Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton

CRIPSR/Cas9 gene editing system is an effective tool for genome modification in plants. Multiple target sites are usually designed and the effective target sites are selected for editing. Upland cotton (Gossypium hirsutum L., hereafter cotton) is allotetraploid and is commonly considered as difficult and inefficient to transform, it is important to select the effective target sites that could result in the ideal transgenic plants with the CRISPR-induced mutations. In this study, Agrobacterium rhizogenes-mediated hairy root method was optimized to detect the feasibility of the target sites designed in cotton phytoene desaturase (GhPDS) gene. A. rhizogenes showed the highest hairy root induction (30%) when the bacteria were cultured until OD600 reached to 0.8. This procedure was successfully applied to induce hairy roots in the other three cultivars (TM–1, Lumian–21, Zhongmian–49) and the mutations were detected in GhPDS induced by CRISPR/Cas9 system. Different degrees of base deletions at two sgRNAs (sgRNA5 and sgRNA10) designed in GhPDS were detected in R15 hairy roots. Furthermore, we obtained an albino transgenic cotton seeding containing CRISPR/Cas9-induced gene editing mutations in sgRNA10. The hairy root transformation system established in this study is sufficient for selecting sgRNAs in cotton, providing a technical basis for functional genomics research of cotton.

[1]  H. Robert,et al.  Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus) , 2022, bioRxiv.

[2]  T. Hoang,et al.  An Efficient Hairy Root System for Validation of Plant Transformation Vector and CRISPR/Cas Construct Activities in Cucumber (Cucumis sativus L.) , 2022, Frontiers in Plant Science.

[3]  Yuxian Zhu,et al.  Breeding cotton with superior fiber quality: identification and utilization of multiple elite loci and exotic genetic resources , 2021, Science China. Life sciences.

[4]  G. Barker,et al.  Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2 , 2021, Plant biotechnology journal.

[5]  Chuanliang Liu,et al.  A Stable Agrobacterium rhizogenes-Mediated Transformation of Cotton (Gossypium hirsutum L.) and Plant Regeneration From Transformed Hairy Root via Embryogenesis , 2020, Frontiers in Plant Science.

[6]  Liangshen Jin,et al.  One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation , 2020, BMC Plant Biology.

[7]  C. Fu,et al.  Efficient Generation of CRISPR/Cas9-Mediated Homozygous/Biallelic Medicago truncatula Mutants Using a Hairy Root System , 2020, Frontiers in Plant Science.

[8]  Wei Gao,et al.  The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton , 2019, Plant biotechnology journal.

[9]  J. Grima-Pettenati,et al.  Hairy Root Transformation: A Useful Tool to Explore Gene Function and Expression in Salix spp. Recalcitrant to Transformation , 2019, Front. Plant Sci..

[10]  Alexandre Cardoso-Taketa,et al.  Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets , 2019, PloS one.

[11]  Yang Tang,et al.  CRISPR/Cas9-mediated targeted mutagenesis of GmLCL genes alters plant height and internode length in soybean , 2019 .

[12]  Bingjun Yu,et al.  The Recretohalophyte Tamarix TrSOS1 Gene Confers Enhanced Salt Tolerance to Transgenic Hairy Root Composite Cotton Seedlings Exhibiting Virus-Induced Gene Silencing of GhSOS1 , 2019, International journal of molecular sciences.

[13]  Wen-qin Song,et al.  Enhancement of tanshinone production in Salvia miltiorrhiza hairy root cultures by metabolic engineering , 2019, Plant Methods.

[14]  R. Stipanovic,et al.  Genes regulating gland development in the cotton plant , 2018, Plant biotechnology journal.

[15]  S. Chakraborty,et al.  Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies , 2018, Plant Methods.

[16]  Caixia Gao,et al.  Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems , 2017, bioRxiv.

[17]  Wei Gao,et al.  Genome Editing in Cotton with the CRISPR/Cas9 System , 2017, Front. Plant Sci..

[18]  Yuqi Hao,et al.  ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton , 2017, Planta.

[19]  Yongping Cai,et al.  Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton , 2017, Science China Life Sciences.

[20]  W. Ye,et al.  Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system , 2017, Scientific Reports.

[21]  P. Goodwin,et al.  Hairy root culture optimization and resveratrol production from Vitis vinifera subsp. sylvesteris , 2017, World journal of microbiology & biotechnology.

[22]  Baohong Zhang,et al.  A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.) , 2017, Scientific Reports.

[23]  H. Lam,et al.  GmCLC1 Confers Enhanced Salt Tolerance through Regulating Chloride Accumulation in Soybean , 2016, Front. Plant Sci..

[24]  Y. Ruan,et al.  The genome sequence of Sea-Island cotton (Gossypium barbadense) provides insights into the allopolyploidization and development of superior spinnable fibres , 2015, Scientific Reports.

[25]  Caiping Cai,et al.  Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites , 2015, Scientific Reports.

[26]  Chaofeng Li,et al.  Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation , 2015, Scientific Reports.

[27]  Yajun Xi,et al.  Targeted mutagenesis in soybean using the CRISPR-Cas9 system , 2015, Scientific Reports.

[28]  He Zhang,et al.  Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution , 2015, Nature Biotechnology.

[29]  Lei Fang,et al.  Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement , 2015, Nature Biotechnology.

[30]  B. Meyer,et al.  Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design , 2015, Genetics.

[31]  张静,et al.  Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening , 2015 .

[32]  P. Kirti,et al.  Current status of tissue culture and genetic transformation research in cotton (Gossypium spp.) , 2015, Plant Cell, Tissue and Organ Culture (PCTOC).

[33]  Z. Lippman,et al.  Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System1 , 2014, Plant Physiology.

[34]  M. Spalding,et al.  Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice , 2014, Nucleic acids research.

[35]  Nivedita Patra,et al.  Enhanced Production of Artemisinin by Hairy Root Cultivation of Artemisia annua in a Modified Stirred Tank Reactor , 2014, Applied Biochemistry and Biotechnology.

[36]  Xun Xu,et al.  Genome sequence of the cultivated cotton Gossypium arboreum , 2014, Nature Genetics.

[37]  R. Qin,et al.  Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice , 2014, Rice.

[38]  Bing Yang,et al.  Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice , 2013, Nucleic acids research.

[39]  Detlef Weigel,et al.  Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[40]  Botao Zhang,et al.  Efficient genome editing in plants using a CRISPR/Cas system , 2013, Cell Research.

[41]  Adi Doron-Faigenboim,et al.  Ecology, Evolution and Organismal Biology Publications Ecology, Evolution and Organismal Biology Repeated Polyploidization of Gossypium Genomes and the Evolution of Spinnable Cotton Fibres , 2022 .

[42]  M. Sienkiewicz,et al.  The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity , 2012, In Vitro Cellular & Developmental Biology - Plant.

[43]  John Z. Yu,et al.  The draft genome of a diploid cotton Gossypium raimondii , 2012, Nature Genetics.

[44]  M. Shibuya,et al.  Antimicrobial polyacetylenes from Panax ginseng hairy root culture. , 2012, Chemical & pharmaceutical bulletin.

[45]  Jianbo Xiao,et al.  Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. , 2011, Metabolic engineering.

[46]  Feng Zhang,et al.  Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA] , 2011, Plant Physiology.

[47]  N. Murai,et al.  Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues. , 2011, Plant science : an international journal of experimental plant biology.

[48]  M. Kumar,et al.  Efficient production of gossypol from hairy root cultures of cotton (Gossypium hirsutum L.). , 2009, Current pharmaceutical biotechnology.

[49]  J. Jenkins,et al.  Phenotypic and molecular evaluation of cotton hairy roots as a model system for studying nematode resistance , 2009, Plant Cell Reports.

[50]  John Z. Yu,et al.  Toward Sequencing Cotton (Gossypium) Genomes , 2007, Plant Physiology.

[51]  N. Trolinder,et al.  Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.) , 1987, Plant Cell Reports.

[52]  A. Lorence,et al.  Camptothecin and 10-hydroxycamptothecin from Camptotheca acuminata hairy roots , 2003, Plant Cell Reports.

[53]  E. F. Smith,et al.  A PLANT-TUMOR OF BACTERIAL ORIGIN. , 1907, Science.