Abstract
Selectable markers help the transformed cell/tissue to survive in an otherwise lethal exposure to an antibiotic or herbicide. Unfortunately, almost all the traditional selectable markers are antibiotic and herbicide resistance genes, which are controversial on human health concerns and environmental impact. Novel plant-derived, non-antibiotic, and non-herbicide selectable markers are urgently needed in plant transformation. Our previous work showed that the seedlings of overexpression Arabidopsis lines of AtGASA6 survived on medium with a high concentration of sugar, which leads to the hypothesis that AtGASA6 could be a selectable marker on media with high or low sugar content. In this study, leaf explants of AtGASA6 overexpression tobacco lines regenerated shoots on sugar-free shooting medium while those of wild type could not. Moreover, the seeds of AtGASA6 overexpression tobacco lines germinated and grew into normal seedlings on sugar-free MS medium while those of WT could not. Attractively, no developmental defects were observed in AtGASA6 transgenic progenies. Using AtGASA6 as a selectable marker, overexpression tobacco lines of GAI, which restrains plant size, were created on sugar-free media. The GAI overexpression lines had a smaller plant size than that of control. Considering its plant-derived and non-antibiotic nature, GASA6 is promising to be used as a selectable marker in plant transformation.
Key message
This research identifies a novel non-antibiotic selectable marker GASA6 using sugar-free selection medium, which could address ecological concerns and food safety issues.
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Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
References
Ben-Nissan G, Lee JY, Borohov A, Weiss D (2004) GIP, a Petunia hybrida GA-induced cysteine-rich protein: a possible role in shoot elongation and transition to flowering. Plant J 37(2):229–238. https://doi.org/10.1046/j.1365-313X.2003.01950.x
Breyer D, Kopertekh L, Reheul D (2014) Alternatives to antibiotic resistance marker genes for in vitro selection of genetically modified plants: scientific developments, current use, operational access and biosafety considerations. Crit Rev Plant Sci 33(4):286–330. https://doi.org/10.1080/07352689.2013.870422
Chen IC, Thiruvengadam V, Lin WD, Chang HH, Hsu WH (2010) Lysine racemase: a novel non-antibiotic selectable marker for plant transformation. Plant Mol Biol 72(1–2):153–169. https://doi.org/10.1007/s11103-009-9558-y
Daniell H, Muthukumar B, Lee SB (2001) Marker tree transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr Genet 39(2):109–116. https://doi.org/10.1007/s002940100185
Deblock M, Debrouwer D, Tenning P (1989) Transformation of Brassica-napus and Brassica-oleracea using Agrobacterium-tumefaciens and the expression of the BAR and NEO genes in the transgenic plants. Plant Physiol 91(2):694–701. https://doi.org/10.1104/pp.91.2.694
Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 94(6):2117–2121. https://doi.org/10.1073/pnas.94.6.2117
Ebmeier A, Allison L, Cerutti H, Clemente T (2004) Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta 218(5):751–758. https://doi.org/10.1007/s00425-003-1129-x
Erikson O, Hertzberg M, Nasholm T (2004) A conditional marker gene allowing both positive and negative selection in plants. Nat Biotechnol 22(4):455–458. https://doi.org/10.1038/nbt946
Erikson O, Hertzberg M, Nasholm T (2005) The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation. Plant Mol Biol 57(3):425–433. https://doi.org/10.1007/s11103-004-7902-9
Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JS, Galluppi GR, Goldberg SB, Hoffmann NL, Woo SC (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80(15):4803–4807. https://doi.org/10.1073/pnas.80.15.4803
Gleave AP, Mitra DS, Mudge SR, Morris BAM (1999) Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol Biol 40(2):223–235. https://doi.org/10.1023/A:1006184221051
Goldsbrough AP, Lastrella CN, Yoder JI (1993) Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato. Nat Biotechnol 11(11):1286–1292. https://doi.org/10.1038/nbt1193-1286
Gonzali S, Loreti E, Solfanelli C, Novi G, Alpi A, Perata P (2006) Identification of sugar-modulated genes and evidence for in vivo sugar sensing in Arabidopsis. J Plant Res 119:115–123. https://doi.org/10.1007/s10265-005-0251-1
Haldrup A, Petersen SG, Okkels FT (1998) The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol Biol 37(2):287–296. https://doi.org/10.1023/A:1005910417789
Hattasch C, Hanke MV, Flachowsky H (2009) Preliminary results to establish the DAAO system as an alternative selection strategy on apple. Acta Hortic 814:267–272. https://doi.org/10.17660/ActaHortic.2009.814.39
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general-method for transferring genes into plants. Science 227(4691):1229–1231. https://doi.org/10.1126/science.227.4691.1229
Huang X, Zou X, Xu Z, Tang F, Shi J, Zheng E, Liu D, Moisyadi S, Urschitz J, Wu Z, Li Z (2020) Efficient deletion of LoxP-flanked selectable marker genes from the genome of transgenic pigs by an engineered Cre recombinase. Transgenic Res 29(3):307–319. https://doi.org/10.1007/s11248-020-00200-3
Joersbo M, Jorgensen K, Brunstedt J (2003) A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose: galactose-1-phosphate uridyltransferase gene as selective gene. Mol Breeding 11(4):315–323. https://doi.org/10.1023/A:1023402424215
Klaus SMJ, Huang FC, Golds TJ, Koop HU (2004) Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat Biotechnol 22(2):225–229. https://doi.org/10.1038/nbt933
Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10(1):165–174. https://doi.org/10.1046/j.1365-313X.1996.10010165.x
LaFayette PR, Kane PM, Phan BH, Parrott WA (2005) Arabitol dehydrogenase as a selectable marker for rice. Plant Cell Rep 24(10):596–602. https://doi.org/10.1007/s00299-005-0015-3
Lai FM, Privalle L, Mei KF, Ghoshal D, Shen YW, Klucinec J, Daeschner K, Mankin LS, Chen N, Cho SH, Jones T (2011) Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation. In Vitro Cell Dev Biol Plant 47(4):467–479. https://doi.org/10.1007/s11627-011-9351-x
Li CW, Lee SH, Chan MT (2013) Utilization of the plant methionine sulfoxide reductase B genes as selectable markers in Arabidopsis and tomato transformation. Plant Cell Tiss Org 113(3):555–563. https://doi.org/10.1007/s11240-013-0296-0
Lindsey K, Gallois P (1990) Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens. J Exp Bot 41(226):529–536. https://doi.org/10.1093/jxb/41.5.529
Liu ZH, Zhu L, Shi HY, Chen Y, Zhang JM, Zheng Y, Li XB (2013) Cotton GASL genes encoding putative gibberellin-regulated proteins are involved in response to GA signaling in fiber development. Mol Biol Rep 40(7):4561–4570. https://doi.org/10.1007/s11033-013-2543-1
Moyano-Canete E, Bellido ML, Garcia-Caparros N, Medina-Puche L, Amil-Ruiz F, Gonzalez-Reyes JA, Caballero JL, Munoz-Blanco J, Blanco-Portales R (2013) FaGAST2, a strawberry ripening-related gene, acts together with FaGAST1 to determine cell size of the fruit receptacle. Plant Cell Physiol 54(2):218–236. https://doi.org/10.1093/pcp/pcs167
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Ray K, Jagannath A, Gangwani SA, Burma PK, Pental D (2004) Mutant acetolactate synthase gene is an efficient in vitro selectable marker for the genetic transformation of Brassica juncea (oilseed mustard). J Plant Physiol 161(9):1079–1083. https://doi.org/10.1016/j.jplph.2004.02.001
Roxrud I, Lid SE, Fletcher JC, Schmidt EDL, Opsahl-Sorteberg HG (2007) GASA4, one of the 14-member Arahidopsis GASA family of small polypeptides, regulates flowering and seed development. Plant Cell Physiol 48(3):471–483. https://doi.org/10.1093/pcp/pcm016
Rubinovich L, Weiss D (2010) The Arabidopsis cysteine-rich protein GASA4 promotes GA responses and exhibits redox activity in bacteria and in planta. Plant J 64(6):1018–1027. https://doi.org/10.1111/j.1365-313X.2010.04390.x
Sun SL, Wang HX, Yu HM, Zhong CM, Zhang XX, Peng JZ, Wang XJ (2013) GASA14 regulates leaf expansion and abiotic stress resistance by modulating reactive oxygen species accumulation. J Exp Bot 64(6):1637–1647. https://doi.org/10.1093/jxb/ert021
Taylor BH, Scheuring CF (1994) A molecular marker for lateral root initiation: The RSI-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia. Mol Gen Genet 243(2):148–157. https://doi.org/10.1007/Bf00280311
Waldron C, Murphy EB, Roberts JL, Gustafson GD, Armour SL, Malcolm SK (1985) Resistance to hygromycin B: a new marker for plant transformation studies. Plant Mol Biol 5(2):103–108. https://doi.org/10.1007/Bf00020092
Wienstroer J, Engqvist MKM, Kunz HH, Flugge UI, Maurino VG (2012) D-Lactate dehydrogenase as a marker gene allows positive selection of transgenic plants. FEBS Lett 586(1):36–40. https://doi.org/10.1016/j.febslet.2011.11.020
Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Nat Biotechnol 12(3):263–267. https://doi.org/10.1038/nbt0394-263
Yu H, Khalid MHB, Lu F, Sun F, Qu J, Liu B, Li W, Fu F (2019) Isolation and identification of a vegetative organ-specific promoter from maize. Physiol Mol Biol Plants 25(1):277–287. https://doi.org/10.1007/s12298-018-0546-z
Zhang SC, Yang CW, Peng JZ, Sun SL, Wang XJ (2009) GASA5, a regulator of flowering time and stem growth in Arabidopsis thaliana. Plant Mol Biol 69(6):745–759. https://doi.org/10.1007/s11103-009-9452-7
Zhao Y, Kim JY, Karan R, Jung JH, Pathak B, Williamson B, Kannan B, Wang D, Fan C, Yu W, Dong S, Srivastava V, Altpeter F (2019) Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane. Plant Mol Biol 100(3):247–263. https://doi.org/10.1007/s11103-019-00856-4
Zhong C, Xu H, Ye S, Wang S, Li L, Zhang S, Wang X (2015) Gibberellic Acid-Stimulated Arabidopsis6 serves as an integrator of gibberellin, abscisic acid, and glucose signaling during seed germination in Arabidopsis. Plant Physiol 169(3):2288–2303. https://doi.org/10.1104/pp.15.00858
Zimmermann R, Sakai H, Hochholdinger F (2010) The Gibberellic Acid Stimulated-Like gene family in maize and its role in lateral root development. Plant Physiol 152(1):356–365. https://doi.org/10.1104/pp.109.149054
Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18(4):442–445. https://doi.org/10.1038/74515
Zuo JR, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol 19(2):157–161. https://doi.org/10.1038/84428
Acknowledgements
This work is supported by the National Natural Science Foundation of China (31870301, 31370350 for S.Z). We thank Dr. Guangbin Luo (University of Copenhagen, Denmark) for polishing the manuscript.
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SZ, XW, and YL designed experiments. YL, JG performed experiments. YL, JG, SZ, and XW performed data analysis. YL wrote the manuscript. All authors approved the submission.
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Luo, Y., Gu, J., Wang, X. et al. A novel non-antibiotic selectable marker GASA6 for plant transformation. Plant Cell Tiss Organ Cult 148, 533–544 (2022). https://doi.org/10.1007/s11240-021-02204-1
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DOI: https://doi.org/10.1007/s11240-021-02204-1