Abstract
Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg L−1 naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology.
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References
Aggarwal PR, Nag P, Choudhary P, Chakraborty N, Chakraborty S (2018) Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies. Plant Methods 14:1–13
Alok A, Sandhya D, Jogam P, Rodrigues V, Bhati KK, Sharma H, Kumar J (2020) The rise of the CRISPR/Cpf1 system for efficient genome editing in plants. Front Plant Sci 11:264
Alok A, Shukla V, Pala Z, Kumar J, Kudale S, Desai N (2016) In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia pallens, an important medicinal plant. Physiol Mol Biol Plants 22:261–269
Bakhsh A (2020) Development of efficient, reproducible and stable Agrobacterium-mediated genetic transformation of five potato cultivars. Food Technol Biotechnol 58:57–63
Bernard G, Gagneul D, Alves Dos Santos H, Etienne A, Hilbert JL, Rambaud C (2019) Efficient genome editing using CRISPR/Cas9 technology in chicory. Intl J Mol Sci 20:1155
Bouis HE, Welch RM (2010) Biofortification-a sustainable agricultural strategy for reducing micronutrient malnutrition in the global south. Crop Sci 50:20
Butler NM, Jansky SH, Jiang J (2020) First-generation genome editing in potato using hairy root transformation. Plant Biotechnol J 18:2201–2209
Chen L, Cai Y, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W (2018) Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation. Crop J 6:162–171
Cheng Y, Wang X, Cao L, Ji J, Liu T, Duan K (2021) Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean. Plant Methods 17:1–12
Devaux A, Kromann P, Ortiz O (2014) Potatoes for sustainable global food security. Potato Res 57:185–199
Habibi P, de Sa MF, da Silva AL, Makhzoum A, da Luz CJ, Borghetti IA, Soccol CR (2016) Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare. Physiol Mol Biol Plants 22:271–277
Hameed A, Zaidi SS, Shakir S, Mansoor S (2018) Applications of new breeding technologies for potato improvement. Front Plant Sci 9:925
Hao Y, Zong X, Ren P, Qian Y, Fu A (2021) Basic Helix-Loop-Helix (bHLH) transcription factors regulate a wide range of functions in Arabidopsis. Intl J Mol Sci 22:7152
Huang Y, He G, Tian W, Li D, Meng L, Wu D, He T (2021) Genome-wide identification of mate gene family in potato (Solanum tuberosum L) and expression analysis in heavy metal stress. Front Genet 12:650500
Irigoyen S, Ramasamy M, Pant S, Niraula P, Bedre R, Gurung M, Rossi D, Laughlin C, Gorman Z, Achor D, Levy A (2020) Plant hairy roots enable high throughput identification of antimicrobials against Candidatus liberibacter spp. Nature Comm 11:1–14
Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA (2015) Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol 15:1–10
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Kaur A, Guleria S, Reddy MS, Kumar A (2020) A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L cultivar ‘Kufri Chipsona 1.’ Physiol Mol Biol Plants 26:367–377
Kaur N, Alok A, Kumar P, Awasthi P, Chaturvedi S, Pandey P, Pandey A, Pandey AK, Tiwari S (2020) CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit. Metab Eng 59:76–86
Kumar P, Kumar P, Sharma D, Verma SK, Halterman D, Kumar A (2021) Genome-wide identification and expression profiling of basic leucine zipper transcription factors following abiotic stresses in potato (Solanum tuberosum L). PLoS ONE 16:e0247864
Long TA, Tsukagoshi H, Busch W, Lahner B, Salt DE, Benfey PN (2010) The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots. Plant Cell 22:2219–2236
Moehninsi, Navarre DA (2018) Optimization of hairy root induction in Solanum tuberosum. Amer J Potato Res 95:650–658
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497
Potato Genome Sequencing Consortium (2011) Genome sequence and analysis of the tuber crop potato. Nature 475:189–195
Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K, Garcha J, Winte S, Masson H, Inagaki S, Federici F (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 Physiol 166:455–469
Stemmer M, Thumberger T, del Sol KM, Wittbrodt J, Mateo JL (2015) CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS ONE 10:e0124633
Upadhyay SK, Kumar J, Alok A, Tuli R (2013) RNA-guided genome editing for target gene mutations in wheat. G3: Genes Genomes Genet 3:2233–2238
Veale MA, Slabbert MM, Van Emmenes L (2012) Agrobacterium-mediated transformation of potato cv Mnandi for resistance to the potato tuber moth (Phthorimaea operculella). S Afr J Bot 80:67–74
Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327
Zaheer K, Akhtar MH (2016) Potato production, usage, and nutrition-a review. Crit Rev Food Sci Nutr 56:711–721
Zhu S, Yu X, Li Y, Sun Y, Zhu Q, Sun J (2018) Highly efficient targeted gene editing in upland cotton using the CRISPR/Cas9 system. Intl J Mol Sci 19:3000
Acknowledgements
HC and Aiana are thankful to the CSIR, India, and UGC, India, respectively, for awarding junior research fellowships.
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KS: conceived the idea, designed the experiments, analyzed the results, and finalized the manuscript. AA: compiled the results and wrote the manuscript. AA and HC: CRISPR construct designing, transformation experiments, and mutation detection, NK and A: molecular cloning, sequencing, and subculturing.
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Alok, A., Chauhan, H., Kaushal, N. et al. Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction. In Vitro Cell.Dev.Biol.-Plant 59, 83–94 (2023). https://doi.org/10.1007/s11627-022-10318-0
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DOI: https://doi.org/10.1007/s11627-022-10318-0