( in Chi-
nese w ith En glish abst ract )
8　LIU J (刘杰) , Liu G-S (刘公社) , Qi D-M (齐冬梅) , Li F-F(李芳
芳) . Cons t ruct ion of Genet ic Fingerp rint s of A neurolep id ium
Chinensis Usin g Microsatel lit e Sequen ces [ J] . Acta Bot S in(植物
学报) , 2000, 42( 9) : 985～987( in Chin ese with Eng lish abst ract )
9　Chen H(陈晖) , Kuang B-J (匡柏健) , Wang J-J (王敬驹) . Quality
improvem ent of A neur ol ep id ium Chinense via cell engin eering
[ J ] . Acta prataclturae S inica (草业学报) , 1995, 4( 1) : 1～ 5( in
Chinese w ith Engl ish abst ract )
10 Ch en H(陈晖) , Kuang B-J (匡柏健) , Wang J-J(王敬驹) . Studies
on am ino acid pool level s and changes of proling an abol ism in
variant lin e HR20-8 of Chinese aneurolepidium [ J] . Acta Bot . Bo-
real . -Occident . Sin(西北植物学报) , 1996, 16( 3) : 208～213
11 M urashige T and Skoog F. A revised medium for rapid grow th
and bioas says with tobacco tis sue cultur es [ J ] . Ph ysiol. Plant .
1962, 15: 473～497
12 Ch en W-P(陈文品) , Du C-X(杜丑新) , Liu T -Y(刘天育) , Wu
Q-S(吴琴生) , Liu D-J(刘大钧) . Direct regener at ion of plant let s
and micr opr op agat ion by imm ature inf lorescence cul ture of Rye-
grass [ J ] . Grass lan d of Ch ina( 中国草地) , 1994, 3: 65～ 67( in
Ch ines e w ith E nglis h abs t ract )
13 Wan g L(王丽) , Zou M -Q(邹明谦) , Li Y-N( 李沿宁) , Zhang
G-F(张根发 ) . T issue culture an d plant reg enerat ion of E lymus
g ig anteus [ J] . Pratacultural Scien ce(草业科学) , 1995, 12( 6) : 42
～44( in Chin es e wi th En glish abst ract )
14 S hi Y-J (时永杰) , Sun X-P(孙晓萍) , Liu X-Q(刘晓强) . Ti ssue
culture of young spikelet s of B othriochloa ischaemum[ J ] . Prata-
cu ltural S cience (草业科学) , 1998, 15 ( 6) : 19～ 20 ( in Chin ese
w ith Engl ish ab st ract )
15 Lu o X(罗璇) , Wang K-P (王克平) , Liu S-J ( 刘四军) . Rapid
propagat ion of Herbages-Tis sue cul tu re of P uccinel lia chinam-
p oensis[ J ] . Grass land of China( 中国草地) , 1994, 4: 46～48 ( in
Ch ines e w ith E nglis h abs t ract )
202 草　地　学　报 第 10卷