Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes

doi:10.16420/j.issn.0513-353x.2024-0690

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (7): 1758-1768.doi: 10.16420/j.issn.0513-353x.2024-0690

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes

SONG Qianna¹^,², SONG Lushuai¹^,³, DUAN Yonghong¹, BAI Xiaodong³^,^*(), and FENG Ruiyun¹^,²^,^*()

¹ College of Agriculture，Shanxi Agricultural University，Taigu，Shanxi 030801，China
² Institute of Crop Sciences，Shanxi Academy of Agricultural Sciences，Taiyuan 030031，China
³ Shanxi Key Laboratory of Potato Genetic Improvement and Germplasm Innovation，Institute of High Altitude Crop of Shanxi Agricultural University，Datong，Shanxi 037008，China

Received:2024-10-14 Revised:2025-04-16 Online:2025-07-23 Published:2025-07-23
Contact: BAI Xiaodong, and FENG Ruiyun

Abstract

Abstract:

The StFBH3，StbHLH52 and StPDS genes were targeted by five CRISPR/Cas9 vectors for genome editing with GFP fluorescence screening marker in tetraploid potato cultivar Desiree，Qingshu 9 and Jinshu 16. The stem segments were infected by Agrobacterium rhizogenes-mediated genetic transformation to induce hairy roots，then the transformation rate and gene editing efficiency were evaluated. Further，whole potato plants were regenerated from hairy roots based on the optimized regeneration system and the mutations were identified. The sequencing showed that five editing vectors were all activity in the three potato varieties. Among them，the knockout efficiency for single target of StPDS were 87.8% and 80.0% in hairy roots of Desiree and Jinshu 16，respectively，while that of StFBH3 was 72.7% in Qingshu 9 hairy roots. The fragment deletion efficiency for dual-target of StbHLH52 was 19.4% in Desiree hairy roots. Moreover，the efficiency of plants regenerated from hairy roots in Desiree，Qingshu 9 and Jinshu 16 were 60%，75% and 50%，respectively. The editing efficiency of regenerated plants was 100%，and the StPDS-knockout line displayed the albino phenotype. The establishment of technology system in this study can quickly verify the target site activity and efficiently obtained edited potato plants，which provides a technical support for gene function research and precise genetic breeding in potatoes.

Key words: potato, GFP, gene editing, hairy root, transformation, plant regeneration

SONG Qianna, SONG Lushuai, DUAN Yonghong, BAI Xiaodong, and FENG Ruiyun. Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes[J]. Acta Horticulturae Sinica, 2025, 52(7): 1758-1768.

Figures/Tables 7

References 27

[1]	Andersson M, Turesson H, Nicolia A, Fält A S, Samuelsson M, Hofvander P. 2017. Efficient targeted multiallelic mutagenesis in tetraploid potato(Solanum tuberosum)by transient CRISPR-Cas9 expression in protoplasts Plant Cell Report, 36 (1):117-128.
[2]	Butler N M, Douches D S. 2016. Sequence-specific nucleases for genetic improvement of potato. American Journal of Potato Research, 93 (4):303-320.
[3]	Butler N M, Jansky S H, Jiang J M. 2020. First-generation genome editing in potato using hairy root transformation. Plant Biotechnology Journal, 18 (11):2201-2209.
[4]	Čermák T, Curtin S J, Gil-Humanes J, Čegan R, Kono T J Y, Konečná E, Belanto J J, Starker C G, Mathre J W, Greenstein R L, Voytas D F. 2017. A multipurpose toolkit to enable advanced genome engineering in plants. The Plant Cell, 29 (6):1196-1217. doi: 10.1105/tpc.16.00922 pmid: 28522548
[5]	Chincinska I A, Miklaszewska M, Soltys-Kalina D. 2023. Recent advances and challenges in potato improvement using CRISPR/Cas genome editing. Planta, 257 (1):25.
[6]	Cui D, Huo S S, Wang X, Zheng Z Q, Zhang Y H, Zhang J L, Zhong F. 2020. Establishment of canine macrophages stably expressing GFP-tagged canine LC 3 protein for effectively detecting autophagy. Molecular and Cellular Probes, 49 (1):101493.
[7]	Dangol S D, Barakate A, Stephens J, Caliskan M E, Bakhsh A. 2019. Genome editing of potato using CRISPR technologies:current development and future prospective. Plant Cell,Tissue and Organ Culture, 139 (2):403-416.
[8]	Du Jingya, Chen Kaiyuan, Pu Jin, Zhou Huiying, Zhu Guangtao, Zhang Chunzhi, Du Hui. 2023. The modification of gene editing vector for efficient GFPuv fluorescence screening and it’s application in potato genetic transformation. Scientia Agriculture Sinica, 56 (11):2223-2236. (in Chinese)
	杜静雅, 陈凯园, 普金, 周会英, 祝光涛, 张春芝, 杜慧. 2023. 高效GFPuv荧光筛选基因编辑载体的改造及其在马铃薯遗传转化中的应用. 中国农业科学, 56 (11):2223-2236.
[9]	Halterman D, Guenthner J, Collinge S, Butler N, Douches D. 2016. Biotech potatoes in the 21st century:20 years since the first biotech potato. American Journal of Potato Research, 93 (1):1-20.
[10]	Honma Y, Yamakawa T. 2019. High expression of GUS activities in sweet potato storage roots by sucrose-inducible minimal promoter. Plant Cell Report, 38 (11):1417-1426.
[11]	Hraška M, Rakouský S, Čurn V. 2006. Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants. Plant Cell,Tissue and Organ Culture, 86 (3):303-318.
[12]	Josefa M A, Cristina M L, Félix J M R, Fernando T, Mustafa B, Saleh A. 2023. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein and hairy roots:a perfect match for gene functional analysis and crop improvement. Current Opinion in Biotechnology, 79 (1):102876.
[13]	Kiryushkin A S, Ilina E L, Guseva E D, Pawlowski K, Demchenko K N. 2021. Hairy CRISPR:genome editing in plants using hairy root transformation. Plants(Basel), 11 (1):51.
[14]	Lin C S, Hsu C T, Yang L H, Lee L Y, Fu J Y, Cheng Q W, Wu F H, Hsiao H C W, Zhang Y S, Zhang R, Chang W J, Yu C T, Wang W, Liao L J, Gelvin S B, Shih M C. 2018. Application of protoplast technology to CRISPR/Cas 9 mutagenesis:from single-cell mutation detection to mutant plant regeneration. Plant Biotechnology Journal, 16 (7):1295-1310.
[15]	Liu Guangyu, Xu Xiaojing, Xia Keke, Sun Haixi, Tao Yueru, Cui Zhen, Gu Ying. 2022. Optimization of CRISPR/Cas 9 genome editing systems in protoplasts of Setaria italica. Journal of Henan Agricultural Sciences, 51 (1):34-42. (in Chinese)
	刘光宇, 徐晓静, 夏科科, 孙海汐, 陶月如, 崔震, 顾颖. 2022. 基于原生质体的谷子CRISPR/Cas9 基因编辑系统优化. 河南农业科学, 51 (1):34-42.
[16]	Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho M J, Scelonge C, Lenderts B, Chamberlin M, Cushatt J, Wang L J, Ryan L, Khan T, Chow-Yiu J, Hua W, Yu M, Banh J, Bao Z M, Brink K, Igo E, Rudrappa B, Shamseer P, Bruce W, Newman L, Shen B, Zheng P Z, Bidney D, Falco C, Register J, Zhao Z Y, Xu D P, Jones T, Kamm W G. 2016. Morphogenic regulators baby boom and wuschel improve monocot transformation. The Plant Cell, 28 (9):1998-2015.
[17]	Lu Chenfei, Gao Yuexia, Huang He, Dai Silan. 2022. Carotenoid metabolism and regulation in plants. Acta Horticulturae Sinica, 49 (12):2559-2578. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0531
	陆晨飞, 高月霞, 黄河, 戴思兰. 2022. 植物类胡萝卜素代谢及调控研究进展. 园艺学报, 49 (12):2559-2578. doi: 10.16420/j.issn.0513-353x.2021-0531
[18]	Ow D W, De Wet J R, Helinski D R, Howell S H, Wood K V, Deluca M. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science, 234 (4778):856-859. doi: 10.1126/science.234.4778.856 pmid: 17758108
[19]	Poddar S, Tanaka J, Cate J H D, Staskawicz B, Cho M J. 2020. Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays. Plant Methods, 16 (1):151. doi: 10.1186/s13007-020-00692-4 pmid: 33292393
[20]	Ramessar K, Peremarti A, Gómez-Galera S, Naqvi S, Moralejo M, Muñoz P, Capell T, Christou P. 2007. Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants:a case of the science not supporting the politics. Transgenic Research, 16 (3):261-280. doi: 10.1007/s11248-007-9083-1 pmid: 17436060
[21]	Ron M, Kajala K, Pauluzzi G, Wang D X, Reynoso M A, Zumstein K, Garcha J, Winte S, Masson H, Inagki S, Federici F, Sinha N, Deal R B, Bailey-Serres J, Brady S M. 2014. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiology, 166 (2):455-469. doi: 10.1104/pp.114.239392
[22]	Song Qianna, Duan Yonghong, Feng Ruiyun. 2024. Establishment of CRISPR/Cas9-mediated highly efficient gene editing system in microtubers of potatoes. Biotechnology Bulletin, 40 (9):33-41. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2024-0536
	宋倩娜, 段永红, 冯瑞云. 2024. CRISPR/Cas9介导的高效四倍体马铃薯试管薯基因编辑体系的建立. 生物技术通报, 40 (9):33-41. doi: 10.13560/j.cnki.biotech.bull.1985.2024-0536
[23]	Stauber R H, Horie K, Carney P, Hudson E A, Tarasova N I, Gaitanaris G A, Pavlakis G N. 1998. Development and applications of enhanced green fluorescent protein mutants. Biotechniques, 24 (3):462-466,468-471. pmid: 9526659
[24]	Sundar I K, Sakthivel N. 2008. Advances in selectable marker genes for plant transformation. Journal of Plant Physiology, 165 (16):1698-1716. doi: 10.1016/j.jplph.2008.08.002 pmid: 18789557
[28]	Wang S J, Wang G, Li H L, Li F, Wang J B. 2023. Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/ Cas9-mediated genome editing in‘Feizixiao’litchi. Horticultural Plant Journal, 9 (5):947-957.
[29]	Yang H M, Baker S F, Gonzalez M E, Topham D J, Martínez-Sobrido L, Zand M, Holden-Wiltse J, Wu H L. 2016. An improved method for estimating antibody titers in microneutralization assay using green fluorescent protein. Journal of Biopharmaceutical Statistics, 26 (3):409-420. doi: 10.1080/10543406.2015.1052475 pmid: 26010892
[30]	Ye M W, Yao M F, Li C H, Gong M. 2023. Salt and osmotic stress can improve the editing efficiency of CRISPR/Cas9-mediated genome editing system in potato. Peer Journal, 11 (1):e15771.
[31]	Yin K Q, Gao C X, Qiu J L. 2017. Progress and prospects in plant genome editing. Nature Plants, 3 (8):17107.
[32]	Zhang Q X, Walawage S L, Tricoli D M, Dandekar A M, Leslie C A. 2015. A red fluorescent protein(DsRED)from Discosoma sp. as a reporter for gene expression in walnut somatic embryos. Plant Cell Report, 34 (5):861-869.
[33]	Zhang Y, Massel K, Godwin I D, Gao C X. 2019. Applications and potential of genome editing in crop improvement. Genome Biology, 20 (1):13. doi: 10.1186/s13059-019-1622-6 pmid: 30651124
[25]	Takahashi Y, Kinoshita T, Matsumoto M, Shimazaki K. 2016. Inhibition of the Arabidopsis bHLH transcription factor by monomerization through abscisic acid-induced phosphorylation. The Plant Journal, 87 (6):559-567. doi: 10.1111/tpj.13217 pmid: 27227462
[26]	Wang H Y, Zheng Y S, Zhou Q, Li Y, Liu T K, Hou X L. 2024. Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots. Horticultural Plant Journal, 10 (2):450-460.
[27]	Wang J, Su H Y, Wu Z B, Wang W S, Zhou Y B, Li M F. 2022. Integrated metabolites and transcriptomics at different growth stages reveal polysaccharide and flavonoid biosynthesis in Cynomorium songaricum. International Journal of Molecular Sciences, 23 (18):10675.

引物名称 Prime name	引物序列（5′-3′） Sequences of primes
StFBH3-sgRNA-F1	ATTGAGTTGAGAGAATAAACCAGC
StFBH3-sgRNA-R1	AAACGCTGGTTTATTCTCTCAACT
StFBH3-sgRNA-F2	ATTGTCCATAGCTGCTATATCAGC
StFBH3-sgRNA-R2	AAACGCTGATATAGCAGCTATGGA
StPDS-sgRNA-F	ATTGAAGGAGCGGGTAAAGCTTCG
StPDS-sgRNA-R	AAACCGAAGCTTTACCCGCTCCTT
StbHLH52-DT1-BsF	ATATATGGTCTCGATTGAATTGTCAATAGTTTCAACGTT
StbHLH52-DT1-F0	TGAATTGTCAATAGTTTCAACGTTTTAGAGCTAGAAATAGC
StbHLH52-DT2-R0	AACGAGTACTCTGTTTATGAGACAATCTCTTAGTCGACTCTAC
StbHLH52-DT2-BsR	ATTATTGGTCTCGAAACGAGTACTCTGTTTATGAGACAA
StbHLH52-DT3-BsF	ATATATGGTCTCGATTGTCTCAAAAGCAGAGTACTCGTT
StbHLH52-DT3-F0	TGTCTCAAAAGCAGAGTACTCGTTTTAGAGCTAGAAATAGC
StbHLH52-DT4-R0	AACGCCACTCACAATTCGTCATCAATCTCTTAGTCGACTCTAC
StbHLH52-DT4-BsR	TTATTGGTCTCGAAACGCCACTCACAATTCGTCATCAA
StFBH3-F1	CACAGTTGCCACCTCAATATCCAAGG
StFBH3-R1	ATAGTCCCTCAGGCTCAGCTCA
StFBH3-F2	CAGCAGCACTCACAGGCCAC
StFBH3-R2	GCTCTTGTAGCTTTCTCATTCGTTCAC
StPDS-F	GCCTTGCTTTTCCTTTCTCATCTTG
StPDS-R	GGCAGGTAGACAATCTAACACCTC
StbHLH52-F	GGAACCAAATGTTGAAGAATTCAAC
StbHLH52-R	CTCCAGCATTATTGCTGCTAAAA
Cas9-F	CCCTCAAAGAAGTTCAAGGTCCT
Cas9-R	AGATCCCCCTCGATCAGGAAATG

引物名称 Prime name	引物序列（5′-3′） Sequences of primes
StFBH3-sgRNA-F1	ATTGAGTTGAGAGAATAAACCAGC
StFBH3-sgRNA-R1	AAACGCTGGTTTATTCTCTCAACT
StFBH3-sgRNA-F2	ATTGTCCATAGCTGCTATATCAGC
StFBH3-sgRNA-R2	AAACGCTGATATAGCAGCTATGGA
StPDS-sgRNA-F	ATTGAAGGAGCGGGTAAAGCTTCG
StPDS-sgRNA-R	AAACCGAAGCTTTACCCGCTCCTT
StbHLH52-DT1-BsF	ATATATGGTCTCGATTGAATTGTCAATAGTTTCAACGTT
StbHLH52-DT1-F0	TGAATTGTCAATAGTTTCAACGTTTTAGAGCTAGAAATAGC
StbHLH52-DT2-R0	AACGAGTACTCTGTTTATGAGACAATCTCTTAGTCGACTCTAC
StbHLH52-DT2-BsR	ATTATTGGTCTCGAAACGAGTACTCTGTTTATGAGACAA
StbHLH52-DT3-BsF	ATATATGGTCTCGATTGTCTCAAAAGCAGAGTACTCGTT
StbHLH52-DT3-F0	TGTCTCAAAAGCAGAGTACTCGTTTTAGAGCTAGAAATAGC
StbHLH52-DT4-R0	AACGCCACTCACAATTCGTCATCAATCTCTTAGTCGACTCTAC
StbHLH52-DT4-BsR	TTATTGGTCTCGAAACGCCACTCACAATTCGTCATCAA
StFBH3-F1	CACAGTTGCCACCTCAATATCCAAGG
StFBH3-R1	ATAGTCCCTCAGGCTCAGCTCA
StFBH3-F2	CAGCAGCACTCACAGGCCAC
StFBH3-R2	GCTCTTGTAGCTTTCTCATTCGTTCAC
StPDS-F	GCCTTGCTTTTCCTTTCTCATCTTG
StPDS-R	GGCAGGTAGACAATCTAACACCTC
StbHLH52-F	GGAACCAAATGTTGAAGAATTCAAC
StbHLH52-R	CTCCAGCATTATTGCTGCTAAAA
Cas9-F	CCCTCAAAGAAGTTCAAGGTCCT
Cas9-R	AGATCCCCCTCGATCAGGAAATG

Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 27

Related Articles 15

Recommended Articles

Metrics

Comments

基因 Gene	载体 Vectors	材料 Materials	总毛状根数 No. of total hairy roots	荧光毛状根数 No. of fluorescent hairy roots	突变毛状根数 No. of mutant hairy roots	编辑效率/% Editing efficiency
StPDS		青薯9号 Qingshu 9	62	36	30	83.3
	StPDS-sgRNA	晋薯16号 Jinshu 6	42	15	12	80.0
		Desiree	60	33	29	87.8
StFBH3	StFBH3-sgRNA1	青薯9号 Qingshu 9	68	17	12	70.6
StFBH3	StFBH3-sgRNA2	青薯9号 Qingshu 9	30	11	8	72.7
StbHLH52	StbHLH52-sgRNA14	Desiree	173	42	7	16.7
StbHLH52	StbHLH52-sgRNA23	Desiree	130	31	6	19.4

[1]	WANG Hongbao, ZHENG Lingjie, DING Ding, MENG Jingrou, DING Fengjie, GUO Yanchao. A New Edible Chrysanthemum morifolium Cultivar‘Binjin 1’ [J]. Acta Horticulturae Sinica, 2025, 52(S1): 143-144.
[2]	YANG Li, QIN Ya, BIAN Baciren, ZHEN Zhen, QIONG Ji, BAI Maquzhen, ZHOU Ya. A New Cultivar of Colored Potato‘Xigezi 2’ [J]. Acta Horticulturae Sinica, 2025, 52(S1): 145-146.
[3]	LIU Lifeng, SONG Yanhong, ZHAO Xia, LI Gang, and ZHOU Houcheng. Analysis of FvPDS Gene Using CRISPR/Cas9 System in Strawberry [J]. Acta Horticulturae Sinica, 2025, 52(7): 1687-1695.
[4]	DOU Xueting, ZHU Xi, ZHANG Ning, and SI Huaijun. Functional Analysis of StHY5 Associated with Low-Temperature Stress in Potato [J]. Acta Horticulturae Sinica, 2025, 52(7): 1745-1757.
[5]	YE Yanran, LIU Jiangang, BIAN Chunsong, GUO Huachun, and JIN Liping. Quantitative Analysis of Potato Growth Under Different Nitrogen Management Practices Based on UAV-Acquired RGB Imagery [J]. Acta Horticulturae Sinica, 2025, 52(7): 1870-1882.
[6]	ZHU Xingzhe, SU Xianyue, SU Wang, ZHANG Honglin, PAN Zhechao, LIU Tao, and XU Xiaoyu. Understanding the Anabolic Differences of Steroidal Glycoalkaloids in Leaf Between Two Potato Varieties Grown at the Winter and Spring Cropping Patterns [J]. Acta Horticulturae Sinica, 2025, 52(7): 1883-1900.
[7]	LI Ziyan, CHEN Weixi, LI Zihan, LI Yin, LIANG Fengming, ZENG Xiangli, JIAN Hongju, and LÜ Dianqiu. Screening Candidate Genes Controlling Potato Maturation Time Based on RNA-Seq [J]. Acta Horticulturae Sinica, 2025, 52(6): 1505-1518.
[8]	WANG Wenlong, LIU Zhaokun, LU Wenjun, WANG Yaolong, LI Xiaofeng, ZHU Hongfang, LIU Tongkun, LI Ying, HOU Xilin, ZHANG Changwei. Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology [J]. Acta Horticulturae Sinica, 2025, 52(5): 1317-1325.
[9]	MA Xi, ZHANG Jinrui, ZHUANG Hongmei, CHEN Wei, ZHANG Mi, YUAN Xiaoqi, YI Xiaofang, WANG Congcong, WANG Haiyun, WANG Yan. Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium rhizogenes in Turnip [J]. Acta Horticulturae Sinica, 2025, 52(5): 1389-1398.
[10]	TIAN Zhuang, FANG Chenxi, LIU Yating, WANG Yutong, XUE Linlei, SHI Jiaxin, REN Tianfang, ZHANG Junwei, BAO Manzhu, ZHANG Jie. Construction of Transient Transformation System of Petals in Prunus mume [J]. Acta Horticulturae Sinica, 2025, 52(4): 908-920.
[11]	LIN Xi, DENG Zhenpeng, YANG Xinyue, ZHOU Keyou, YI Xiaoping, WANG Jichun. Effects of Reducing Chemical Fertilizer Combined with Organic Fertilizer on Potato Yield and Nutrient Utilization [J]. Acta Horticulturae Sinica, 2025, 52(4): 1007-1019.
[12]	WANG Zhongyi, LIU Yi, HU Bowen, ZHU Fan, LIU Feng, YANG Sha, XIONG Cheng, OU Lijun, DAI Xiongze, ZOU Xuexiao. Construction of a High-Efficiency Genetic Transformation System in Pepper Leveraging RUBY and CaREF1 [J]. Acta Horticulturae Sinica, 2025, 52(4): 1093-1104.
[13]	ZHOU Jinhua, BAI Lei, ZHANG Rui, GUO Huachun. The Effect of Reducing Soil Temperature with Straw Mulching Cultivation on Potato Growth [J]. Acta Horticulturae Sinica, 2025, 52(2): 453-466.
[14]	LI Min, LI Siyu, SHI Zihan, CHEN Shuang, XU Yan, LIU Guotian. Studies on the Efficiency of GRFs/GIFs for Genetic Transformation and Regeneration in Grapevine [J]. Acta Horticulturae Sinica, 2025, 52(1): 51-65.
[15]	HUANG Xun, LIU Xia, DENG Linmei, WANG Xingguo, XU Yajin, YANG Yanli. Identification and Study on Its Biocontrol and Growth Promoting Properties of Bacillus velezensis 1X1Y Against Potato Common Scab [J]. Acta Horticulturae Sinica, 2025, 52(1): 229-246.