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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (12): 2987-2993.doi: 10.3724/SP.J.1006.2022.13068

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Establishment of genotype-independent high-efficiency transformation system in maize

XU Jie-Ting1,2,3(), LIU Xiang-Guo4(), JIN Min-Liang1,2, PAN Hong2, HAN Bao-Zhu2, LI Meng-Jiao2, YAN Shuo5, HU Guo-Qing5, YAN Jian-Bing1()   

  1. 1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2WIMI Biotechnology Co., Ltd., Changzhou 213000, Jiangsu, China
    3WIMI Biotechnology Co., Ltd., Sanya 572000, Hainan, China
    4Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, Jilin, China
    5Institute of Agricultural Sciences of Xishuangbanna Prefecture of Yunnan Province, Jinghong 666100, Yunnan, China
  • Received:2021-11-26 Accepted:2022-03-25 Online:2022-12-12 Published:2022-04-20
  • Contact: YAN Jian-Bing E-mail:xjt@wimibio.com;lxgyyj@cjaas.com;yjianbing@mail.hzau.edu.cn
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Major Project for Developing New GM Crops(2018ZX08010-04B);National Major Project for Developing New GM Crops(2019ZX08010003-002-013);Science and Technology Innovation Program of Jilin Province(20190201290JC)

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Abstract:

The reliance on receptor genotype of genetic transformation made it difficult for the transformation of commercial maize lines. Expression regulation of two important genes in plant stem cell development, Baby boom (Bbm) and Wuschel2 (Wus2), was revealed to significantly improve transformation efficiency. Several Chinese core maize inbred lines were used as receptor materials to test the transformation efficiency. Although the overexpression of Bbm and Wus2 could significantly improve the transformation efficiency, it had a negative impact on the growth and development of T0 plants. Here, a new assisted transformation technology was developed, in which a lethal gene element was added to the assist vector and the spatial expression of the two genes were regulated. The results revealed that the hybrid transformation of the assist vector and the target vector could not only successfully obtain high-quality transformation seedlings without Bbm and Wus2 assist vector in T0 generation, but also significantly improve the transformation efficiency with an average 19.5%. The application of this improved genotype-independent genetic transformation system promises maize precise improvement with higher efficiency.

Key words: maize, genetic transformation, Baby boom, Wuschel2, assist vector

Fig. 1

Schematic diagram for assist vectors and gene-targeting vectors"

Fig. 2

Generation (A-C) and differentiation (D) of callus in Zheng 58 and abnormal phenotype of T0 plant in IL3 (E) The red arrow refers to an abnormal maize tassel."

Table 1

Transformation efficiency of pWMDR001 and p193412 in maize elite inbred and receptor lines"

试验编号
Test ID		 玉米自交系
Maize inbred line		 载体
Vector name		 起始胚数量
No. of embryos		 转化阳性苗数量
No. of positive transgenic seedlings		 转化效率
Transformation frequency (%)
1 郑58 Zheng 58 pWMDR001 100 24 24.0
2 X923-1 pWMDR001 50 3 6.0
3 IL3 pWMDR001 50 5 10.0
4 IL4 pWMDR001 50 9 18.0
5 京724 Jing 724 pWMDR001 50 6 12.0
6 KN5585 pWMDR001 50 9 18.0
7 B104 pWMDR001 100 18 18.0
8 郑58 Zheng 58 p193412 200 0 0
9 X923-1 p193412 100 0 0
10 IL3 p193412 100 0 0
11 IL4 p193412 100 0 0
12 京724 Jing 724 p193412 100 0 0
13 KN5585 p193412 200 21 10.5
14 B104 p193412 100 5 5.0

Fig. 3

Genotype identification after mixture transformation (A, B) and sequencing results for Waxy mutants (C) M: DL2000 marker; P: the positive control; N: the negative control."

Table 2

Transformation efficiency by mixing pWMDR002 and gene editing vector in three maize lines"

试验编号
Test ID		 玉米自交系
Maize
inbred line		 起始胚数量
No. of
embryos		 转化阳性苗数量
No. of positive
transgenic seedlings		 优质转化苗数量
No. of high-quality transformed
seedlings		 优质转化苗分离率
Separation rate of high-quality transformed seedling (%)		 优质转化效率
High-quality
transformation
frequency (%)
1 郑58 Zheng 58 313 24 8 33.3 2.6
2 郑58 Zheng 58 535 107 2 1.9 0.4
3 郑58 Zheng 58 828 129 56 43.4 6.8
4 郑58 Zheng 58 446 103 14 13.6 3.1
5 KN5585 210 40 5 12.5 2.4
6 KN5585 256 65 18 27.7 7.0
7 KN5585 260 51 16 31.4 6.2
8 B104 189 59 13 22.0 6.9
9 B104 242 70 11 15.7 4.5
10 B104 150 48 10 20.8 6.7

Table 3

Transformation efficiency by mixing pWMDR003 and gene editing vector in four maize inbred lines"

试验编号
Test ID		 玉米自交系
Maize inbred
line		 起始胚数量
No. of
embryos		 转化阳性苗数量
No. of positive transgenic
seedlings		 优质转化苗数量
No. of high-quality transformed seedlings		 优质转化苗分离率
Separation rate of high-quality
transformed
seedling (%)		 优质转化效率
High-quality transformation frequency (%)		 平均优质转化效率
Average high-quality
transformation frequency (%)
1 B104 430 133 123 92.5 28.6 31.0
2 B104 100 39 39 100.0 39.0
3 B104 150 45 38 84.4 25.3
4 郑58
Zheng 58		 160 18 14 77.8 8.8 16.4
5 郑58
Zheng 58		 200 56 48 85.7 24.0
6 京724
Jing 724		 50 6 5 83.3 10.0 11.5
7 京724
Jing 724		 100 15 13 86.7 13.0
8 糯IN12
Nuo IN12		 50 7 7 100.0 14.0 13.3
9 糯IN12
Nuo IN12		 200 27 25 92.6 12.5
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