全 文 ：　文章编号 :100028551 (2001) 0520274208
IRRADIATION MUTATION TECHNIQUES
COMBINED WITH BIOTECHNOLOGY
FOR SOYBEAN BREEDING
WANG Lian2zheng 　WANG Lan 　ZHAO Rong2juan
PEI Yan2long 　FU Yu2qing 　YAN Qing2shang 　LI Qiang
( Crop Breeding and Cultivation Institute , Chinese Academy of Agricultural Sciences , Beijing 　100081)
ABSTRACT : By using 60 Coγ2irradiation to 30 adapted Chinese cultivars of soybean and
selection of high yield , good quality , early maturity resistance to diseases and insects ,
several lines have been evaluated as good lines over the control , Zhonghuang 4 ,an im2
portant soybean variety in Beijing area . After 10 generations of individual selection , 5
high yield mutants are obtained and 1～2 mutants from these mutants will be released in
2001. 3 mutants selected have the crude oil content of over 22. 00 %. The crude oil cont2
ent of 39722 is 23. 52 %and the crude protein content of 34824 is 47167 %. 30 lines were
evaluated for their genetic transformation ability and Heinong 35 has been identif ied as
very suitable material for genetic transformation.
Key words :soybean breeding ;60 Coγ irradiation ;biotechnology
收稿日期 :2000207201
基金项目 :国际原子能机构资助课题 ( IAEA8292ΠR4) ,国家九五攻关项目农作物辐射育种技术研究专题 (96 - B12 - 02 -
01)资助
作者简介 :王连铮 (1930～) ,男 ,辽宁人 ,中国农业科学研究院作物所研究员 ,博士生导师 ,主要从事大豆育种研究
Soybean is the fourth largest crop after rice ,wheat and corn in China. Soybean meal occupies 60 % of
the world protein consumption and its oil occupies 20 %～30 % of the total plant oil production in the
world. In China soybean production reached about 16 million tons in 1994 and 15million tons in
1998[1 - 3 ] . The main problems with soybean production in China are drought in spring ,low yield ,diseases
and insects. The pod borer ,cyst nematode and weeds also cause serious damage in some area. The objec2
tives of this project are : ①to develop high yield mutants of soybean with high oil or protein content andΠor
resistance to cyst nematode ,pod borer and soybean mosaic virus ; ②to screen soybean germaplasm includ2
ing mutants for molecular markers and transformation ability[4 - 6 ] .
472 　核 农 学 报　2001 ,15 (5) :274～281Acta Agriculturae Nucleatae Sinica
1 　Materials and methods
Mutation treatments of soybean were carried out in 1994 using 3 doses(100 ,120 and 150Gy) of 60 Co
irradiation. The M1 generation of 10 cultivars including Linzhen No. 1 ,F5 (Sidou 11 ×Jilin22) ,F5 (Sidou
11 ×Kefeng No. 6) ,F4 ( Youdon 8 ×D90) ,Juifeng ,Youchou No. 4 and Hanyin No. 1 ,CG661 ×9121 ,Za2
kang F6 ×Ludou 4 , Thailand soybean and H. P. F6 ×Z. Z852095 were grown at Aicheng experimental
farm of Cotton Research Institute , CAAS.
2 mutant populations were planted on the experimental farm near CAAS in May 1995 (M2 ) and 1996
(M3 ) to select for suitability to spring sown. All other mutant populations were planted at the Changping
experimental farm of CAAS in the suburb of Beijing in June 1995 (M2 ) ,1996 (M5 ) in order to select the
lines suitable for summer sown. M3 and M4 populations were grown in Oct . 1995～May 1996 at Hainan Is2
land Selection for semi2dwarf types. For early maturity it was started in M2 and continued in subsequent
generations. Plants with high protein and oil content were tested and selected from M3 . M6 ,M7 and M8
generations were grown in different locations. Individual selection for mutation breeding was used in this
experiment[7 - 10 ] .
2 　Results
211 　High yield
Through 11 generations of mutation breeding and selection from 1994 to 1999 , 34 soybean mutants
were selected. According to the regional yield tests in 1997 ,the yields increases from 12 % to 25 % in
comparison with Zhonghuang 4 (CK with yield of 2100kgΠhm2 ) ,which is an important variety in Beijing
area.
These soybean mutants are now under the regional test and production test at 10 provinces. Some of
them will be released this year or next year. According to the rules of Seed Management , regional test
needs 2～3 years , and production test needs 1～2 years.
Table 1 　Good soybean lines in northern China (1997 Beijing)
soybean lines yield (kgΠhm2) increase compared
with CK( %)
maturity
time
lodging
Zhongzuo973 (M5003) 2621. 5 24. 83 Oct . 10 0
Zhongzuo96 - M5028 (Nangyin) 2432. 5 21. 81 Oct . 10 1
Zhongzuo965 (M55 - 56 Youdou 8 ×D90) 2429. 2 21. 75 Oct . 12 0
Zhongzuo962 2310. 1 19. 75 Oct . 28 0
Zhongzuo M17 2234. 2 12. 00 Oct . 8 0
Zhonghuang4(CK) 2100. 0 0 Oct . 5 0
According to the yield tests ,following lines are considered as good lines of soybean.
Zhongzuo 965 : Yield of this cultivar increases by 15167 % compared with CK and has a plant height
572　5 期 辐射和生物技术相结合进行大豆育种 (英文)
of 80 cm. It was suitable to grow in northern part of Henan province after winter wheat . In 1999 ,the yield
of Zhongzuo 965 was 1112 % higher than Longchung No. 1 in Gansu province.
Zhongzuo 962 :The yield of Zhongzuo 962 was 321715kgΠhm2 in Teiling Soybean Institute ,Liaoning
province and increased by 1219 % in comparison with Teifeng 27 (CK) in 1999. This line has character of
early maturity and is suitable to grow as two crops per year near Beijing ,Tianjin and northern part of He2
bei Province. It has plant height of 65～70 cm ,so it is semi2dwarf line and needs high density.
Zhongzuo 973 (962M25003) : Its yield (262115kgΠhm2 ) increases by 24183 % compared with CK. It
is suitable to grow in northern parts of Henan and Shandong provinces with two crops per year. It is resis2
tant to some diseases.
Zhongzuo 962M25028 : It is mutant from Hanyin No11 and its yield (243215 kgΠhm2 ) increases by
15183 % over CK. It is suitable to grow in the southern part of Shanxi province and northen part of Shan2
don province. It is resistant to lodging.
Zhongzuo M17 : It is a good line with high protein content of 45111 % ,oil content of 19115 % and
large seed. The weight of 100 seeds is 26～28g and it is resistant to lodging.
212 　Quality analysis
Table 2 　The crude oil and protein of mutants
lines crude oil ( %) crude protein ( %)
Zhongzuo 965(M55
-
56) 18. 20 43. 55
M53 16. 30 43. 98
M19 18. 55 46. 07
M17 19. 15 45. 11
Zhaoshu 18(CK) 18. 78 43. 98
　 　Some soybean lines selected by mutation
breeding on their crude oil and protein content are
shown in Table 2. Oil content of CG661 ×9121 is
21116 % , H. P. F6 ×22 852059 is only 18189 % ;
protein content of Zakang F6 ×Ludou4 is 42118 %
and Hanyin No11 is only 34196 %.
The results of treatment with H. PF6 ×Z. Z852
095 are shown in table 3. From the table it is seen that crude protein content of 34824 is 47167 %(aver2
age of two years) and crude protein content of 34924 is 47109 %.
Table 3 　Variance of crude oil and protein content in M3 and M4 of H. PF6 ×Z. Z852095.
plants
M3 (1995) M4 (1996)
crude oil content ( %) crude protein content ( %) crude oil content ( %) crude protein content ( %)
34821 (CK) 18. 89 41. 19
34823 17. 58 45. 31 17. 84 43. 48
34824 18. 22 45. 81 16. 18 49. 54
34922 17. 30 45. 68 16. 18 48. 84
34923 17. 15 47. 08 16. 52 44. 23
34924 16. 30 47. 00 17. 04 47. 18
The results of treatment with CG661 ×9921 are shown in table 4. Crude oil content of CK(CG661 ×
9921) is 21116 %. The crude oil content of 39722 is 23152 % , showing that it is a plant with high oil
content .
672 核　农　学　报 15 卷
Table 4 　Variance of oil and protein in M3 of CG661 ×9921 (1995 ,Beijing)
plants crude oil content ( %) crude protein content ( %)
39121 (CK) CG6661 ×9921 21. 16 37. 88
39722 M3 23. 52 36. 15
39723 M3 21. 61 36. 95
39724 M3 21. 04 38. 72
39725 M3 22. 64 37. 82
39726 M3 22. 27 38. 01
39821 M3 21. 88 39. 03
39822 M3 20. 72 40. 87
39823 M3 21. 44 36. 29
39824 M3 21. 94 36. 39
The results of treatment with Sidou ×Jilin22 M3 and M4 are shown in table 5. The crude protein con2
tent of plant 38822 is 46102 % (average of two year) . The crude oil content of 37523 is 21100 %.
Table 5 　Variance of crude protein content in M3 and M4 of Sidou ×Jilin22
plants
M3 (1995) M4 (1996)
crude oil content ( %) crude protein content ( %) crude oil content ( %) crude protein content ( %)
CK - 38. 68
37523 21. 00 37. 56 16. 77 44. 28
38723 19. 18 44. 14 17. 95 45. 76
38729 19. 11 44. 80 18. 48 45. 02
38822 19. 06 46. 16 17. 97 45. 88
38823 18. 72 44. 40 17. 34 45. 57
38824 18. 59 44. 83 16. 20 46. 03
38924 17. 24 46. 87 18. 44 43. 90
39224 20. 19 45. 23 19. 01 43. 16
Crude oil and crude protein content of 145 plants of Sidou ×Jilin 22 M3 were analyzed. The crude oil
content of CK(Sidou ×Jilin 22) is 20106 % and the 6 plants have the crude oil content of over 2115 % ,
and plant 38622 has crude oil content of 21193 %.
From 145 plants ,it is found that 19 plants have crude protein content of over 43 % ,9 plants are over
44 % and 3 plants are over 45 %. The crude protein content of plant 38924 (M3 ) is the highest one
(46187 %) in the treatment .
The crude oil content for 220 plants of M3 and crude protein content for 220 plants were analyzed. It
is found that 3 plants have crude oil content of over 22100 %(high oil sample) and 7 plants have protein
content of over 46100 %(high protein sample) . M3 of CG661 ×9921 is very good material for increasing
oil content as oil content of CK was 21116 %. 3 plants with crude oil of over 22100 % by this treatment
were obtained.
M3 and M4 of H. P F6 ×22852095 are good materials for increasing protein content . 2 samples with
protein content of over 47 % were found in M3 and 4 samples with protein content of over 47 % were found
772　5 期 辐射和生物技术相结合进行大豆育种 (英文)
in M4 . Analyzing quality at early generation of mutation is very important for improving quality.
213 　Mutation in growth period.
After treatment by 60 Co lots of early maturing mutants from Lingzhen (No. 1) were obtained. Some
mutants had growth period of 15～20days shorter than that of control (CK) .
Plate I　Early maturing type (left) and Lingzhen No. 1 (CK,right)
214 　Resistance to nematode
Soybean cyst nematode (SCN) is very serious soybean disease in China. Using the randomly ampli2
fied polymorphic DNA(RAPD) technique ,we analyzed 8 soybean cultivars and lines including two resis2
tant lines ,1259Ywith yellow seed coat and 1259B with bicolor seed coat ,and their resistant parent Huip2
izhiheidou ,susceptible parent2Jinyi 9 ,other two resistant varieties , Yuanboheidou and PI 437654 ,other
two resistant varieties. Yuanboheidou and PI 437654 ,other two susceptible cultivars ,Ludou 1 and Ludou
7 ,to race 4 of Heterodera glycines for their amplified products. Among 33 primers ,one primer ,OPG 04
(Operon Company) ,amplified a DNA frangment which specifically existed in the products of all 5 resis2
tant varieties and lines , and not found in those of 3 susceptible cultivars ( including susceptible par2
ents) [11 ,12 ] .
Plate Ⅱ　RAPD products amplified by primer OPG04
1～8 are the RAPD products of PI 437654 ,Yuanboheidou ,Huipizhiheidou ,1259Y(yellow color seed coat) ,1259B (Bicol2
or seed coat) ,Jinyi No. 9 ,Ludou No. 7 and Ludou No. 1 , respectively ,indicating the fragments specifically amplificated in
　　　　　 resistant cultivars or lines.
872 核　农　学　报 15 卷
215 　Transformation of foreign genes to mutants
The transformation of foreign genes to soybean mutants , Zhongzuo 962 , Zhongzuo 965 , Zhongzuo
M17 ,Heinong 35 (Hybrid with mutant) ,Zhongzuo 975 , Iudou 10 and others was tested. under the guid2
ance of Dr. Thomas H. Clemente from Biotechnology Centre at University of Nelraska Lincoln.
During the past 15 to 20 years ,genetic engineering has played an important role in improving crops
such as rice ,cotton ,wheat ,corn ,tomato and soybean. However , regeneration of soybean is difficult . Our
laboratory tried to select good genotypes for regeneration , and started soyean gene transformation by using
cotyledon node mediated Agrobacterium tumif aciens .
It is found that some of soybean cultivars , such as Heinong 35 , Zhongzuo975 , Hefeng35 , Hei2
nong37 ,are good genotypes for regeneration. We selected different media for germination and B5 is proved
to be very good medium ,PA(potato juice and agar) also gets good result for germination. Different methods
of sterilization of seeds were compared and chlorine is shown very well for sterilization. After germination
we put them in shoot initiation medium. By protoplast culture and tissue culture more than 100 germa2
plasms were tested for regeneration and more than 2838 germplasms of soybean were tested to response to
the different strains of Agrobacterium tumif aciens . It is found that some cultivars were easy to noduration
by injection of several strains of Agrobacterium [13 ,14 ] .
Table 6 　Regeneration rate and susceptibility to EHA101 with PPTN140
experiment 2nd :wk(regeneration rate) 4wk(susceptibiity to EHA101with PPTN 140)
2nd :7 - 8 number of exp regular axillary no regener plants gus sect gus diff
Heinong35 92 75 4 13 61 11
962 147 116 13 18 2 0
Thorne 44 32 3 9 17 5
experiment 2nd :wk 4wk
2nd :wk 6 - 23 number of exp regular axillary no regener plants gus sect gus diff
ZhongzuoM17 18 14 7 17 5 2
ZhongzuoM965 20 12 0 8 13 2
Thorne 35 29 3 3 17 0
experiment 2nd :wk 4wk
2nd :wk 3 - 27 Number of exp regular axillary no regener plants gus sect gus diff
Heinong 37 73 55 1 17 139 9
Thorne 35 34 1 15 44 2
　　(By Wang Lan ,1999 at Lincoln Nebraska University)
3 　Discussions and conclusions
It is found that M2 and M3 have different variation in growth period ,plant height ,seed size ,resistance
to disease. Different variations in crude oil content and crude protein content are observed. The crude oil
972　5 期 辐射和生物技术相结合进行大豆育种 (英文)
content of plant 39722 (M3 ) is 23152 % and the crude protein content of plant 34824 is 47167 %(average
of two years) . Selected soybean lines have good yield ,large seed and resistance to lodging.
Stable lines at M5～M6 generations ,sometimes at M4 can be selected through mutation breeding.
Combination of mutation breeding and cross breeding can achieve good results. In cross breeding we
have a lot of progenies with late maturity ,which can be treated and easily selected for best lines with best
comprehensive characteristics and early maturity.
By using biotechnology in mutation breeding foreign genes can transfer into soybean muants and mu2
tants resistant to diseases and nematodes might be selected.
According to our works a superyielding type of soybean has following charactristics : (1) . 3～17
branches with convergence ; (2) determinate or subdeterminate types ,which have much more pods ,espe2
cially in top ; (3) plant height of 65～85 cm with strong stem and short internode without lodging ; (4)
much more dry matter with higher harvest index of over 515 ; (5) much more pods on each internode with
large seeds ; (6) many small leaves with small shade density but more seeds and higher photosynthesis ; (7)
high resistance to diseases and insects.
Identification of RAPD maker associated with resistance to race 4 of Heterodera glycines is very use2
ful .
References :
[ 1 ] 　Wang Lan ,Wang Lianzheng ,Zhao Rongjuan , Pei yanlong. Soybean mutation breeding and biotechnology ,A presentation at second
FAOΠIAEA research coordination meeting on improvement of new and tradiational industrial crops by induced mutation and related
biotechnology ,Giessen ,Germany ,June 30～July 4 ,1997
[ 2 ] 　Wang Lianzheng ,Fu Youqing ,Pei Yanlong ,Wang Lan , Zhao Rongjuan. Some problems of soybean breeding in Northern China ,in
“Proceeding of Sixth National Soybean Symposium”,Chengde Hebei Province ,China. 1997 ,1～2
[ 3 ] 　Wang Lianzheng ,Wang Lan ,Liu Zhifang ,Zhao Rongjuan. Current situation of soybean production and its way of increasing ,Soybean
Bulletin. 1999 , (2) :42～45
[ 4 ] 　Wang Lianzheng , Pei Yanlong , Fu Yuqing and Wang Lan. Soybean mutation breeding and tumor formation by Agrobacterium tumefa2
ciens . IAEA SM340Π202p , FAOΠIAEA International symposium on the use of induced mutations and molecular techniques for crop
improvement . Vienna ,June 19223 1995
[ 5 ] 　Wang Lianzheng. Combining radiation mutation techniques with biotechnology for soybean breeding , a presentation on the First FAOΠ
IAEA Research Corrdination Meeting on Improvement of New and Tradiational Industrial Crops by Induced Mutation and Related Bio2
technology. Romm CO7V1 ,IAEA ,Vienna ,Austria ,April 3～7 ,1995
[ 6 ] 　Wang Lianzheng. Soybean improvement using nuclear techneques. in Plant Mutation Breeding in Asia Beijing. China Agricultural
Press for Science and Technology. 1996 ,80～102
[7 ] 　Maluszynski M ,Van Zanten L ,Ashri A ,Brunner H. Ahlooawaili B. Zapata FJ . Weck E ,Mutation techniques in plant breeding , IAEA2
SM2340Π029. FAOΠΠAEA International Symposium on the Use of Induced Mutation and Molecular Techniques for Crop Improvement ,
1995
[8 ] 　Walter R Fehr ,Breeding methods for cultivar divelopment . In book“Soybeans :improvement ,production and Uses”. Madison ,Wiscon2
son ,USA. 1987 :249～294
[ 9 ] 　Wilcox J R. Soybean improvement ,production and uses ,second edition ,Agronomy No. 16. ASA ,CAAS ,SSSA ,Publishers Madison ,Wis2
conson ,USA. 1987
[10 ] 　Wang Lianzheng ,Wang Jinling. Soybean genetics and breeding ,Science Press ,Beijing. 1992
[11 ] 　Yan Qingshang ,Wang lan ,Li Ying ,Wang Lianzheng ,Chen Pinsan. Priliminary study on identification of RAPD Marker associated with
resistance to race 4 of Heterodela glycines . Soybean Sicence 1996. 15 (2) :126～129
082 核　农　学　报 15 卷
[12 ] 　Pei Yanlong ,Wang Lan ,Wang Lizanhzeng. Studies on genetic diversity of Glycine soja lsozyme variation in four population. Soybean
Sicence 1996. 15 (4)
[13 ] 　Wang Lan , Wang Lianzheng , Liu Zhifang , Zhao Rongjuan. Soybean transformation of foreign gene mediated Agrobacterium tumifa2
ciens . Abstract submmited to the Proceedings of World Soybean Research Conference VI ,Chicageo ,USA. 4 - 7 August 1999. 448
[ 14 ] 　Zhanyuan Zhang ,Aiqiu Xing ,Paul Staswick ,Thomas E Clemente. The use of glusfosinate as a selective agent is Agrobacterium2mediat2
ed transformation of soybean. Plant Cell ,Tissue and Organ Culture 1999. 56 :37～46
Acknowledgement :Dr. Thomas H. Clemente visited our laboratory , gave us a series of lectures and helped us a lot at soybean transformation
mediated Agrobacterium .
辐射和生物技术相结合进行大豆育种
王连铮　王　岚　赵荣娟　裴颜龙　傅玉清　颜清上　李　强
(中国农业科学院作物育种栽培研究所 ,北京　100081)
摘　要 :从 1991 年开始 ,利用60 Coγ射线对 30 个中国大豆品种进行处理 ,通过南繁北
育 10 代的个体选育 ,已育成 30 余个大豆辐射品系。其中中作 965、中作 962、中作
973、中作 M15 和中作 M17 产量性状表现较好。同时对大豆品质进行了筛选 ,含油量
超过 22 %的有 3 份。其中中作 39722 含油量达 23152 % , 34824 粗蛋白含量在
47167 % ;对 30 个大豆品系分析了再生能力和研究了对农杆菌的敏感性 ,发现黑农
35、黑农 37、Throne 和中作 97 再生能力较强。
关键词 :大豆育种 ;60 Coγ射线 ;生物技术
(上接 315 页)
参考文献 :
[1 ] 　Oscar J , et al . Inhibition of Trehalase Activity Enhances Trehalose Accumulation in Transgenic Plants. Physiol 1997 ,113 :181～190
[2 ] 　陈红曼 ,董志扬. 海藻糖的生物合成及抗逆研究进展. 农业生物技术学报. 1999 ,3 (增刊) :108～113
THE INITIAL RESEARCH REPORT OF WHEAT TRANSFOMATION OF
TREHALOSE SYNTHASE GENE
LIU Lu2xiang1 　ZHAO Lin2shu1 　ZHENG Qi2cheng1 　Dong Zhi2yang2
WANGJing1 　ZHAO Shi2rong1 　CHEN Wen2hua1
(11 Institute for Application of Atomic Energy , Chinese Academy of Agricultural Sciences , Beijing 　100094 ;
21 Institute of Microbiology , Chinese Academy of Science , Beijing 　100080)
182Acta Agriculturae Nucleatae Sinica
2001 ,15 (5) :274～281