Abstract
Transformation technology is a powerful technique to study the role of gene function in improving peanut tolerance to abiotic stress. Thus, developing an effective genetic transformation method for peanut is a very interesting goal. Here, we describe a pollen tube pathway-mediated Agrobacterium tumefaciens transformation method in peanut. It is a simple, efficient, low cost and tissue culture-independent method of peanut transformation, involving injection of a fresh suspension of transformed A. tumefaciens carrying AhBI-1 encoding Bax inhibitor-1 from Arachis hypogaea L. into pollen tubes, integration of the gene into the host plant genome, and rapid screening for transformants using quantitative real-time PCR. The findings confirmed that this strategy provided an efficient (50% frequency of positive transformants) gene transformation technique in peanut. Furthermore, overexpression of the AhBI-1 transgene resulted in increased root and shoot growth even in the absence of exogenous abiotic stress.
Key message
AhBI-1 was transformed into peanut via PTT with high efficiency, and overexpressed AhBI-1 caused enhanced growth of root and shoot in normal condition.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
Abbreviations
- PCR:
-
Polymerase chain reaction
- PTT:
-
The pollen tube transformation
- PCD:
-
Programmed cell death
- YEP:
-
Yeast Extract Peptone Medium
- OD:
-
Optical density
- AS:
-
Acetosyringone
- MES:
-
2-Ethane sulfonic acid
- CTAB:
-
Cetyltrimethyl ammonium bromide
- WT:
-
Wild-type
- qPCR:
-
Quantitative real-time PCR
- OE:
-
Over expression
- NPBTs:
-
New plant breeding tools
References
Bhatnagar-Mathur P, Devi MJ, Reddy DS, Lavanya M, Vadez V, Serraj R, Yamaguchi-Shinozaki K, Sharma KK (2007) Stress-inducible expression of AT DREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions. Plant Cell Rep 26:2071–2082. https://doi.org/10.1007/s00299-007-0406-8
Bikash R, Senjuti S (2022) An improvised hairy root transformation method for efficient gene silencing in roots and nodules of Arachis hypogaea. Methods Mol Biol 2408:303–316. https://doi.org/10.1007/978-1-0716-1875-2_20
Brar GS, Cohen BA, Vick CL, Johnson GW (1994) Recovery of transgenic peanut (Arachis hypogaea L.) plants from elite cultivars utilizing ACCELL® technology. Plant J 5:745–753. https://doi.org/10.1111/j.1365-313X.1994.00745.x
Cao S, Niu J, Cao Q, Li H, Meng Y, Xue H, Chen L, Zhang F, Zhao D, Xie S (2019) Transformation of the roLB gene via the pollen-tube pathway to obtain transgenic pomegranate plants. Acta Hortic 1254:19–26. https://doi.org/10.17660/actahortic.2019.1254.4
Chen MN, Yang QL, Wang T, Chen N, Pan LJ, Chi XY, Yang Z, Wang M, Yu SL (2015) Agrobacterium-mediated genetic transformation of peanut and the efficient recovery of transgenic plants. Can J Plant Sci 95(4):735–744. https://doi.org/10.4141/CJPS-2014-012
Chen Y, Sun KY, Zhang T (2012) Transformation of Lagerstroemia indica by improved Agrobacterium-mediated floral-dip and pollen-tube pathway method. J Zhejiang Univ Sci 38(3):250–255. https://doi.org/10.3785/j.issn.1008-9209.2012.03003
Chu Y, Bhattacharya A, Wu CL, Knoll JE, Ozias Akins P (2013) Improvement of peanut (Arachis hypogaea L.) transformation efficiency and determination of transgene copy number by relative quantitative real-time PCR. In Vitro Cell Dev Biol Plant 49(3):266–275. https://doi.org/10.1007/s11627-013-9518-8
Cuc LM, Mace ES, Crouch JH, Quang VD, Long TD, Varshney RK (2008) Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea). BMC Plant Biol 8(55):1. https://doi.org/10.1186/1471-2229-8-55
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21. https://doi.org/10.1007/BF02712670
Détain A, Bhowmik P, Leborgne-Castel N, Ochatt S (2022) Latest biotechnology tools and targets for improving abiotic stress tolerance in protein legumes. Environ Exp Bot 197:104824. https://doi.org/10.1016/j.envexpbot.2022.104824
Ding XX, Zhang XG, Olsen OA, Otegui MS (2021) Transient expression of fluorescently tagged proteins in developing maize aleurone cells. Methodsx 8(3):101446. https://doi.org/10.1016/j.mex.2021.101446
Eapen S, George L (1993) Plant regeneration from leaf discs of peanut and pigeonpea: Influence of benzyladenine, indoleacetic acid and indoleacetic acid-amino acid conjugates. Plant Cell Tissue Organ Cult 35(3):223–227. https://doi.org/10.1007/BF00037274
FAOSTAT (2022) http://www.fao.org/faostat/en/#data/QC/visualize [accessed on April 2022]
Gantait S, Mondal S (2018) Transgenic approaches for genetic improvement in groundnut (Arachis hypogaea L.) against major biotic and abiotic stress factors. J Genet Eng Biotechnol 16(2):537–544. https://doi.org/10.1016/j.jgeb.2018.08.005
Hao J, Niu Y, Yang B, Gao F, Zhang L, Wang J, Hasi A (2011) Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway. Biotechnol Lett 33:55–61. https://doi.org/10.1007/s10529-010-0398-2
Hu CY, Wang LZ (1999) In planta soybean transformation technologies developed in China: Procedure, confirmation and field performance. In Vitro Cell Dev Biol Plant 35:417–420. https://doi.org/10.1007/s11627-999-0058-1
Huang GC, Dong YM, Sun JS (1999) Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter. Chin Sci Bull 44:698–701. https://doi.org/10.1007/BF02909705
Hwang HH, Yu MD, Lai EM (2017) Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book 15: e0186. https://doi.org/10.1199/Table
Iqbal MM, Nazir F, Ali S, Asif MA, Zafar Y, Iqbal J, Ali GM (2012) Over expression of rice chitinase gene in transgenic peanut (Arachis hypogaea L.) improves resistance against leaf spot. Mol Biotechnol 50:129–136. https://doi.org/10.1007/s12033-011-9426-2
Karthik S, Pavan G, Sathish S, Sathish S, Siva R, Kumar PS, Manickavasagam M (2018) Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)]. 3 Biotech 8(4):202. https://doi.org/10.1007/s13205-018-1231-1
Keshavareddy G, Kumar A, Ramu VS (2018) Methods of plant transformation- a review. Int J Curr Microbiol 7(7):2656–2668. https://doi.org/10.20546/ijcmas.2018.707.312
Keshavareddy G, Rohini S, Ramu SV, Sundaresha S, Kumar ARV, 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
Krishna G, Singh BK, Kim EK, Morya VK, Ramteke PW (2015) Progress in genetic engineering of peanut (Arachis hypogaea L.) — a review. Plant Biotechnol J 13(2):147–162. https://doi.org/10.1111/pbi.12339
Li HQ, Xu J, Chen L, Li MR (2007) Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of Thellungiella halophila.. Plant Cell Rep 26(10):1785–1789. https://doi.org/10.1007/s00299-007-0391-y
Li Z, Nelson RL, Widholm JM, Bent A (2002) Soybean transformation via the pollen tube pathway. Soybean genet newsl 29:1–11
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Mace ES, Phong DT, Upadhyaya HD (2006) SSR analysis of cultivated groundnut (Arachis hypogaea L.) germplasm resistant to rust and late leaf spot diseases. Euphytica 152:317–330. https://doi.org/10.1007/s10681-006-9218-0
Magbanua ZV, Wilde HD, Roberts JK, chowdhuryk, Abad J, Moyer JW, Wetzstein HY, Parrott WA (2000) Field resistance to tomato spotted wilt virus in transgenic peanut (Arachis hypogaea L.) expressing an antisense nucleocapsid gene sequence. Mol Breed 6:227–236. https://doi.org/10.1023/A:1009649408157
Mallikarjuna G, Rao T, Kirti PB (2016) Genetic engineering for peanut improvement: current status and prospects. Plant Cell Tissue Organ Cult 125(3):417–417. https://doi.org/10.1007/s11240-016-0966-9
Martin N, Forgeois P, Picard E (1992) Investigations on transforming Triticum aestivum via pollen tube pathway. Agronomy 12:537–544. https://doi.org/10.1051/agro:19920705
Mehta R, Radhakrishnan T, Kumar A, Yadav R, Dobaria JR, Thirumalaisamy PP, Jain RK, Chigurupati P (2013) Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation. Indian J Virol 24:205–213. https://doi.org/10.1007/s13337-013-0157-9
Ozias-Akins P, Schnall JA, Anderson WF, Singsit C, Clemente TE, Adang MJ, Weissinger AK (1993) Regeneration of transgenic peanut plants from stably transformed embryogenic callus. Plant Sci 93:185–194. https://doi.org/10.1016/0168-9452(93)90048-5
Pilaisangsuree V, Anuwan P, Supdensong K, Lumpa P, Kongbangkerd A, Limmongkon A (2020) Enhancement of adaptive response in peanut hairy root by exogenous signalling molecules under cadmium stress. J Plant Physiol 254:153278. https://doi.org/10.1016/j.jplph.2020.153278
Prasad K, Bhatnagar-Mathur P, Waliyar F, Sharma KK (2013) Overexpression of a chitinase gene in transgenic peanut confers enhanced resistance to major soil borne and foliar fungal pathogens. J Plant Biochem Biotechnol 22:222–233. https://doi.org/10.1007/s13562-012-0155-9
Pruthvi V, Narasimhan R, Nataraja KN (2014) Simultaneous expression of abiotic stress responsive transcription factors, atdreb2a, athb7 and atabf3 improves salinity and drought tolerance in peanut (Arachis hypogaea L.). Plos One 9 (12): e111152. https://doi.org/10.1371/journal.pone.0111152
Rohini VK, Sankara Rao K (2000) Transformation of peanut (Arachis hypogaea L.): a non-tissue culture-based approach for generating transgenic plants. Plant Sci 150(1):41–49. https://doi.org/10.1016/S0168-9452(99)00160-0
Rohini VK, Sankara Rao K (2001) Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease. Plant Sci 160:883–892. https://doi.org/10.1016/S0168-9452(00)00462-3
Shou HX, Palmer RG, Wang K (2002) Irreproducibility of the soybean pollen-tube pathway transformation procedure. Plant Mol Biol Rep 20:325–334. https://doi.org/10.1007/BF02772120
Tiwari S, Mishra DK, Singh A, Singh PK, Tuli R (2008) Expression of a synthetic cry1ec gene for resistance against Spodoptera litura in transgenic peanut (Arachis hypogaea L.). Plant Cell Rep 27(6):1017–1025. https://doi.org/10.1007/s00299-008-0525-x
Tuttle J, Haigler CH, Robertson D (2012) Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system. Plant Methods 8(1):27–27. https://doi.org/10.1186/1746-4811-8-27
Wang M, Zhang BH, Wang QL (2013) Cotton transformation via pollen tube pathway. Methods Mol Biol 958(958):71–77. https://doi.org/10.1007/978-1-62703-212-4-6
Yang A, Su Q, An L, Liu J, Wu W, Qiu Z (2009) Detection of vector- and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway. J Biotechnol 139:1–5. https://doi.org/10.1016/j.jbiotec.2008.08.012
Yang S, Li G, Li M, Wang J (2011) Transgenic soybean with low phytate content constructed by Agrobacterium transformation and pollen-tube pathway. Euphytica 177:375382. https://doi.org/10.1007/s10681-010-0262-4
Yao SC, Zhan J, Pan CL, Xiong WJ, Xiao D, Wang YL, Shen H, Wang AQ, He LF (2019) Identification and validation of reference genes for real-time qPCR normalization during Al-induced programmed cell death in peanut. Biol Plant 63:237–246. https://doi.org/10.32615/bp.2019.027
Yuan M, Zhu J, Gong L, He L, Lee C, Han S, Chen C, He G (2019) Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing. BMC Biotechnol 19:24. https://doi.org/10.1186/s12896-019-0516-8
Zhan J, He HY, Wang TJ, Wang AQ, Li CZ, He LF (2013) Aluminum-induced programmed cell death promoted by ahsag, a senescence-associated gene in Arachis hypoganea L. Plant Sci 210:108–117. https://doi.org/10.1016/j.plantsci.2013.05.012
Zhang YS, Yin XY, Yang AF, Li GS, Zhang JR (2005) Stability of inheritance of transgenes in maize (Zea mays L.) lines produced using different transformation methods. Euphytica 144:11–22. https://doi.org/10.1007/s10681-005-4560-1
Zheng QS, Ju B, Liang LK, Xiao XH (2005) Effects of antioxidants on the plant regeneration and GUS expressive frequency of peanut (Arachis hypogaea) explants by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 81:83–90. https://doi.org/10.1007/s11240-004-3176-9
Zhou G, Weng J, Zheng Y, Huang J, Qian S, Liu G (1983) Introduction of exogenous DNA into cotton embryos. Methods Enzymol 101:433–481. https://doi.org/10.1007/BF02909705
Zhuang W, Chen H, Yang M, Wang J, Pandey MK, Zhang C, Chang WC, Zhang L, Zhang X, Tang R, Garg V, Wang X, Tang H, Chow CN, Wang J, Deng Y, Wang D, Khan AW, Yang Q, Cai T, Bajaj P, Wu K, Guo B, Zhang X, Li J, Liang F, Hu J, Liao B, Liu S, Chitikineni A, Yan H, Zheng Y, Shan S, Liu Q, Xie D, Wang Z, Khan SA, Ali N, Zhao C, Li X, Luo Z, Zhang S, Zhuang R, Peng Z, Wang S, Mamadou G, Zhuang Y, Zhao Z, Yu W, Xiong F, Quan W, Yuan M, Li Y, Zou H, Xia H, Zha L, Fan J, Yu J, Xie W, Yuan J, Chen K, Zhao S, Chu W, Chen Y, Sun P, Meng F, Zhuo T, Zhao Y, Li C, He G, Zhao Y, Wang C, Kavikishor PB, Pan RL, Paterson AH, Wang X, Ming R, Varshney RK (2021) The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication. Nat Genet 51:865–876. https://doi.org/10.1038/s41588-019-0402-2
Acknowledgements
This work was financially supported by the Guangxi Natural Science Foundation (Grant No. 2022GXNSFAA035437), Central Guidance for Local Science and Technology Development Projects (Grant No. ZY22096013), the National Natural Science Foundation of China (Grant No. 31776190), and Training Program for 1000 Young and Middle-aged Key Teachers in Guangxi at 2019.
Author information
Authors and Affiliations
Contributions
JZ conceived the experiments and revised this manuscript; MZ designed the experiments and wrote the manuscript; MZ and JL performed the data analysis; MZ, LJ, AQW, DX, LFH and JZ analyzed the data. All authors have read and agreed to the published version of the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflicts of interest.
Additional information
Communicated by Sergio J. Ochatt.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zhou, M., Luo, J., Xiao, D. et al. An efficient method for the production of transgenic peanut plants by pollen tube transformation mediated by Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 152, 207–214 (2023). https://doi.org/10.1007/s11240-022-02388-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-022-02388-0