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作物学报 ›› 2024, Vol. 50 ›› Issue (11): 2674-2683.doi: 10.3724/SP.J.1006.2024.43014

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米无基因型限制遗传转化体系建立和应用

杨雅文1,2,3,4,6(), 朱东杰3, 潘弘3, 张云涛5,6, 夏梦吟5,6, 韩宝柱3,6, 金敏亮3, 李梦娇3, 董鲁朋3, 杨宁1,2,6, 周英5,6, 许洁婷3,6,*(), 严建兵1,2,4,6,*()   

  1. 1华中农业大学 / 作物遗传改良全国重点实验室, 湖北武汉 430070
    2湖北洪山实验室, 湖北武汉 430070
    3新米生物科技有限公司, 江苏常州 213000
    4崖州湾国家实验室, 海南三亚 572000
    5西双版纳傣族自治州农业科学研究所, 云南景洪 666100
    6云南省严建兵专家工作站, 云南景洪 666100
  • 收稿日期:2024-03-31 接受日期:2024-06-20 出版日期:2024-11-12 网络出版日期:2024-07-11
  • 通讯作者: *严建兵, E-mail: yjianbing@mail.hzau.edu.cn; 许洁婷, E-mail: xjt@wimibio.com
  • 作者简介:E-mail: yyw.hzau.edu.cn@webmail.hzau.edu.cn
  • 基金资助:
    国家自然科学基金项目(32321005);云南省严建兵专家工作站(202305AF150111);江苏省农业科技自主创新资金项目(CX(21)1003)

Genotype-independent transformation technique development and application in maize

YANG Ya-Wen1,2,3,4,6(), ZHU Dong-Jie3, PAN Hong3, ZHANG Yun-Tao5,6, XIA Meng-Yin5,6, HAN Bao-Zhu3,6, JIN Min-Liang3, LI Meng-Jiao3, DONG Lu-Peng3, YANG Ning1,2,6, ZHOU Ying5,6, XU Jie-Ting3,6,*(), YAN Jian-Bing1,2,4,6,*()   

  1. 1National Key Laboratory of Crop Genetic Improvement / Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2Hubei Hongshan Laboratory, Wuhan 430070, Hubei, China
    3XINMI Biotechnology Co., Ltd., Changzhou 213000, Jiangsu, China
    4Yazhouwan National Laboratory, Sanya 572024, Hainan, China
    5Institute of Agricultural Sciences of Xishuangbanna Prefecture of Yunnan Province, Jinghong 666100, Yunnan, China
    6Yan Jianbing Expert Workstation of Yunnan Province, Jinghong 666100, Yunnan, China
  • Received:2024-03-31 Accepted:2024-06-20 Published:2024-11-12 Published online:2024-07-11
  • Contact: *E-mail: yjianbing@mail.hzau.edu.cn; E-mail: xjt@wimibio.com
  • Supported by:
    National Natural Science Foundation of China(32321005);Yan Jianbing Expert Workstation of Yunnan Province(202305AF150111);Independent Innovation Fund for Agricultural Science and Technology of Jiangsu Province(CX(21)1003)

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摘要：

农杆菌介导的玉米自交系遗传转化具有基因型依赖性。形态发生基因Baby boom (Bbm)和Wuschel2 (Wus2)显著提高了转化效率, 拓宽了可转化自交系的范围。然而, 多数玉米自交系仍然难以得到转基因苗, 且潜在机制尚不清楚。本研究发现, 目标载体与Bbm和Wus2的辅助载体按10∶1比例混合能使大部分自交系产生体细胞胚。瞬时侵染效率和筛选是影响体细胞胚形成和成苗的关键因素。通过利用Bbm和Wus2混转以及优化侵染和延迟筛选的方式, 建立了一个快速、不受基因型限制的玉米遗传转化体系。利用该技术体系对131个自交系进行遗传转化, 其中104个自交系获得阳性转基因植株。

关键词: 玉米, 遗传转化, 形态发生基因, 侵染效率, 筛选

Abstract:

The genetic transformation of maize inbred lines via Agrobacterium tumefaciens is highly genotype-dependent. The morphogenetic genes Baby boom (Bbm) and Wuschel2 (Wus2) significantly enhance transformation efficiency and expand the range of amenable inbred lines. However, achieving transgenic seedlings remain challenging for many maize inbred lines, and the underlying mechanism remains unclear. In this study, we found that mixing the target vector with Bbm and Wus2 in a 10:1 ratio facilitates the generation of somatic embryos in most inbred lines. Transient transfection efficiency and the timing of selection are critical factors influencing the formation of somatic embryos and subsequent seedling development. By optimizing infection conditions and delaying selection, we established an efficient and rapid genetic transformation system that is not restricted by genotype. Using this system, we conducted genetic transformation on 131 inbred lines, resulting in successful transgenic plants in 104 of these lines.

Key words: maize, genetic transformation, morphogenic genes, infection efficiency, selection

表1

131个自交系转化详情"

玉米自交系
Maize inbred lines		 起始幼胚数
Number of embryos		 阳性苗数
Number of positive transgenic seedlings		 平均阳性率
Average positive rate
(%)		 平均转化效率
Average transformation frequency (%)
L649 301 20 74.07 66.67
FL218 1501 46 68.66 30.67
Jing2416KX 2692 8 53.33 26.67
21N39-1 3992 66 75.77 15.36
HI-ll 3631 55 94.83 15.15
cg03 3642 51 100.00 14.56
T038 2601 44 97.78 12.69
T387 1501 17 100.00 11.33
JT38 173613 37 41.11 11.28
T392 1752 25 79.33 10.77
HUANGC 2752 25 41.69 9.60
AB6 7576 61 36.23 9.43
NS16967-S53 7002 72 70.59 9.15
cg01/02 13115 117 100.00 9.03
Mintian-1 7675 54 100.00 8.93
ZONG3 3683 26 25.00 8.05
L661 251 2 50.00 8.00
C1 3402 27 100.00 7.95
C4 10095 70 95.52 7.23
T031 3501 32 88.89 7.14
T026 6502 51 92.14 6.64
Jing72464 3864 17 75.00 6.41
C6 4703 27 78.57 6.30
rgl-76 2001 16 76.19 6.00
KN5585-KO 10204 74 50.00 5.87
S4 579529 197 54.87 5.82
C7 16958 82 90.76 5.60
VT12 8023 46 42.75 5.47
NG9577 2372 14 44.19 5.39
TS017*18599 14568 125 63.04 5.31
BY815 485917 78 23.22 5.29
C9 2502 14 96.15 5.00
C3 12826 57 90.87 4.92
C5 27206 133 91.71 4.78
DF2 29,489109 1185 59.62 4.70
Y73 4664 9 69.05 4.50
T037 4501 27 79.41 4.44
605M-2 24477 80 55.74 4.35
HZW-1 466237 55 21.87 4.08
JT18 320426 73 61.00 4.03
AB2 382824 81 25.49 4.00
BY807 2001 10 50.00 4.00
JI1037 2622 8 57.14 4.00
AB3 18468 47 37.64 3.70
超早熟1
Chaozaoshu 1		 138611
 56
 76.61
 3.70
Jing724K 187714 51 61.25 3.76
L647 455824 37 77.01 3.45
C8 9606 31 97.22 3.38
605FK 12505 25 75.76 3.33
C2 4924 16 90.00 3.25
AB15 10886 13 76.47 3.18
VT11(1902HN) 971 3 30.00 3.09
PH6WC 8865 21 35.00 3.08
C10 4163 13 85.19 2.91
S91 32255 92 96.85 2.82
JT19 868750 88 39.88 2.80
C11 14787 41 90.58 2.72
Jing724 607421 115 63.56 2.65
SY048 3001 7 77.78 2.33
Y70 1232 2 66.67 2.22
Y71 6002 2 28.57 2.22
958F 5687 7 43.65 2.21
NG8589 113013 8 55.55 2.13
超早熟2
Chaozaoshu 2		 812
 1
 33.33
 2.08
HCL645 4364 2 40.00 2.06
超早熟3
Chaozaoshu 3		 2343
 15
 10.93
 2.04
P178 501 1 10.00 2.00
JING147 9002 18 88.10 2.00
AB5 4203 4 56.00 1.88
359 6555 12 47.69 1.83
A188 8402 17 100.00 1.80
B547 10516 17 20.00 1.76
SU1611 1201 1 16.67 1.70
D1798Z 1201 2 10.53 1.67
JING725 3502 6 50.00 1.59
AB1 170317 26 58.64 1.53
f1i1 368823 108 49.98 1.50
Yun001 12,66542 151 56.09 1.45
JT14 17068 18 41.33 1.32
Y31 483621 40 57.14 1.22
605M-1 8389 9 46.73 1.21
828F 13,92671 136 28.59 1.08
SWHC1826 10079 1 100.00 1.05
WM2793 991 1 16.67 1.01
335M 10171 9 100.00 0.98
吉V203 Ji V203 1181 1 100.00 0.85
JN6 5544 3 30.00 0.81
MO17 1301 1 25.00 0.77
RC 7154 5 78.57 0.75
YM-KH3 12187 6 16.54 0.75
AB13 5626 1 25.00 0.72
Zheng58 48763202 221 62.78 0.68
335F 4501 3 100.00 0.67
B73 4792 3 33.33 0.64
12糯 12 Nuo 3291 3 100.00 0.61
YM-KH4 20452 6 50.00 0.58
Y69 21586 7 58.33 0.53
MC01 1932 1 50.00 0.50
Jing2416 2351 1 100.00 0.43
Y68 15554 1 33.33 0.37
21N29-3 7272 1 33.00 0.36
Y33 3001 1 100.00 0.33
Jing92 324228 10 28.00 0.31
605F 6781 1 100.00 0.15
AB4 251711 0 0 0
AB7 6927 0 0 0
AB9 111510 0 0 0
AB10 3992 0 0 0
AB12 301 0 0 0
KW4M029 5273 0 0 0
JingX005 3762 0 0 0
KWS49 2392 0 0 0
DH101 1232 0 0 0
DH351 4182 0 0 0
JI853 1001 0 0 0
F19 1311 0 0 0
CHANG7-2 7525 0 0 0
YE478 3002 0 0 0
247 10502 0 0 0
CAU5 3001 0 0 0
S46 1561 0 0 0
S85 2001 0 0 0
80007 11886 0 0 0
Jing92k 339016 0 0 0
605MK 13545 0 0 0
JT135 5485 0 0 0
H3-DFP 5385 0 0 0
69 1921 0 0 0
NG8588 5903 0 0 0
NG7017 501 0 0 0
YM-KH2 8277 0 0 0

图1

不同类型自交系诱导体细胞胚情况 A: KN5585; B: Jing724; C: Jing92诱导的体细胞胚; D: Jing92没能诱导出体细胞胚状态。标尺: 1 mm。"

图2

不同农杆菌菌株侵染KN5585和Jing92瞬时荧光 KN5585 (A~D); Jing92 (E~H)。标尺: 1 mm。"

图3

不同温度处理幼胚瞬时荧光 A~F: KN5585, 45℃ (A, B), 42℃ (C, D), 25℃ (E, F); G~L: Jing724, 45℃ (G, H), 42℃ (I, J), 25℃ (K, L)。标尺: 1 mm。"

图4

Jing724和Jing92筛选与不筛选再生对比 A: Jing724在5 mg L-1草丁膦分化培养基中分化; B: Jing92在5 mg L-1草丁膦分化培养基中分化; C: Jing724在无筛选分化培养基中分化; D: Jing92在无筛选分化培养基中分化。"

图5

苗期筛选"

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