Abstract
Main conclusion
The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton.
Abstract
Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4–10 weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method’s success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.
Similar content being viewed by others
References
ABRAPA (2020) Estatísticas: O algodão no mundo. Associação Brasileira de Produtores de Algodão. https://www.abrapa.com.br/Paginas/dados/algodao-no-mundo. Accessed 13 Jan 2021
Akbar W, Gowda A, Ahrens JE et al (2019) First transgenic trait for control of plant bugs and thrips in cotton. Pest Manag Sci 75:867–877. https://doi.org/10.1002/ps.5234
Aragão FJL, Sarokin L, Vianna GR, Rech EL (2000) Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L.) Merril] plants at a high frequency. Theor Appl Genet 101:1–6. https://doi.org/10.1007/s001220051441
Aragao FJ, Vianna GR, Carvalheira SB, Rech EL (2005) Germ line genetic transformation in cotton (Gossypium hirsutum L.) by selection of transgenic meristematic cells with a herbicide molecule. Plant Sci 168(5):1227–1233. https://doi.org/10.1016/j.plantsci.2004.12.024
Asad S, Mukhtar Z, Nazir F et al (2008) Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants. Mol Biotechnol 40:161–169. https://doi.org/10.1007/s12033-008-9072-5
Basso MF, Costa JA, Ribeiro TP, Arraes FBM, Lourenço-Tessutti IT et al (2021) Overexpression of the CaHB12 transcription factor in cotton (Gossypium hirsutum) improves drought tolerance. Plant Physiol Biochem 165:80–93. https://doi.org/10.1016/j.plaphy.2021.05.009
Bibi N, Fan K, Yuan S et al (2013) An efficient and highly reproducible approach for the selection of upland transgenic cotton produced by pollen tube pathway method. Aust J Crop Sci 7:1714–1722. https://doi.org/10.3316/informit.644871058289920
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Bravo A, Gómez I, Porta H et al (2013) Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microb Biotechnol 6:17–26. https://doi.org/10.1111/j.1751-7915.2012.00342.x
Chen X, Wang Y, Lv B et al (2014) The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway. Plant Cell Physiol 55:604–619. https://doi.org/10.1093/pcp/pct204
Chen Y, Lange A, Vaghchhipawala Z et al (2020) Direct germline transformation of cotton meristem explants with no selection. Front Plant Sci 11:575283. https://doi.org/10.3389/fpls.2020.575283
Chlan CA, Lin J, Cary JW, Cleveland TE (1995) A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue. Plant Mol Biol Rep/ISPMB 13:31–37. https://doi.org/10.1007/BF02668391
Cousins Y, Lyon B, Llewellyn D (1991) Transformation of an Australian cotton cultivar: prospects for cotton improvement through genetic engineering. Funct Plant Biol 18:481. https://doi.org/10.1071/PP9910481
Duncan DR (2010) Cotton transformation. In: Zehr UB (ed) Cotton: biotechnological advances. Biotechnology agriculture. Springer Berlin Heidelberg, pp 65–77. https://doi.org/10.1007/978-3-642-04796-1_4
El-Esawi MA, Alayafi AA (2019) Overexpression of rice Rab7 gene improves drought and heat tolerance and increases grain yield in rice (Oryza sativa L.). Genes 10(1):56. https://doi.org/10.3390/genes10010056
Emani C (2016) Transgenic cotton for agronomical useful traits. In: Ramawat KG, Ahuja MR (eds) Fiber plants. Springer Intern Publ, Cham, pp 201–216
Finer JJ, McMullen MD (1990) Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep 8:586–589. https://doi.org/10.1007/BF00270059
Firoozabady E, Deboer DL, Merlo DJ et al (1987) Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol Biol 10:105–116. https://doi.org/10.1007/BF00016148
Ganesan S, George M, Jap S et al (2009) Supply chain management and retailer performance: emerging trends, issues, and implications for research and practice. J Retail 85:84–94. https://doi.org/10.1016/j.jretai.2008.12.001
Gaspar YM, McKenna JA, McGinness BS et al (2014) Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1. J Exp Bot 65(6):1541–1550. https://doi.org/10.1093/jxb/eru021
Gurusaravanan P, Vinoth S, Jayabalan N (2020) An improved Agrobacterium-mediated transformation method for cotton (Gossypium hirsutum L. ‘KC3’) assisted by microinjection and sonication. Vitro Cell Dev Biol Plant 56:111–121. https://doi.org/10.1007/s11627-019-10030-6
Hashmi JA, Zafar Y, Arshad M et al (2011) Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences. Virus Genes 42:286–296. https://doi.org/10.1007/s11262-011-0569-9
Hayat K, Bardak A, Parlak D et al (2020) Biotechnology for cotton improvement. In: Ahmad S, Hasanuzzaman M (eds) Cotton production and uses: agronomy, crop protection, and postharvest technologies. Springer Singapore, Singapore, pp 509–525
ICAC (2020) International Cotton Advisory Committee. https://www.icac.org/DataPortal/DataPortal?Units=Area&Year=2019/20%20for. Accessed 13 Jan 2021
IMAmt (2015) Manual de boas práticas de manejo do algodoeiro em Mato Grosso. Editores: Ima-MT e AMPA, Cuiabá–Mato Grosso, Brasil (ISBN 978-85-66457-06-3)
ISAAA (2018) International Service for the Acquisition of Agri-biotech Applications, GM ApprovalDatabase. https://www.isaaa.org/resources/publications/biotech_crop_annual_update/download/biotech-crop-annual-update-cotton-2019.pdf. Accessed 13 Jan 2021
ISAAA (2020) International Service for the Acquisition of Agri-biotech Applications, GM Approval Database Approval Database (http://www.isaaa.org/gmapprovaldatabase/advsearch/default.asp?CropID=7&TraitTypeID=Any&DeveloperID=Any&CountryID=Any&ApprovalTypeID=Any). Accessed 13 Jan 2021
Juturu VN, Mekala GK, Kirti PB (2015) Current status of tissue culture and genetic transformation research in cotton (Gossypium spp.). Plant Cell Tissue Organ Cult 120:813–839. https://doi.org/10.1007/s11240-014-0640-z
Keshamma E, Rohini S, Rao KS, Madhusudhan B, Kumar MU (2008) Tissue culture-independent in planta transformation strategy: an Agrobacterium tumefaciens-mediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.). J Cotton Sci 12:264–272. http://cotton.org/journal/2008-12/3/upload/JCS12-264.pdf. Accessed 13 Jan 2021
Kumar S, Dhingra A, Daniell H (2004) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216. https://doi.org/10.1007/s11103-004-2907-y
Leelavathi S, Sunnichan V, Kumria R et al (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22:465–470. https://doi.org/10.1007/s00299-003-0710-x
Li X, Wang XD, Zhao X, Dutt Y (2004) Improvement of cotton fiber quality by transforming the acsA and acsB genes into Gossypium hirsutum L. by means of vacuum infiltration. Plant Cell Rep 22:691–697. https://doi.org/10.1007/s00299-003-0751-1
Li S, Cong Y, Liu Y et al (2017) Optimization of Agrobacterium-mediated transformation in soybean. Front Plant Sci 8:246. https://doi.org/10.3389/fpls.2017.00246
Lisei-de-Sa ME, Arraes FB, Brito G et al (2017) AtDREB2A-CA influences root architecture and increases drought tolerance in transgenic cotton. Agric Sci 8:1195–1225. https://doi.org/10.4236/as.2017.810087
Liu JF, Wang XF, Li QL et al (2011) Biolistic transformation of cotton (Gossypium hirsutum L.) with the phyA gene from Aspergillus ficum. Plant Cell Tissue Organ Cult 106:207–214. https://doi.org/10.1007/s11240-010-9908-0
McCabe DE, Martinell BJ (1993) Transformation of elite cotton cultivars via particle bombardment of meristems. Nat Biotechnol 11:596–598. https://doi.org/10.1038/nbt0593-596
Meng Z-H, Liang A-H, Yang W-C (2007) Effects of hygromycin on cotton cultures and its application in Agrobacterium-mediated cotton transformation. Vitro Cell Dev Biol Plant 43:111–118. https://doi.org/10.1007/s11627-007-9031-z
Nandeshwar SB, Moghe S, Chakrabarty PK et al (2009) Agrobacterium-mediated transformation of cry1Ac gene into shoot-tip meristem of diploid cotton Gossypium arboreum cv. RG8 and regeneration of transgenic plants. Plant Mol Biol Rep 27:549–557. https://doi.org/10.1007/s11105-009-0102-7
Paes de Melo B, Lourenço-Tessutti IT, Morgante CV et al (2020) Soybean embryonic axis transformation: combining biolistic and Agrobacterium-mediated protocols to overcome typical complications of in vitro plant regeneration. Front Plant Sci 11:1228. https://doi.org/10.3389/fpls.2020.01228
Perlak FJ, Deaton RW, Armstrong TA et al (1990) Insect resistant cotton plants. Biotechnology 8:939–943. https://doi.org/10.1038/nbt1090-939
Pitzschke A (2013) Agrobacterium infection and plant defense-transformation success hangs by a thread. Front Plant Sci 4:519. https://doi.org/10.3389/fpls.2013.00519
Rajasekaran K, Hudspeth RL, Cary JW et al (2000) High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures. Plant Cell Rep 19:539–545. https://doi.org/10.1007/s002990050770
Rech EL, Vianna GR, Aragão FJL (2008) High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3:410–418. https://doi.org/10.1038/nprot.2008.9
Ribeiro TP, Arraes FBM, Lourenço-Tessutti IT et al (2017) Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil. Plant Biotechnol J 15:997–1009. https://doi.org/10.1111/pbi.12694
Ribeiro TP, Basso MF, Carvalho MH et al (2020) Stability and tissue-specific Cry10Aa overexpression improves cotton resistance to the cotton boll weevil. Biotechnol Res Innov 3:27–41. https://doi.org/10.1016/j.biori.2019.12.003
Sunilkumar G, Rathore KS (2001) Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Mol Breed 8:37–52. https://doi.org/10.1023/A:1011906701925
Umbeck P, Johnson G, Barton K, Swain W (1987) Genetically transformed cotton (Gossypium hirsutum L.) plants. Nat Biotechnol 5:263–266. https://doi.org/10.1038/nbt0387-263
Wilkins TA, Rajasekaran K, Anderson DM (2000) Cotton biotechnology. Crit Rev Plant Sci 19:511–550. https://doi.org/10.1080/07352680091139286
Yang X (2012) Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-△△CT method. Afr J Biotechnol 11. https://doi.org/10.5897/AJB11.4117
Zapata C, Park SH, El-Zik KM, Smith RH (1999) Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex. Theor Appl Genet 98:252–256. https://doi.org/10.1007/s001220051065
Zhang B, Feng R, Wang Y, Wang Q (2001) High frequency somatic embryogenesis and plant regeneration of an elite Chinese cotton variety. Bot Bull Acad Sin 42:9–16. https://doi.org/10.1023/B:BIOP.0000047142.07987.e1
Zhang XD, Jenkins JN, Callahan FE et al (2003) Molecular cloning, differential expression, and functional characterization of a family of class I ubiquitin-conjugating enzyme (E2) genes in cotton (Gossypium). Biochem Biophys Acta 1625:269–327. https://doi.org/10.1016/s0167-4781(02)00623-1
Zhao F-Y, Li Y-F, Xu P (2006) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker. Biotechnol Lett 28:1199–1207. https://doi.org/10.1007/s10529-006-9078-7
Zhu S-W, Gao P, Sun J-S et al (2006) Genetic transformation of green-colored cotton. Vitro Cell Dev Biol Plant 42:439–444. https://doi.org/10.1079/IVP2006777
Funding
This work was supported by CAPES, CNPq, FAP-DF, UCB, INCT PlantStress Biotech, and ABRAPA.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be interpreted as a potential conflict of interest.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Additional information
Communicated by Dorothea Bartels.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ribeiro, T.P., Lourenço-Tessutti, I.T., de Melo, B.P. et al. Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods. Planta 254, 20 (2021). https://doi.org/10.1007/s00425-021-03666-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00425-021-03666-5