Abstract
Radish (Raphanus sativus) is a rich source of glucosinolates (GSLs) and their hydrolytic products such as isothiocyanates (ITCs). GSLs and ITCs enhance plant defense responses to biotic and abiotic stresses and are health promoting effect in human. The branched-chain aminotransferase 4 (BCAT4) gene encode an enzyme catalyzing the deamination of methionine in the first step in the chain elongation of aliphatic GSL biosynthesis. Previously, plant transformation in radish has been successfully performed using several methods such as floral dipping, vacuum infiltration and sonic infiltration, protoplast transformation and microspore culture. However, the recalcitrant of regeneration in radish affects the transformation efficiency remain relatively low. Therefore, there is still a need to improve the transformation methods for radish. In this study, we used a simple method for the efficient transformation of radish using Agrobacterium tumefaciens strain GV3101 and tested it with the radish BCAT4 (RsBCAT4) transgene. The PCR, RT-qPCR, Southern blot, GFP fluorescence, and HPLC analyses were used to confirm the transgene integration. Positive correlations between the expression of RsBCAT4 and downstream genes (i.e., CYP79F1, CYP83A1, and GRS1) were also observed in selected T2 transgenic lines. RsBCAT4 transgenic lines exhibited significantly increased levels of aliphatic GSLs compared to the levels in wild type plants, particularly glucoraphasatin. This needle perforation technique is simple in plant transformation method significantly enhancing transformation efficiency in radish, which could be utilized for molecular breeding of radish to improve its traits.
Key message
An efficient protocol for stable transformation of apical meristems of radish (Raphanus sativa L.) seedlings using a needle perforation and Agrobacterium tumefaciens incubation method. We developed a needle perforation and Agrobacterium tumefaciens incubation method for in planta transformation
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bae H, Kim YB, Il PN et al (2012) Agrobacterium rhizogenes-mediated genetic transformation of radish (Raphanus sativus L. Cv. Valentine) for accumulation of anthocyanin. Plant OMICS 5:381–385
Baenas N, Ferreres F, García-Viguera C, Moreno DA (2015) Radish sprouts-characterization and elicitation of novel varieties rich in anthocyanins. Food Res Int 69:305–312. https://doi.org/10.1016/j.foodres.2015.01.009
Barillari J, Iori R, Broccoli M et al (2007) Glucoraphasatin and glucoraphenin, a redox pair of glucosinolates of brassicaceae, differently affect metabolizing enzymes in rats. J Agricul Food Chem 55:5505–5511. https://doi.org/10.1021/jf070558r
Beaujean A, Sangwan RS, Lecardonnel A, Sangwan-Norreel BS (1998) Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. J Exp Bot 49:1589–1595. https://doi.org/10.1093/jxb/49.326.1589
Brown PD, Tokuhisa JG, Reichelt M, Gershenzon J (2003) Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 62:471–481. https://doi.org/10.1016/S0031-9422(02)00549-6
Burow M, Halkier BA (2017) How does a plant orchestrate defense in time and space? Using glucosinolates in Arabidopsis as case study. Curr Opin Plant Biol 38:142–147. https://doi.org/10.1016/j.pbi.2017.04.009
Cartea ME, Velasco P (2008) Glucosinolates in Brassica foods: bioavailability in food and significance for human health. Phytochem Rev 7:213–229. https://doi.org/10.1007/s11101-007-9072-2
Chhajed S, Mostafa I, He Y et al (2020) Glucosinolate biosynthesis and the glucosinolate-myrosinase system in plant defense. Agronomy 10:1786. https://doi.org/10.3390/agronomy10111786
Cho MA, Min SR, Ko SM et al (2008) Agrobacterium-mediated genetic transformation of radish (Raphanus sativus L.). Plant Biotechnol 25:205–208. https://doi.org/10.5511/plantbiotechnology.25.205
Ciska E, Honke J, Kozłowska H (2008) Effect of light conditions on the contents of glucosinolates in germinating seeds of white mustard, red radish, white radish, and rapeseed. J Agricult Food Chem 56:9087–9093. https://doi.org/10.1021/jf801206g
Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743. https://doi.org/10.1046/j.1365-313X.1998.00343.x
Curtis IS (2011) Genetic engineering of radish: current achievements and future goals. Plant Cell Rep 30:733–744. https://doi.org/10.1007/s00299-010-0978-6
Curtis IS, Nam HG (2001) Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method—plant development and surfactant are important in optimizing transformation efficiency. Transgenic Res 10:363–371. https://doi.org/10.1023/A:1016600517293
Curtis IS, Nam HG, Sakamoto K (2004) Optimized shoot regeneration system for the commercial Korean radish “Jin Ju Dae Pyong.” Plant Cell Tissue Organ Cult 77:81–87. https://doi.org/10.1023/B:TICU.0000016536.80238.ef
De Jonge J, Kodde J, Severing EI et al (2016) Low temperature affects stem cell maintenance in Brassica oleracea seedlings. Front Plant Sci 7:1–14. https://doi.org/10.3389/fpls.2016.00800
Fahey JW, Zalcmann AT, Talalay P (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates amoung plants. Phytochemistry 56:5–51. https://doi.org/10.1016/S0031-9422(00)00316-2
Fimognari C, Turrini E, Ferruzzi L et al (2012) Natural isothiocyanates: genotoxic potential versus chemoprevention. Mutat Res 750:107–131. https://doi.org/10.1016/j.mrrev.2011.12.001
Foster CSP (2016) The evolutionary history of flowering plants. J Proc R Soc N S W 149:65–82
Fu JJ, Liu J, Yang LY et al (2017) Effects of low temperature on seed germination, early seedling growth and antioxidant systems of the wild Elymus nutans Griseb. J Agricult Sci Technol 19:1113–1125
Gelvin SB (2003) Agrobacterium—mediated plant transformation: the biology behind the agrobacterium—mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37. https://doi.org/10.1128/MMBR.67.1.16
Gelvin SB (2017) Integration of agrobacterium T-DNA into the plant genome. Annu Rev Genet 51:195–217. https://doi.org/10.1146/annurev-genet-120215-035320
Hegazi HH, Matsubara S (1992) Callus formation and plant regeneration from protoplast derived from cotyledons and hypocotyls of radish (Raphanus sativus L.) and other cruciferous plants. Engei Gakkai zasshi 61:63–68. https://doi.org/10.2503/jjshs.61.63
Hwang H-H, Yu M, Lai E-M (2017) Agrobacterium—mediated plant transformation: biology and applications. Arabidopsis Book 15:e0186. https://doi.org/10.1199/tab.0186
Jeong WJ, Min SR, Liu JR (1995) Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.). Plant Cell Rep 14:648–651. https://doi.org/10.1007/BF00232731
Kakizaki T, Kitashiba H, Zou Z et al (2017) A 2-oxoglutarate-dependent dioxygenase mediates the biosynthesis of glucoraphasatin in Radish. Plant Physiol 173:1583–1593. https://doi.org/10.1104/pp.16.01814
Kitajima S, Miura K, Yasuda J (2020) Radish sprouts as an efficient and rapidly available host for an agroinfiltration-based transient gene expression system 92:89–92. https://doi.org/10.5511/plantbiotechnology.19.1216a
Kumar P, Augustine R, Singh AK, Bisht NC (2017) Feeding behaviour of generalist pests on Brassica juncea: implication for manipulation of glucosinolate biosynthesis pathway for enhanced resistance. Plant Cell Environ 40:2109–2120. https://doi.org/10.1111/pce.13009
Lacroix B, Citovsky V (2019) Pathways of DNA transfer to plants from agrobacterium tumefaciens and related bacterial species. Annu Rev Phytopathol 57:231–251. https://doi.org/10.1146/annurev-phyto-082718-100101
Li D, Wei X, Liu T et al (2020) Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida. J Microbiol Methods 169:105810. https://doi.org/10.1016/j.mimet.2019.105810
Lim S, Ahn JC, Lee EJ, Kim J (2020) Antiproliferation effect of sulforaphene isolated from radish (Raphanus sativus L.) seeds on A549 cells. Appl Biol Chem. https://doi.org/10.1186/s13765-020-00561-7
Liu W, Yang Y, Liu Q (2018) Establishment of an efficient regeneration system using heading leaves of Chinese cabbage (Brassica rapa L.) and its application in genetic transformation. Hortic Environ Biotechnol 59:583–596. https://doi.org/10.1007/s13580-018-0064-5
Matveeva TV, Lutova LA (2014) Horizontal gene transfer from agrobacterium to plants. Front Plant Sci 5:1–11. https://doi.org/10.3389/fpls.2014.00326
Mi AC, Sung RM, Suk MK et al (2008) Agrobacterium-mediated genetic transformation of radish (Raphanus sativus L.). Plant Biotechnol 25:205–208. https://doi.org/10.5511/plantbiotechnology.25.205
Murashige T, Skoog F (1962) Murashige1962Revised.Pdf. Physiol Plant 15:474–497
Nugroho ABD, Han N, Pervitasari AN et al (2019) Differential expression of major genes involved in the biosynthesis of aliphatic glucosinolates in intergeneric Baemoochae (Brassicaceae) and its parents during development. Plant Mol Biol. https://doi.org/10.1007/s11103-019-00939-2
Park BJ, Liu Z, Kanno A, Kameya T (2005) Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with agrobacterium harboring a group 3 LEA gene from B. napus. Plant Cell Rep 24:494–500. https://doi.org/10.1007/s00299-005-0973-5
Pua EC, Lee JEE (1995) Enhanced de novo shoot morphogenesis in vitro by expression of antisense 1- aminocyclopropane-1-carboxylate oxidase gene in transgenic mustard plants. Planta: Int J Plant Biol 196:69–76. https://doi.org/10.1007/BF00193219
Pua EC, Sim GE, Chi GL, Kong LF (1996) Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro. Plant Cell Reports 15:685–690. https://doi.org/10.1007/BF00231925
Rao KS, Sreevathsa R, Sharma PD et al (2008) In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro. Physiol Mol Biol Plants 14:321–328. https://doi.org/10.1007/s12298-008-0030-2
Rivera AL, Gómez-Lim M, Fernández F, Loske AM (2012) Physical methods for genetic plant transformation. Phys Life Rev 9:308–345. https://doi.org/10.1016/j.plrev.2012.06.002
Sasaki M, Nonoshita Y, Kajiya T et al (2020) Characteristic analysis of trigonelline contained in Raphanus sativus cv. Sakurajima daikon and results from the first trial examining its vasodilator properties in humans. Nutrients 12:1–11. https://doi.org/10.3390/nu12061872
Schuster J, Knill T, Reichelt M et al (2006) BRANCHED-CHAIN AMINOTRANSFERASE4 is part of the chain elongation pathway in the biosynthesis of methionine-derived glucosinolates in arabidopsis. Plant Cell 18:2664–2679. https://doi.org/10.1105/tpc.105.039339
Shah SH, Ali S, Jan SA et al (2015) Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.). Plant Cell Tissue Organ Cult 120:1139–1157. https://doi.org/10.1007/s11240-014-0670-6
Sønderby IE, Geu-Flores F, Halkier BA (2010) Biosynthesis of glucosinolates—gene discovery and beyond. Trends Plant Sci 15:283–290. https://doi.org/10.1016/j.tplants.2010.02.005
Supartana P, Shimizu T, Shioiri H et al (2005) Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using agrobacterium tumefaciens. J Biosci Bioeng 100:391–397. https://doi.org/10.1263/jbb.100.391
Takahata Y, Komatsu H, Kaizuma N (1996) Microspore culture of radish (Raphanus sativus L.): Influence of genotype and culture conditions on embryogenesis. Plant Cell Rep 16:163–166. https://doi.org/10.1007/s002990050198
Ting HM, Cheah BH, Chen YC et al (2020) The role of a glucosinolate-derived nitrile in plant immune responses. Front Plant Sci 11:1–18. https://doi.org/10.3389/fpls.2020.00257
Tobeh A, Jamaati-E-Somarin S (2012) Low temperature stress effect on wheat cultivars germination. Afr J Microbiol Res 6:1265–1269
Trieu AT, Burleigh SH, Kardailsky IV et al (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with agrobacterium. Plant J 22:531–541. https://doi.org/10.1046/j.1365-313X.2000.00757.x
Weigel D, Glazebrook J (2006) Transformation of agrobacterium using the freeze-thaw method. Cold Spring Harbor Protoc 2006:pdb.prot4666. https://doi.org/10.1101/PDB.PROT4666
Xu S, Lai E, Zhao L et al (2020) Development of a fast and efficient root transgenic system for functional genomics and genetic engineering in peach. Sci Rep 10:1–9. https://doi.org/10.1038/s41598-020-59626-8
Yasmeen A, Mirza B, Inayatullah S et al (2009) In planta transformation of tomato. Plant Mol Biol Rep 27:20–28. https://doi.org/10.1007/s11105-008-0044-5
Yi G, Lim S, Chae WB et al (2016) Root glucosinolate profiles for screening of radish (Raphanus sativus L.) genetic resources. J Agric Food Chem 64:61–70. https://doi.org/10.1021/acs.jafc.5b04575
Yu R, Xu L, Zhang W et al (2016) De novo taproot transcriptome sequencing and analysis of major genes involved in sucrose metabolism in radish (Raphanus sativus L.). Front Plant Sci 7:1–12. https://doi.org/10.3389/fpls.2016.00585
Zhang Y, Huai D, Yang Q et al (2015) Overexpression of three glucosinolate biosynthesis genes in Brassica napus identifies enhanced resistance to Sclerotinia sclerotiorum and Botrytis cinerea. PLoS ONE 10:1–17. https://doi.org/10.1371/journal.pone.0140491
Acknowledgements
This research was supported by the CAYSS Program of Chung-Ang University to A.N.P. and a research grant (Grant No. PJ01566203) from the Rural Development Administration (RDA) and a Grant (2021R1A5A1032428) from the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) to D-H. K.
Author information
Authors and Affiliations
Contributions
ANP and ABDN planted all plant materials and performed the molecular experiments; ANP and ABDN performed the UHPLC experiments. ANP, WHJ and JK planned the experiments; ANP, D-HK, and JK analyzed the data and wrote the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Goetz Hensel.
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
Pervitasari, A.N., Nugroho, A.B.D., Jung, W.H. et al. An efficient Agrobacterium tumefaciens-mediated transformation of apical meristem in radish (Raphanus sativus L.) using a needle perforation. Plant Cell Tiss Organ Cult 148, 305–318 (2022). https://doi.org/10.1007/s11240-021-02190-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-021-02190-4