Abstract
Rye is extremely recalcitrant to tissue culture and genetic transformation. We describe the efficient and reproducible production of stably expressing transgenic rye plants after biolistic gene transfer to callus tissue derived from immature embryos. Key factors in the production of transgenic rye plants include the identification of biolistic gene transfer parameters and a selection protocol, which does not affect its regeneration ability. The bar gene was used as a selectable marker and selection was performed by spraying the regenerated shoots with 0.05% Basta solution without any previous selection of tissue cultures. Based on Southern blot analysis, a total of 21 transgenic rye plants with independent transgene integration patterns were produced. A low transgene copy number was observed in most transgenic plants and 40% of the plants had a single transgene copy insert. The high frequency of single transgene copy inserts might be a consequence of the selection system, which is based on the identification of stably expressing transgenic plantlets rather than stably expressing tissue cultures. All transgenic rye lines with single transgene inserts showed stable transgene expression in sexual progenies, but indications of transcriptional and post-transcriptional gene silencing were observed in few transgenic lines with multiple transgene inserts. Tissue culture-based selection was not necessary for the generation of transgenic rye. The identification of 17 transgenic rye plants without using any selectable marker gene by PCR amplification of transgene sequences is also demonstrated. Instant generation of selectable marker-free transgenic rye avoids a negative impact of selective agents on the transgenic tissue cultures, responds to public concerns on the safety of selectable markers and will support multiple transformation cycles for transgene pyramiding.
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References
Altpeter F, Vasil V, Srivastava V, Stöger E and Vasil IK (1996) Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep 16: 12–17.
Armaleo D, Ye GN, Klein TM, Shark KB, Sanford JC and Johnston SA (1990) Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi. Curr Genet 17: 97–103.
Barro F, Cannell ME, Lazzeri PA and Barceló P (1998) The in-fluence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants. Theoret Appl Genet 97: 684–695.
Baulcombe DC and English JJ (1996) Ectopic pairing of homologous DNA and post-transcriptional gene silencing in transgenic plants. Curr Opin Biotechnol 7: 173–180.
Bilang R, Zhang S, Leduc N, Iglesias VA, Gisel A and Simmonds J (1993) Transient gene expression in vegetative shoot apical meristems of wheat after ballistic microtargeting. Plant J 4: 735–744.
Castillo AM, Vasil V and Vasil IK (1994) Rapid production of fertile transgenic plants of rye (Secale cereale L.). Bio/Technology 12: 1366–1371.
Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM and Duncan DR (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971–980.
Chibbar RN, Kartha KK, Leung N, Qureshi J and Caswell K (1991) Transient expression of marker genes in immature zygotic embryos of spring wheat (Triticum aestivum) through microparticle bombardment. Genome 34: 453–460.
Christou P and Ford T (1995) Parameters influencing stable transformation of rice immature embryos and recovery of transgenic plants using electric discharge particle acceleration. Ann Bot 75: 407–413.
Dale EC and Ow DW(1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88: 10558–10562.
De Framond AJ, Back EW, Chilton WS, Kayes L and Chilton MD (1986) Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet 202: 125–131.
Dellaporta SL, Wood J and Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 4: 19–21.
Depicker A, Herman L, Jacobs A, Schell J and Van Montagu M (1985) Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant interaction. Mol Gen Genet 201: 477–484.
Ebinuma H, Sugita K, Matsunaga E, Endo S, Yamada K and Komamine A (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20: 383–392.
Ebinuma H and Komamine A (2001) MAT (multi-autotransformation) vector system. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants. In vitro Cell Dev Biol Plant 37: 103–113.
Fang Y-D, Akula C and Altpeter 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.
Finer JJ, Vain P, Jones MW and McMullen MD (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11: 323–328.
Goldsbrough AP, Lastrella CN and Yoder JI (1993) Transposition mediated re-positioning and sequence elimination of marker genes from transgenic tomato. Bio/Technology 11: 1286–1292.
Hajdukiewicz P, Svab Z and Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25: 989–994
Iser M, Fettig S, Scheyhing F, Viertel K and Hess D (1999) Genotype-dependent stable genetic transformation in German spring wheat varieties selected for high regeneration potential. J Plant Physiol 154: 509–516.
Jackson SA, Zhang P, Chen WP, Phillips RL, Friebe B and Muthukrishnan S (2001) High-resolution structural analysis of biolistic transgene integration into the genome of wheat. Theoret Appl Genet 103: 56–62.
Kohli A, Leech M, Vain P, Laurie DA and Christou P (1998) Transgene integration 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.
Koprek T, Hänsch R, Nerlich A, Mendel RR and Schulze J (1996) Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response. Plant Sci 119: 79–91.
Li Z, Upadhyaya NM, Meena S, Gibbs AJ and Waterhouse PM (1997) Comparison of promoters and selectable marker genes for use in Indica rice transformation. Mol Breed 3: 1–14.
Matzke MA and Matzke AJM (1995) How and why do plants inactivate homologous (Trans)genes? Plant Physiol 107: 679–685.
Murashige T and Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15: 473–479.
Pawlowski WP and Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc Natl Acad Sci USA 95: 12106–12110.
Pederson C, Zimny J, Becker D, Jahne-Gartner A and Lorz H (1997) Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization. Theoret Appl Genet 94: 749–757.
Perl A, Kless H, Blumenthal A, Galili G and Galun E (1992) Improvement of plant regeneration and GUS expression in scutellar wheat by optimization of culture conditions and DNA-microparticle delivery procedures. Mol Gen Genet 235: 279–284.
Popelka JC and Altpeter F (2001) Interactions between genotypes and culture media components for improved in vitro response of rye (Secale cereale L.) inbred lines. Plant Cell Rep 20: 575–582.
Rasco-Gaunt S, Riley A, Barcelo P and Lazzeri PA (1999) Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues. Plant Cell Rep 19: 118–127.
Rasco-Gaunt S, Riley A, Cannell M, Barcelo P and 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: 865–874.
Sanford JC, De Vit MJ, Russel JA, Smith FD, Harpening PR and Roy MK (1991) An improved, helium-driven biolistic device. Technique 3: 3–16.
SAS Institute (1990) SAS/STAT Guide for Personal Computers, Version 6.03. SAS Institute, Inc., Cary, NC.
Shimada T (1978) Plant regeneration from the callus induced from wheat embryo. Jpn J Genet 53: 371–374.
Spencer TM, Gordon-Kamm WJ, Daines RJ, Start WG and Lemaux PG (1990) Bialophos selection of stable transformants from maize cell culture. Theoret Appl Genet 79: 625–631.
Stoger E, Williams S, Keen D and Christou P (1998) Molecular characterisation of transgenic wheat and the effect on transgene expression. Transgenic Res 7: 463–471.
Vain P, McMullen MD and Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12: 84–88.
Varshney A and Altpeter F (2001) Stable transformation and tissue culture response in current European winter wheats (Triticum aestivum L.). Mol Breed 8: 295–309.
Wuytswinkel Van O, Savino G and Briat JF (1995) Purification and characterisation of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro. Biochem J 305: 253–261.
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Popelka, J.C., Xu, J. & Altpeter, F. Generation of Rye (Secale cereale L.) Plants With Low Transgene Copy Number After Biolistic Gene Transfer and Production of Instantly Marker-Free Transgenic Rye. Transgenic Res 12, 587–596 (2003). https://doi.org/10.1023/A:1025822731661
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DOI: https://doi.org/10.1023/A:1025822731661