全 文 ：第 28 卷 第 6 期 作　物　学　报 V ol. 28, N o. 6
2002 年 11 月　 852～ 856 页 A CTA A GRONOM ICA S IN ICA pp. 852～ 856　N ov. , 2002
Study on M a in Factors Inf luenc ing Soma tic Embryo Inducting from Imma ture
Cotyledon of Soybean (G lycine m ax L. )
L I H ai2Yan　ZHU Yan2M ing3 　L IU Bei2Dong　ZHAN G B in2B in　ZHAN G Shu2Zhen
(L aboratory of P lant B ioeng ineering , College of L if e S cience, N ortheast A g ricultural U niversity , H eilong j iang H arbin 150030, China)
Abstract　U sing imm ature co tyledon of four m ain soybean cultivars of Dongbei district, th is paper focused on
the influence of m edium componen t of n itrogen、aux in and sucrose, pH and low temperature p retreatm en t on
som atic em bryo induction; Fo r the first tim e, low temperature p retreatm en t and pH = 7. 0 m edium w ere app lied
to som atic em bryo induction. T he results show ed that, H igh efficien t m edium of inducing som atic em bryo w as
M SB + 40 m göL 2, 42D + 6% sucrose w h ich attained the h ighest percen tage of som atic em bryo induction and
the efficiency of induction, 49% and 2. 94% respectively in th iswork. Imm ature co tyledon p retreated w ith 4℃
and induced on pH = 7. 0 m edium ach ieved 4. 46% efficiency of induction, w h ich is 1% sign ifican t difference
w ith the result from con tro l. T he in teraction betw een geno types and duration of low temperature p retreatm en t
ex its and the op tim al low temperature p retreatm en t duration of differen t geno types varied from 1 to 3 days.
Key words　Soybean (G ly cine m ax L. ) ; Som atic em bryo induction; T he componen t of n itrogen、A uxin and
sucrose; pH; L ow temperature p retreatm en t
大豆幼胚子叶体细胞胚诱导主要影响因素的研究Ξ
李海燕　朱延明3 　刘北东　张彬彬　张淑珍
(东北农业大学生命科学学院植物生物工程研究室, 黑龙江哈尔滨 150030)
摘　要　本研究选用东北地区 4 个主栽大豆品种的幼胚子叶, 探讨了培养基中氮源、生长素和蔗糖, pH 值和低温预处
理对体细胞胚诱导的影响; 并首次将低温预处理和 pH = 7. 0 的培养基应用到体细胞胚诱导中。结果表明, M SB 培养基
附加 40 m göL 2, 42D 和 6% 蔗糖, 其体细胞胚诱导率和诱导效率最高, 分别为 49% 和 2. 94%。于 4℃低温预处理的材
料, 在 pH = 7. 0 的培养基上诱导效率为 4. 46% , 与对照的结果达到 1% 的极显著差异。基因型与低温预处理的时间存
在相互作用, 不同基因型低温预处理的最适时间为 1～ 3 天。
关键词　大豆 (G ly cine m ax L. ) ; 体细胞胚诱导; 氮源、生长素和蔗糖; pH; 低温预处理
中图分类号: S565　　　文献标识码: A
　　 In recen t years, genetic engineering has becom e
one of the new and effective m ethods fo r p lan t genet2
ic imp rovem en t, among w h ich the h igh ly effective
regeneration system is the foundation fo r genetic
transfo rm ation. D ue to the fact that soybean has a u2
n ique and differen t model of regeneration, that is, it
has little distinction betw een de2differen tiation and
re2differen tiation, the work to determ ine the influ2 ence facto rs on som atic em bryo induction and regen2eration becom es very comp licated. T herefo re, ah igh ly effective and stable system fo r regeneration ofsom atic em bryo has no t yet established. Induction ofsom atic em bryo is the key to the so lution of regenera2tion, w h ile soybean geno type, imm ature em bryosize, inoculation m ethod, m edium componen t andculture conditions are key facto rs that determ ine theΞ Foundation item: Supported by the national item s of research and industrial developm ent of transgenic p lants(国家转基因植物研究与产业化
开发专项) (J992B2013)
Author in troduction: L I Hai2yan ( 19722 ) , fem ale, Harbin, doctor student, specialization: p lant bio logy engineering. Tel: + 8624512
5390734, E2m ail: lhy72@hotm ail. com
　3 A uthor of correspondence: ZHU Yan2m ing (19552 ) , m ale, Harbin, doctor, p rofessor, specialization: p lant bio logy engineering. TelöFax: +
86245125390161, E2m ail: ym zhu2001@hotm ail. com

percen tage of induction [ 1～ 4 ]. A cadem ic study show s
that app rop riate low temperature p retreatm en t can
obviously raise the yield of the callus and percen tage
of em bryo induction of an ther fo r H airy D atura
(D atu ra inox ia M ill. ) , barley (H ord eum vu lg are
L. ) and rice (O ry za sativa L. ) [ 5 ]. U p till now , w e
haven′t found any research repo rt about soybean on
th is m atter. O n the basis of generally recogn ized re2
search results, w e focused our research on the fo llow 2
ing m atter: influence of the concen tration of n itro2
gen, aux in and sucrose in the m edium , the pH and
the low temperature p retreatm en t on the induction of
som atic em bryo. T h is research laid a foundation fo r
further study of the grow th of soybean som atic em 2
bryo and the establishm en t of regeneration system.
1　M a ter ia ls and m ethods
1. 1　M a ter ia ls
T he m aterials selected fo r experim en t are h igh ly
qualified soybeans of four m ajo r varieties available in
no rtheast district of Ch ina, e. g. , H efeng 35,
H einong 35, Dongnong 42 and J ilin 30, w h ich
w ere offered by Soybean L aborato ry, N ortheast A 2
griculturalU n iversity. T he experim en t w as conducted
in labo rato ry of P lan t B io logy Engineeri2
ng, N ortheast A gricultural U n iversity in 2000～
2001.
1. 2　M ethods
1. 2. 1　M ethod of induction and culture of som atic
em bryo from imm ature co tyledon
Imm ature pods w ere co llected 2～ 3 w eek s after
blo ssom , and p ick the in itial pods on each stalk every
3～ 4 days, fo r th ree tim es at most. R inse the pods
fo r 10～ 30 m inutes, then surface sterilized in 75%
ethano l fo r one m inute fo llow ed by imm ersion in
0. 1% m ercuric ch lo ride con tain ing 0. 1% Tw een
- 20 fo r 10～ 15 m inutes and then rin se w ith sterile
distilled w ater fo r 3～ 4 tim es. Select 4～ 5 mm long
(green ish and translucen t) seeds, use fo rcep s to ex2
trude the co tyledon, cut off the hypoco tyls, p lace
two co tyledons abax ial side dow n on the m edium. A ll
cultures w ere m ain tained at 26 ± 1℃ in comp lete
darkness. T he first subculture should select ro sette2
like cell clump s (P late A ) w ith h igh ly structural and
th ick cytop lasm , adjunct w ith co tyledon exp lan ts.
1. 2. 2　D esign of m ajo r facto rs influencing the in2
duction of som atic em bryo
W e conducted research m ain ly in the m edium
componen t and pH value, as w ell as the influence of
low temperature p retreatm en t on som atic em bryo in2
duction.
Induction m edium : T he basal m edium is M SB
(M S inorgan ic salts+ B 5 organ ic m atters) , add up or2
gan ic n itrogen of Glutam ine and A sparagine, aux in
and sucrose, 0. 8% agar. T he n itrogen, aux in and
sucrose invo lve w ith th ree differen t concen trations re2
spectively. A lso, w e designed L 934 O rthogonal T able
(T able 1).
pH: 5. 8, 7. 0.
L ow temperature treatm en t: p retreat imm ature
co tyledon in a 4℃ refrigerato r fo r 1, 2, 3, 4 respec2
tive days.
F irst of all, under the condition of pH = 5. 8 and
w ithout low temperature treatm en t, w e find out the
ideal componen t of m edium w ith n itrogen, aux in and
sucrose. O n th is basis, the nex t step is to find the
difference of som atic em bryo induction affected by
pH and low temperature.
1. 2. 3　 Index of investigation
Four w eek s later, in spect the Percen tage of So2
m atic Em bryo Induction (num ber of exp lan ts w ith
som atic em bryosöto tal num ber of exp lan ts×100% ) ,
Percen tage of N orm al Som atic Em bryo (num ber of
no rm al som atic em bryosönum ber of exp lan tsw ith so2
m atic em bryos×100% ) , Induction Efficiency (per2
cen tage of som atic em bryo induction×percen tage of
no rm al som atic em bryo). N orm al som atic em bryo is
defined as the criterion of L azzeri[ 6 ]: A norm al so2
m atic em bryo should has obvious shoo t and roo t
grow th po in ts and w ill develop in to at least one nor2
m al co tyledon (P late B ).
T he experim en t result should be m ade a sign ifi2
cance analysis w ith SPSS D ata P rocesso r softw are.
Op tim ize the induction m edium for the nex t inocula2
tion.
3586 期　　　L I H 2Yan et al. : Study on M ain Facto rs Influencing Som atic⋯⋯　　　　　　　

Explana tion of Pla tes
A 　Rosette2like cell clump s (bar= 0. 5 mm ) 　B　Cotyledon som atic em bryo developed from norm al global som atic em bryo　C　Horn shape
abnorm al em bryo　D　Norm al global em bryos (bar= 0. 5 mm ) 　E　Response of som atic em bryos initiation under low temperature p retreatm ent,
pH = 7 m edium　F　Response of som atic em bryos initiation under pH = 5. 8 m edium , no p retreatm ent
Table 1　　D ifferent levels of factors inf luenc ing
somatic embryo induction
M edium N itrogen A uxin (m göL ) Sucrose (% )
1 a N 103 1. 5
2 a D 20 3
3 a D 40 6
4 b N 10 3
5 b D 20 6
6 b D 40 1. 5
7 c N 10 6
8 c D 20 1. 5
9 c D 40 3
　Note: a m eansM S nitrogen; 　b m eans 1ö2M S nitrogen+ 5 mmolö
L Glutam ine+ 5 mmolöL A sparagine; 　c m eans 1ö2M S ni2
trogen+ 10 mmolöL Glutam ine+ 10 mmolöL A sparagine; 3
m eans the k ind and concentration of auxin, N indicates
NAA , D indicates 2, 42D.
2　Results and discussion
2. 1　 Inf luence of n itrogen , aux in and sucrose on
soma tic em bryo induction
T he influence of n itrogen, aux in and sucrose on
som atic em bryo induction w as sim ilar to each o ther a2
mong differen t geno types, so w e go t the datas of
m ean values of all varieties in table 2. F rom the o r2
thogonal table 2, it show ed that N o. 3 m edium w as
most suitable fo r the induction of som atic em bryo,
nam ely, the m edium of M SB + 40 m göL 2, 42D +
6% sucrose attained the h ighest percen tage of som at2
ic em bryo induction and the efficiency of induction,
49% and 2. 94% respectively in th is work. It show s
that the o rgan ic n itrogen is no t a m atter abso lutely
needed in the induction of som atic em bryo. How ev2
er, the composition of aux in and sucrose are facto rs
govern ing the induction result. In th is research, the
induction of som atic em bryo needed a h igher concen2
tration of 2, 42D and sucrose, w h ich reached the
sam e result as obtained by F iner in the induction of
som atic em bryo from soybean imm ature co tyledon [ 7 ].
T he result differed from L azzeri and o ther inductions
m ade of 3% sucrose. A lso, som atic em bryo induc2
tion efficiency declined along w ith the low ering of 2,
42D and sucrose. Fo r in stance, 1, 6 and 8 m edium s
w ith 1. 5% sucrose, couldn′t bring about the som atic
em bryos. So, during the p rocess of som atic em bryo
induction, there is a co rrelation betw een the con ten ts
of sucrose and aux in [ 8 ] , that is, each of them shall
have an app rop riate percen tage in the composition of
458　　　　　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　 　　　　　　　　　　　　28 卷

m edium , once the equilibrium is out of con tro l, such
as one is ex trem ely h igher than the o ther, the effi2
ciency of em bryo induction w ill also sharp ly declined.
　　T ak ing 2, 42D and NAA for examp le, the per2
cen tage of som atic em bryo induction, induction speed
and percen tage of no rm al em bryo varied from differ2
en t aux ins. T he induction percen tage increased after
invo lvem en t of 2, 42D , how ever, the induction speed
w as slow and percen tage of no rm al em bryo w as
rather low , most of w h ich grew in to abnorm al em 2
bryos like ho rn o r sim ilar to hypoco tyls shape (P late
C). T he norm al em bryos largely stayed on the global
stage, loose and smoo th ly (P late D ). M edium w ith
NAA could hardly induce som atic em bryos,
how ever , the em bryos grew at a fast speed and a
Table 2　　Inf luence of n itrogen, aux in and sucrose on
somatic embryo induction from immature cotyledons
in soybean (L 9 34)
M edium
No. of
co tyledons
　
Percen tage of
som atic em bryo
induction (% )
Percen tage of
no rm al som atic
em bryo (% )
Induction
efficiency
(% ) 3
1 400 0 0 0
2 380 21 5 1. 05 D
3 400 49 6 2. 94 A
4 400 4 10 0. 40 F
5 400 36 4 1. 44 C
6 390 0 0 0
7 350 6 9 0. 54 E
8 300 0 0 0
9 370 38 5 1. 90 B
　　No te: 3 m eans 1% difference sign ificance level. 　　D atas in th is table are
m ean values of all varieties.
h igh percen tage of no rm al em bryos w ere resulted
from. P ractically speak ing, 2, 42D m edium w as re2
garded as the comparatively ideal induction system
because of the h igh efficiency of em bryo induction
that w as considered as a comp rehensive index, re2
gardless of h igh percen tage of abnorm al em bryo.
2. 2　 Inf luence of pH and low tem pera ture pretr-
ea tm en t on soma tic em bryo induction
Experim en ts w ith the op tim ized m edium have
p roved that pH and low temperature treatm en t have
an obvious influence on em bryo induction (F ig. 1) ,
also w ith the sam e influence trend among differen t
geno types. 4. 46% induction efficiency ( the m ean
value of differen t geno types) w as the h ighest one ob2
tained under the conditions of 4℃ p retreatm en t and
w ith pH = 7. 0 m edium , w h ich w as 51. 7% h igher
than the result (2. 94% ) w ith pH = 5. 8 and w ithout
low temperature treatm en t. Bo th experim en ts result2
ed in 1% sign ifican t difference. M oreover, the fo r2
m er had more em bryos on each exp lan ts (P late E,
F ). U p till now , w e haven′t read any repo rt about
the comp rehensive influence of two facto rs of pH and
low temperature treatm en t on som atic em bryo induc2
tion, excep t M r. San tarem exp lained the relations
betw een pH and som atic em bryos induction, w h ich
believe that pH m ay con tro l the nutritional abso rp tion
of cultured exp lan ts, and a p roper h igh pH can p ro2
mote the startup of em bryo and it′s later develop2
m ent[ 9 ]. T he m edium w ith pH = 7. 0 w as also used in
the induction of som atic em bryos by T rick and
F ig. 1　　 Influence of pH and low temperature
on som atic em bryos induction
F ig. 2　　 Influence of genotypes and duration
of low temperature p retreatm ent on
som atic em bryos induction
5586 期　　　L I H 2Yan et al. : Study on M ain Facto rs Influencing Som atic⋯⋯　　　　　　　

Bailey [ 10, 11 ] , how ever, they didn′t unfo ld the exp la2
nations from the angle of molecular m echan ism.
L ow temperature p retreatm en t p lays an acceler2
ating ro le in the som atic em bryo induction and callus
fo rm ation. T h is ro le has been p roved in the an ther
culture of various p lan ts such as tobacco, rice, barley
and w heat, etc. T here are still d ifferen t view s on the
m echan ism of low temperature treatm en t. Pechan
believes that, the low temperature treatm en t of an2
ther is, to som e ex ten t, a k ind of stress treatm en t,
that is, th rough stim ulation of adverse environm en t,
accelerate the de2differen tiation start of separated
spo re in side an thers. T he w ays of exp ression vary
from differen t geno types in the face of stress treat2
m ent; how ever, all of them activize the p rocess of
em bryo induction [ 12 ]. It has been show n that, low
temperature treatm en t obviously imp roves the
m etabo lism of active oxygen and hormone con ten t on
the tim e of seed germ ination in soybean, thereby,
enhance the ability of mRNA transcrip tion and p ro2
tein syn thesis; A lso, low temperature treatm en t in2
creases the temp late activity of seed DNA , RNA
transcrip tion activity and the activities of RNA poly2
m erase and som e hydro lytic enzym es[ 13 ]. T hat imm a2
ture em bryos of soybean are inoculated th rough p re2
treatm en t w ith 4℃ after separated from m aternal
p lan t is p rop itious to cell d ifferen tiation, so as to p ro2
mote the induction efficiency sign ifican tly. T h is re2
sult has been p roved by our experim en t. W e are fur2
ther studying the in terrelation betw een som atic em 2
bryo induction and low temperature treatm en t, gene
exp ression of m etabo lism enzym es and hormone con2
tro l.
2. 3　 Inf luence of soybean genotype and tim e of low
tem pera ture pretrea tm en t on em bryo induction
F igure 2 show s the induction effciency of em 2
bryos of four differen t soybean geno types at 4℃
treatm en t fo r 0, 1, 2, 3 and 4 days respectively.
T he induction efficiency varied from differen t geno2
types and w as in the fo llow ing descending sequence:
H efeng 35, H einong35, J ilin 30 and Dongnong 42.
A lso, the op tim al tim e taken fo r low temperature
treatm en t varied from geno type to geno type w ith in
the lim it of 123 days, fo r in stance, it took two days
fo r H efeng 35 and H einong 35, one day fo r J ilin 30
and th ree days fo r Dongnong 42. D ifference of tim e
taken fo r treatm en t and its influence on the induction
efficiency of geno type w as determ ined by in ternal and
m ultip le facto rs, no t by the genetic facto r alone[ 14 ].
Generally speak ing, the in teraction betw een geno2
types and tim e of low temperature p retreatm en t ex its
and the op tim al low temperature p retreatm en t length
is differen t from differen t geno types.
References
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[ 2 ]　Ghazi T D , Cheem a H V , N aborsM W. Som atic em bryogene2
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658　　　　　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　 　　　　　　　　　　　　28 卷