Abstract
The feasibility of map-based cloning in wheat has been demonstrated recently, opening new perspectives for a better understanding of wheat plant biology and for accelerating wheat improvement in the coming decades. To validate the function of candidate genes, an efficient transformation system is needed. Here, we have performed two methods for wheat transformation using particle bombardment that ensures the production of transgenic plants with simple integration patterns for research purposes and stable transgene expression for accurate and rapid validation of gene function. To establish this method, we used the bar and pmi selectable genes either as part of whole plasmids, gene cassettes (obtained by PCR or purified on agarose gels), or as dephosphorylated cassettes. The analysis of about 300 transgenic plants showed that the use of gene cassettes or dephosphorylated gene cassettes leads to a majority (50–60 %) of simple integration events. This is significantly higher than the number of simple events obtained with whole plasmids (9–25 %). Moreover, the decrease of the quantity of DNA from 500 to 5 ng/µl for PCR-amplified cassettes used for transformation increased the number of single integration events. The transformation efficiency remained stable at 2.5 %, and a higher number of plants expressing the transgenes were obtained with the dephosphorylated cassette. No correlation was observed between the complexity of the events and stability of expression of the transgene, suggesting that plasmid sequences could be involved on transgene silencing. The inheritability of the transgene was demonstrated in T1 and T2 generations. These results show that biolistic transformation of dephosphorylated gene cassettes provides an easy and efficient route to produce backbone vector-free transgenic wheat carrying and expressing intact and single transgenes.
Similar content being viewed by others
References
Agrawal PK, Kohli A, Twyman RM, Christou P (2005) Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels. Mol Breed 16:247–260
Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL, Simpson MA, Cao Z, Caroll C, Pawelczak KS, Blue R, West K, Rowland LM, Perkins D, Samuel P, Dewes CM, Shen L, Sriram S, Evans SL, Rebar EJ, Zhang L, Gregory PD, Urnov FD, Webb SR, Petolino JF (2013) Trait stacking via targeted genome editing. Plant Biotechnol J 11:1126–1134
Alfares W, Bouguennec A, Balfourier F, Gay G, Bergès H, Vautrin S, Sourdille P, Bernard M, Feuillet C (2009) Fine mapping and marker development for the crossability gene Skr on chromosome 5BS of hexaploid wheat (Triticum aestivum L.). Genetics 183(2):469–481
Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta S, Dix PJ, Fauquet C, Huang N, Kohli A, Mooibroek H, Nicholson L, Nguyen TT, Nugent G, Raemakers K, Romano A, Somers DA, Stoger E, Taylor N, Visser R (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breed 15:305–327
Anand A, Trick HN, Gill BS, Muthukrishnan S (2003) Stable transgene expression and random gene silencing in wheat. Plant Biotechnol J 1:241–251
Barret P, Delourme R, Renard M, Domergue F, Lessire R, Delseny M, Roscoe TJ (1998) A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid. Theor Appl Genet 96:177–186
Bhalla PL, Ottenhof HH, Singh MB (2006) Wheat transformation—an update of recent progress. Euphytica 149:353–366
Breitler JC, Labeyrie A, Meynard D, Legavre T, Guiderdoni E (2002) Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes. Theor Appl Genet 104:709–719
Camargo CEdeO, Neto AT, Filho AWPF, Felicio JC (2000) Genetic control of aluminium tolerance in mutant lines of the wheat cultivar Anahuac. Euphytica 114:47–53
Chen ZY, Yant SR, He CY, Meuse L, Shen S, Kay MA (2001) Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 3:403–410
Chen ZY, He CY, Meuse L, Kay MA (2004) Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo. Gene Ther 11:856–864
Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980
Christensen AH, Quail PH (1996) Ubiquitin promoter based vectors for high level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218
De Buck S, Windels P, De Loose M, Depicker A (2004) Single copy of T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable beta-glucuronidase accumulation levels. Cell Mol Life Sci 61:2632–2645
Demeke T, Huci P, Baga M, Caswell K, Leung N, Chibbar RN (1999) Transgene inheritance and silencing in hexaploid spring wheat. Theor Appl Genet 99:947–953
Fang YD, Akula C, Alpeter F (2002) Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA: genomic DNA junctions. J Plant Physiol 159:1131–1138
Feuillet C, Travella S, Stein N, Albar L, Nublat A, Keller B (2003) Map-based isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome. Proc Natl Acad Sci USA 100:15253–15258
Fu X, Duc LT, Fontana S, Bong BB, Tinjuangjun P, Sudhakar D, Twyman RM, Christou P, Kohli A (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res 9:11–19
Gupta PK, Varshney RK (2004) Cereal genomics: an overview. In: Gupta PK, Varshney RK (eds) Cereal genomics. Kluwer Academic Publishers, Netherlands, pp 1–18
Hammond SM, Caudy AA, Hannon GJ (2001) Post transcriptional gene silencing double stranded RNA. Nat Rev Genet 2:110–119
Hardwood WA (2012) Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot 63(5):1791–1798
He DG, Mouradov A, Yang YM, Mouradeva E, Scott KJ (1994) Transformation of wheat (Triticum eastivum L.) through electroporation of protoplasts. Plant Cell Rep 14:192–196
Holme IB, Wendt T, Holm PB (2013) Intragenesis and cisgenesis as alternatives to transgenic crop development. Plant Biotechnol J. doi:10.1111/pbi.12055
Howarth JR, Jacquet JN, Doherty A, Jones H, Cannell ME (2005) Molecular genetic analysis of silencing in two lines of Triticum aestivum transformed with the reporter gene construct pAHC25. Ann Appl Biol 146:311–320
Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F, Fry J (2003) Agrobacterium-mediated transformation large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21:1010–1019
Huang L, Brooks SA, Li WL, Fellers JP, Trick HN, Gill BS (2003) Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploidy genome of bread wheat. Genetics 164:655–664
Jagga-Chugh S, Kachhawaha S, Sharma M, Kothari-Chajer A, Kothari SL (2012) Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn. Plant Cell, Tissue Organ Cult 109:401–410
Khanna HK, Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436
Kim S-R, An G (2012) Bacterial transposons are co-transferred with T-DNA to rice chromosomes during Agrobacterium-mediated transformation. Mol Cells 33:583–589
Kim JY, Gallo M, Alpeter F (2012) Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (Saccharum spp. Hybrids). Plant Cell, Tissue Organ Cult 108:297–302
Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95:7203–7208
Lange M, Vincze E, Moller MG, Holm PB (2006) Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation. Plant Cell Rep 25:815–820
Loc NT, Tinjuangjun P, Gatehouse AMR, Christou P, Gatehouse JA (2002) Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance. Mol Breed 9:231–244
Lowe BA, Prakash NS, Way M, Mann MT, Spencer TM, Boddupalli RS (2009) Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA. Transgenic Res 18:831–840
Makarevitch I, Svitashev SK, Somers DA (2003) Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment. Plant Mol Biol 52:421–432
Marone D, Russo MA, Laido G, De Vita P, Papa R, Blanco A, Gadaleta A, Rubiales D, Mastrangelo A (2013) Genetic basis of qualitative and quantitative resistance to powdery mildew in wheat: from consensus regions to candidate genes. BMC Genom 14:562
Nandadeva YL, Lupi CG, Meyer CS, Devi PS, Potrykus I, Bilang R (1999) Microprojectile-mediated transient and integrative transformation of rice embryogenic suspension cells: effects of osmotic cell conditioning and of the physical configuration of plasmid DNA. Plant Cell Rep 18:500–504
Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Conesa DP, Ros G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci USA 106(19):7762–7767
Pellegrineschi A, Noguera LM, Skovmand B, Brito RM, Velazquez L, Salgado MM, Hernandez R, Warburton M, Hoisington D (2002) Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Genome 45:421–430
Popelka JC, Altpeter F (2003) Agrobacterium tumefasciens-mediated genetic transformation of rye (Secale cereale L.). Mol Breed 11:203–211
Przetakiewicz A, Karas A, Orczyk W, Nadolska-Orczyk A (2004) Agrobacterium-mediated transformation of polyploidy cereals, the efficiency of selection and transgene expression in wheat. Cell Mol Biol Lett 9:903–917
Rasco-Gaunt S, Riley A, Cannell M, Barcelo P, Lazzeri PA (2001) Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment. J Exp Bot 52:461–473
Romano A, Raemakers K, Bernardi J, Visser R, Mooibroek H (2003) Transgene organisation in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes. Transgenic Res 12:461–473
Russel Kikkert J (1993) The Biolistic® PDS-1000/He device. Plant Cell, Tissue Organ Cult 33:221–226
Sanjurjo L, Vidal JR, Segura A, de la Torre F (2013) Genetic transformation of grapevine cells using the minimal cassette technology: the need of 3′-end protection. J Biotechnol 163:386–390
Schouten HJ, Jacobsen E (2008) Cisgenesis and intragenesis, sisters in innovative plant breeding. Trends Plant Sci 13:260–261
Schouten HJ, Krens FA, Jacobsen E (2006) Cisgenic plants are similar to traditionally bred plants: international regulations for genetically modified organisms should be altered to exempt cisgenesis. EMBO Rep 7:750–753
Scofield SR, Huang L, Brandt AS, Gill BS (2005) Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138:2165–2173
Shou H, Frame BR, Whitham SA, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208
Simmonds J, Stewart P, Simmonds D (1992) Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA. Physiol Plant 85:197–206
Simons KJ, Fellers JP, Trick HN, Zhang Z, Tai YS, Gill BS, Faris JD (2006) Molecular characterization of the major wheat domestication gene Q. Genetics 172:547–555
Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D (2004) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23:75–81
Sorokin AP, Ke X, Chen D, Elliott MC (2000) Production of fertile transgenic wheat plants via tissue electroporation. Plant Sci 156:227–233
Sparks CA, Jones HD (2009) Biolistic transformation of wheat. In: Jones HD, Shewry PR (eds) Humana Press. Methods in molecular biology, transgenic wheat, barley and oats, vol 478, ch 4, pp 71–92
Srivastava V, Anderson OD, Ow DW (1999) Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA 96:11117–11121
Stoger E, Williams S, Keen D, Christou P (1998) Molecular characteristics of transgenic wheat and the effect on transgene expression. Transgenic Res 7:463–471
Stokstad E (2004) Monsanto pulls the plug on genetically modified wheat. Science 304:1088–1089
Stoykova P, Stoeva-Popova P (2011) PMI (manA) as a nonantibiotic selectable marker gene in plant biotechnology. Plant Cell, Tissue Organ Cult 105:141–148
Takumi S, Murai K, Mori N, Nakamura C (1999) Trans-activation of a maize Ds transposable element in transgenic wheat plants expressing the Ac transposase gene. Theor Appl Genet 98:947–953
Taparia Y, Fouad WM, Gallo M, Altpeter F (2012) Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis. In Vitro Cell Dev Biol Plant 48:15–22
Tassy C, Feuillet C, Barret P (2006) A method for the mid-term storage of plant tissue samples at room temperature and successive cycles of DNA extraction. Plant Mol Biol Rep 24:247a–247f
Torney F, Partier A, Says-Lesage V, Nadaud I, Barret P, Beckert M (2004) Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize adh-1 matrix attachment regions: tissue and developmental specificity in maize transgenic plants. Plant Cell Rep 22:931–938
Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789
Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J (2006) A NAC regulating senescence improves grain protein zinc, and iron content in wheat. Science 314:1298–1301
Ulker B, Li Y, Rosso MG, Logemann E, Somssich IE, Weisshaar B (2008) T-DNA-mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants. Nat Biotechnol 26:1015–1017
Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology 10:667–674
Vidal JR, Kikkert JR, Donzelli BD, Wallace PG, Reisch BI (2006) Biolistic transformation of grapevine using minimal gene cassette technology. Plant Cell Rep 25:807–814
Weeks JT, Anderson OD, Blechl AE (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol 102:1077–1084
Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim-Moore L (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20:429–436
Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659–668
Wu H, Sparks CA, Jones HD (2006) Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation. Mol Breed 18:195–208
Yahiaoui N, Srichumpa P, Dudler R, Keller B (2004) Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat. Plant J 37:528–538
Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J (2003) Positional cloning of the wheat vernalization gene VRN1. Proc Natl Acad Sci USA 100:6263–6368
Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M, Sanchez A, Valarik M, Yasuda S, Dubcovsky J (2006) The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc Natl Acad Sci USA 103:19581–19586
Yao Q, Cong L, Chang JL, Li KX, Yang GX, He GY (2006) Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. J Exp Bot 57:3737–3746
Yao Q, Cong L, He G, Chang J, Li K, Yang G (2007) Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency. Mol Biol Rep 34:61–67
Zhu Z, Sun B, Liu C, Xiao G, Li X (1993) Transformation of wheat protoplasts mediated by cationic liposome and regeneration of transgenic plantlets. Chin J Biotechnol 9:257–261
Acknowledgments
This work was supported by INRA Innovation Projects grants and by the program Investments for the Future (Grant ANR-11-BTBR-0006-GENIUS) managed by the French National Research Agency. We are grateful to Francois Torney and Natasha Glover for critical reading of the manuscript. We thank Stephane Benedit for his help in the DNA extractions. Special thanks to the greenhouse team for taking care of the plants.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Tassy, C., Partier, A., Beckert, M. et al. Biolistic transformation of wheat: increased production of plants with simple insertions and heritable transgene expression. Plant Cell Tiss Organ Cult 119, 171–181 (2014). https://doi.org/10.1007/s11240-014-0524-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-014-0524-2