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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (11): 2674-2683.doi: 10.3724/SP.J.1006.2024.43014

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

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 Online:2024-11-12 Published: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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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

Table 1

Details of transformation of 131 inbred lines"

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

Fig. 1

Induction of somatic embryos for different inbred lines A: KN5585; B: Jing724; C: Jing92 induced somatic embryos; D: failed to induce somatic embryo. Bar: 1 mm."

Fig. 2

Transient fluorescence in KN5585 and Jing92 infected by different Agrobacterium strains KN5585 (A-D); Jing92 (E-H). Bar: 1 mm."

Fig. 3

Transient fluorescence in immature embryos under different temperature treatments A-F: KN5585, 45℃ (A, B), 42℃ (C, D), 25℃ (E, F); G-L: Jing724, 45℃ (G, H), 42℃ (I, J), 25℃ (K, L). Bar: 1 mm."

Fig. 4

Comparison of regeneration with selection and non- selection for Jing724 and Jing92 A: regeneration of Jing724 in 5 mg L-1 glufosinate medium; B: regeneration of Jing92 in 5 mg L-1 glufosinate medium; C: regeneration of Jing724 in no selection medium; D: regeneration of Jing92 in no selection medium."

Fig. 5

Seedling selection"

[1] 仇焕广, 李新海, 余嘉玲. 中国玉米产业: 发展趋势与政策建议. 农业经济问题, 2021, (7): 4-15.
Chou H G, Li X H, Yu J L. China maize industry: development trends and policy suggestions. Issues Agric Econ, 2021, (7): 4-15 (in Chinese with English abstract).
[2] Ishida Y, Hiei Y, Komari T. Agrobacterium-mediated transformation of maize. Nat Protoc, 2007, 2: 1614-1621.
doi: 10.1038/nprot.2007.241 pmid: 17585302
[3] Zhang Q, Zhang Y, Lu M H, Chai Y P, Jiang Y Y, Zhou Y, Wang X C, Chen Q J. A novel ternary vector system united with morphogenic genes enhances CRISPR/Cas delivery in maize. Plant Physiol, 2019, 181: 1441-1448.
doi: 10.1104/pp.19.00767 pmid: 31558579
[4] Sylvie De Buck C D W, Montagu M V, Depicker A. Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants. Mol Plant Microbe Interact, 2000, 13: 658-665.
[5] Lowe K, La Rota M, Hoerster G, Hastings C, Wang N, Chamberlin M, Wu E, Jones T, Gordon-Kamm W. Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis. In Vitro Cell Dev Biol Plant, 2018, 54: 240-252.
[6] Boutilier K, Offringa R, Sharma V K, Kieft H, Ouellet T, Zhang L, Hattori J, Liu C M, van Lammeren A A M, Miki B L A, Custers J B M, van Lookeren Campagne M M. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell, 2002, 14: 1737-1749.
doi: 10.1105/tpc.001941 pmid: 12172019
[7] Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho M J, Scelonge C, Lenderts B, Chamberlin M, Cushatt J, Wang L, Ryan L, Khan T, Chow-Yiu J, Hua W, Yu M, Banh J, Bao Z, Brink K, Igo E, Rudrappa B, Shamseer P M, Bruce W, Newman L, Shen B, Zheng P, Bidney D, Falco C, Register J, Zhao Z Y, Xu D, Jones T, Gordon-Kamm W. Morphogenic regulators Baby Boom and wuschel improve monocot transformation. Plant Cell, 2016, 28: 1998-2015.
[8] Keith Lowe G H, Sun X F, Sonriza R G, Paul L, Sam E, Shane A, Kimberly G, Bill G K. Maize LEC1 improves transfromation in both maize and wheat. Plant Biotechnol, 2002: 283-284.
[9] McFarland F L, Collier R, Walter N, Martinell B, Kaeppler S M, Kaeppler H F. A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.). Plant Biotechnol J, 2023, 21: 1860-1872.
doi: 10.1111/pbi.14098 pmid: 37357571
[10] Liu X, Bie X M, Lin X, Li M, Wang H, Zhang X, Yang Y, Zhang C, Zhang X S, Xiao J. Uncovering the transcriptional regulatory network involved in boosting wheat regeneration and transformation. Nat Plants, 2023, 9: 908-925.
doi: 10.1038/s41477-023-01406-z pmid: 37142750
[11] Zhai N, Xu L. Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration. Nat Plants, 2021, 7: 1453-1460.
doi: 10.1038/s41477-021-01015-8 pmid: 34782770
[12] Khanday I, Santos-Medellin C, Sundaresan V. Somatic embryo initiation by rice BABY BOOM1 involves activation of zygote-expressed auxin biosynthesis genes. New Phytol, 2023, 238: 673-687.
[13] Ogura N S Y, Ito T, Tameshige T, Kawai S, Sano M, Doll Y, Iwase A, Kawamura A, Suzuki T, Nikaido I, Sugimoto K, Ikeuchi M. WUSCHEL-RELATED HOMEOBOX 13 suppresses de novo shoot regeneration via cell fate control of pluripotent callus. Sci Adv, 2023, 9: 1-13.
[14] Mendez-Hernandez H A, Ledezma-Rodriguez M, Avilez- Montalvo R N, Juarez-Gomez Y L, Skeete A, Avilez-Montalvo J, De-la-Pena C, Loyola-Vargas V M. Signaling overview of plant somatic embryogenesis. Front Plant Sci, 2019, 10: 77.
[15] 许洁婷, 刘相国, 金敏亮, 潘弘, 韩宝柱, 李梦娇, 岩说, 胡国庆, 严建兵. 不依赖基因型的高效玉米遗传转化体系的建立. 作物学报, 2022, 48: 2987-2993.
doi: 10.3724/SP.J.1006.2022.13068
Xu J T, Liu X G, Jin M L, Pan H, Han B Z, Li M J, Yan S, Hu G Q, Yan J B. Establishment of genotype-independent high efficiency transformation system in maize. Acta Agron Sin, 2022, 48: 2987-2993 (in Chinese with English abstract).
[16] Sidorov V, Duncan D. Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods Mol Biol, 2009. pp 47-58.
[17] Liu S, Shi Y, Liu F, Guo Y, Lu M. LaCl3 treatment improves Agrobacterium-mediated immature embryo genetic transformation frequency of maize. Plant Cell Rep, 2022, 41: 1439-1448.
[18] Jha P, Kumar V. BABY BOOM (BBM): a candidate transcription factor gene in plant biotechnology. Biotechnol Lett, 2018, 40: 1467-1475.
[19] Hoerster G, Wang N, Ryan L, Wu E, Anand A, McBride K, Lowe K, Jones T, Gordon-Kamm B. Use of non-integrating Zm-Wus2 vectors to enhance maize transformation. In Vitro Cell Dev Biol Plant, 2020, 56: 265-279.
[20] Yadav R K, Perales M, Gruel J, Girke T, Jonsson H, Reddy G V. WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex. Genes Dev, 2011, 25: 2025-2030.
[21] Mookkan M, Nelson-Vasilchik K, Hague J, Zhang Z J, Kausch A P. Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2. Plant Cell Rep, 2017, 36: 1477-1491.
[22] Aregawi K, Shen J, Pierroz G, Sharma M K, Dahlberg J, Owiti J, Lemaux P G. Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing. Plant Biotechnol J, 2022, 20: 748-760.
[23] Masters A, Kang M, McCaw M, Zobrist J D, Gordon-Kamm W, Jones T, Wang K. Agrobacterium-mediated immature embryo transformation of recalcitrant maize inbred lines using morphogenic genes. J Vis Exp, 2020, 156: e60782.
[24] Sun C, Lei Y, Li B, Gao Q, Li Y, Cao W, Yang C, Li H, Wang Z, Li Y, Wang Y, Liu J, Zhao K T, Gao C. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat Biotechnol, 2023, 42: 316-327.
doi: 10.1038/s41587-023-01769-w pmid: 37095350
[25] Wang F X, Shang G D, Wu L Y, Xu Z G, Zhao X Y, Wang J W. Chromatin accessibility dynamics and a hierarchical transcriptional regulatory network structure for plant somatic embryogenesis. Dev Cell, 2020, 54: 742-757.
[26] Chen Z, Debernardi J M, Dubcovsky J, Gallavotti A. The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency. bioRxiv, 2022, doi: 101101/2022.09.02.506370.
[27] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126: 663-676.
doi: 10.1016/j.cell.2006.07.024 pmid: 16904174
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