Abstract
Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8–6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.
This is a preview of subscription content, log in via an institution to check access.
References
Aydin Y, Ipekci Z, Talas-Oğraş T, Zehir A, Bajrovic K, Gozukirmizi N (2004) High frequency somatic embryogenesis in cotton. Biol Plant 48:491–495. https://doi.org/10.1023/B:BIOP.0000047142.07987.e1
Birch RG (1997) Plant transformation: problems and strategies for practical application. Annu Rev Plant Biol 48:297–326. https://doi.org/10.1146/annurev.arplant.48.1.297
Directorate of Cotton Development Government of India (2017) Status paper of Indian cotton. Nagpur. Accessed on July 27, 2019 https://www.nfsm.gov.in/StatusPaper/Cotton2016.pdf
Entoori K, Sreevathsa R, Arthikala MK, Kumar PA, Kumar ARV, Madhusudhan B, Makarla U (2008) A chimeric cry1X gene imparts resistance to Spodoptera litura and Helicoverpa armigera in the transgenic groundnut. Eur Asia J BioSc 2:53–65
Guo WF, Wang KY, Wang N, Jun LI, Li GQ, Liu DH (2018) Rapid and convenient transformation of cotton (Gossypium hirsutum L.) using in planta shoot apex via glyphosate selection. J Integr Agric 17:2196–2203. https://doi.org/10.1016/S2095-3119(17)61865-3
Hussain SS, Rao AQ, Husnain T, Riazuddin S (2009) Cotton somatic embryo morphology affects its conversion to plant. Biol Plant 53:307–311. https://doi.org/10.1007/s10535-009-0055-6
** SX, Liu GZ, Zhu HG, Yang XY, Zhang XL (2012) Transformation of upland cotton (Gossypium hirsutum L.) with gfp gene as a visual marker. J Integr Agric 11:910–919. https://doi.org/10.1016/S2095-3119(12)60081-1
Kalbande BB, Patil AS (2016) Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum). J Genet Eng Biotechnol 14:9–18. https://doi.org/10.1016/j.jgeb.2016.05.003
Karthik K, Nandiganti M, Thangaraj A, Singh S, Mishra P, Sharma M, Sreevathsa R (2020) Transgenic cotton (Gossypium hirsutum L.) to combat weed vagaries: utility of an apical meristem-targeted in planta transformation strategy to introgress a modified CP4-EPSPS gene for glyphosate tolerance. Front Plant Sci. https://doi.org/10.3389/fpls.2020.00768
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 hirsutumL.). J Cotton Sci 12:264–272
Keshavareddy G, Rohini S, Ramu SV, Sundaresha S, Kumar AR, Kumar PA, Udayakumar M (2013) Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.). Physiol Mol Biol Plants 19:343–352. https://doi.org/10.1007/s12298-013-0182-6
Kesiraju K, Sreevathsa R (2017) Apical meristem-targeted In Planta transformation strategy: an overview on its utility in crop improvement. Agri Res and Techol. https://doi.org/10.19080/ARTOAJ.2017.08.555734
Kumar AM, Reddy KN, Manjulatha M, Arellano ES, Sreevathsa R, Ganeshan G (2011) A rapid, novel and high-throughput identification of putative bell pepper transformants generated through in planta transformation approach. Sci Hortic 129:898–903. https://doi.org/10.1016/j.scienta.2011.05.001
Kumar AM, Reddy KN, Sreevathsa R, Ganeshan G, Udayakumar M (2009) Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A: hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach. Sci Hortic 119:362–370. https://doi.org/10.1016/j.scienta.2008.08.034
Kumar M, Tuli R (2004) Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis. Vitro Cell Dev Biol-Pl 40:294–298. https://doi.org/10.1079/IVP2004531
Kumar NR, Rathinam M, Singh S, Kesiraju K, Muniyandi V, Singh NK, Dash PK, Sreevathsa R (2019) Assessment of pigeonpea (Cajanus cajan L.) transgenics expressing Bt ICPs, Cry2Aa and Cry1AcF under nethouse containment implicated an effective control against herbivory by Helicoverpa armigera (Hübner). Pest Manag Sci. https://doi.org/10.1002/ps.5722
Ma W, Zhang T (2019) Next-generation transgenic cotton: pyramiding RNAi with Bt counters insect resistance. In: Zhang B (ed) Transgenic cotton methods in molecular biology, 1st edn. Humana Press, New York, pp 245–256. https://doi.org/10.1007/978-1-4939-8952-2_21
Maher MF, Nasti RA, Vollbrecht M, Starker CG, Clark MD, Voytas DF (2020) Plant gene editing through de novo induction of meristems. Nat Biotechnol 38:84–89. https://doi.org/10.1038/s41587-019-0337-2
Mogali SC, Khadi BM, Kategeri IS (2013) High efficiency transformation protocol for two Indian cotton (Gossypium hirsutum) varieties via pollen tube pathway. Ind J Agric Sci 83:949–952
Mohapatra L, Saha G (2019) Cotton farming in India: alternative perspectives and paradigms. In: Nayak AJRN (ed) Transition strategies for sustainable community systems, The anthropocene: Politik—Economics—Society—Science, 1st edn. Springer, Switzerland, pp 195–213
Pathi KM, Tuteja N (2013) High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex. Plant Signal Behav 8:e22763. https://doi.org/10.4161/psb.22763
Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15:8–15. https://doi.org/10.1007/BF02772108
Rajasekaran K, Cary JW, Jaynes JM, Cleveland TE (2005) Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton (Gossypium hirsutum L.) plants. Plant Biotechnol J 3:545–554. https://doi.org/10.1111/j.1467-7652.2005.00145.x
Rohini VK, Rao KS (2002) In planta strategy for gene transfer into plants: embryo transformation. Physiol Mol Biol Plants 8:161–169
Sivasupramaniam S, Moar WJ, Ruschke LG, Osborn JA, Jiang C, Sebaugh JL, Greenplate JT (2014) Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests. J Econ Entomol 101:546–554. https://doi.org/10.1093/jee/101.2.546
Tian G, Cheng L, Qi X, Ge Z, Niu C, Zhang X, ** S (2015) Transgenic cotton plants expressing double-stranded RNAs target HMG-CoA reductase (HMGR) gene inhibits the growth, development and survival of cotton bollworms. Int J Biol Sci 11:1296–1305
Tian ZC, Shen JW, Jun Z, Wang ZG, Tian ZZ (2010) Pistil drip following pollination: a simple in planta Agrobacterium-mediated transformation in cotton. Biotechnol Lett 32:547–555. https://doi.org/10.1007/s10529-009-0179-y
Vajhala CS, Sadumpati VK, Nunna HR, Puligundla SK, Vudem DR, Khareedu VR (2013) Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests. PLoS ONE 8:e72542. https://doi.org/10.1371/journal.pone.0072542
Wu SJ, Wang HH, Li FF, Chen TZ, Zhang J, Jiang YJ, Zhang TZ (2008) Enhanced Agrobacterium-mediated transformation of embryogenic calli of upland cotton via efficient selection and timely subculture of somatic embryos. Plant Mol Biol Rep 26:174–185. https://doi.org/10.1007/s11105-008-0032-9
Zhu SJ, Li L, Chen JH, He QL, Fang XX, Ye CY, Mei L (2011) Advance in research and utilization of cotton biotechnology in China. Plant Omics 4:329–338
Author information
Authors and Affiliations
Contributions
KK developed the transgenic plants. KK, PM, AB performed molecular analyses. KK wrote the manuscript. MS critically edited the manuscript. UR provided the fluorescence microscope for GFP expression studies and critically edited the manuscript. RS designed experiments, edited and revised the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this article.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Kesiraju, K., Mishra, P., Bajpai, A. et al. Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker. Physiol Mol Biol Plants 26, 2319–2327 (2020). https://doi.org/10.1007/s12298-020-00887-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12298-020-00887-y