Abstract
The efficiency of cell-penetrating peptide (CPP)-mediated dsDNA transfection in triticale microspores was investigated through transient and stable integration of the β-glucoronidase (GUS) reporter gene and expression assays in microspore-derived embryos and plantlets. The RecA protein, usually associated with prokaryote homologous recombination, was also tested for its capacity to protect the linear transgene from degradation. Transfections mediated by the CPP nanocarriers Tat2 and Pep1 reduced the number of regenerated embryos from 158 in the control to 122 and 100, respectively. The co-delivery of CPP-dsDNA with RecA protein also resulted in fewer embryos, 87 and 104 for Tat2 and Pep1, respectively. Delivery of dsDNA with Tat2 or Pep1, without RecA, resulted in the highest frequencies of GUS activity in regenerated embryos, at 26%. Co-delivery with RecA decreased the percentage of GUS-positive embryos to 16%. Interestingly, co-delivered RecA-dsDNA reduced the loss of integrity of inserted genetic construct, as observed by polymerase chain reaction (PCR) amplification of the 5′ and 3′ ends. GUS activity was also detected in mature haploid and diploid plants. Of all treatments, 31 T0 plants tested positive for the GUS gene by quantitative PCR, although 50% were derived from the single treatment dsDNA-Tat2. The estimated copy number of the GUS transgene varied between four and eight. This study provides the foundations for CPP-mediated co-delivery of dsDNA and protein RecA in haploid microspore nuclei for functional genomic studies in crop species.
Similar content being viewed by others
References
Astriab-Fisher A, Sergueev D, Fisher M, Shaw BR, Juliano RL (2002) Conjugates of antisense oligonucleotides with the Tat and Antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences and biologic actions. Pharm Res 19:744–754
Biet E, Maurisse R, Dutreix M, Sun JS (2001) Stimulation of RecA-mediated D-loop formation by oligonucleotide-directed triple-helix formation: guided homologous recombination (GOREC). Biochemistry 40:1779–1786
Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F, Chauvin S, Bechtold N, Cruaud C, DeRose R, Pelletier G, Lepiniec L, Caboche M, Lecharny A (2002) T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Rep 3:1152–1157
Chen CP, Chou JC, Liu BR, Chang M, Lee HJ (2007) Transfection and expression of plasmid DNA in plant cells by arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Lett 581:1891–1897
Cho MJ, Yano H, Okamoto D, Kim HK, Jung HR, Newcomb K, Le VK, Yoo HS, Langham R, Buchanan BB, Lemaux PG (2004) Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed. Plant Cell Rep 22:483–489
Chugh A, Eudes F (2007) Translocation and nuclear accumulation of monomer and dimmer of HIV-1Tat basic domain in triticale mesophyll protoplasts. Biochim Biophys Acta 1768:419–426
Chugh A, Eudes F (2008a) Studies of uptake of cell-penetrating peptides and their cargoes in wheat immature embryos. FEBS J 275:2403–2414
Chugh A, Eudes F (2008b) Cellular uptake of cell-penetrating peptides pVEC and transportan in plants. J Pept Sci 14:477–481
Chugh A, Amundsen E, Eudes F (2009) Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores. Plant Cell Rep 28:801–810
Chugh A, Eudes F, Shim Y-S (2010) Cell-penetrating peptides: nanocarrier for macromolecule delivery in living cells. IUBMB Life 62:183–193
Clark AJ, Margulies AD (1965) Isolation and characterization of recombination-deficient mutants of Escherichia coli K12. Proc Natl Acad Sci 53:451–459
Doyle JJ, Dickson EE (1987) Preservation of plant samples for DNA restriction endonuclease analysis. Taxon 36:715–772
Eggenberger K, Birtalan E, Schröder T, Bräse S, Nick P (2009) Passage of Trojan peptides into plant cells. ChemBioChem 10:2504–2512
El-Andaloussi S, Johansson HJ, Holm T, Langel U (2007) A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids. Mol Theor 15:1820–1826
Eudes F, Acharya S, Laroche A, Selinger LB, Cheng K-J (2003) A novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants. Plant Cell Tiss Organ Cult 73:147–157
Eudes F, Amundsen E (2005) Isolated microspore culture of 6× triticale cultivars. Plant Cell Tiss Organ Cult 82:233–241
Folling L, Olesen A (2002) Genetic transformation and hybridization. Plant Cell Rep 20:1098–1105
Fominaya J, Gasset M, Garcia R, Roncal F, Albar JP, Bernad A (2000) An optimised amphiphilic cationic peptide as an efficient non-viral gene delivery vector. J Gene Med 2:455–464
Gardner RV, Voloshin ON, Camerini-Otero RD (1995) The identification of the single-stranded DNA-binding domain of the Escherichia coli RecA protein. Eur J Biochem 233:419–425
Haliloglu K, Baenziger PS (2003) Agrobacterium tumefaciens-mediated wheat transformation. Cereal Res Comm 31:9–16
Henriques ST, Costa J, Castanho MA (2005) Translocation of betagalactosidase mediated by cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential. Biochemistry 44:10189–10198
Heslop-Harrison JS (1992) Cytological techniques to assess pollen quality. In: Cresti M, Tiezzi A (eds) Sexual plant reproduction. Springer, Heidelberg, pp 41–48
Hoebeeck J, Speleman F, Vandesompele J (2007) Real-time quantitative PCR as an alternative to Southern blot or fluorescence in situ hybridization for detection of gene copy number changes. In: Hilario E, Mackay J (eds) Protocols for nucleic acid analysis by nonradioactive probes. Methods in molecular biology, vol 353. Humana, New York, pp 205–226
Jähne A, Becker D, Lörz H (1995) Genetic engineering of cereal plants: a review. Euphytica 85:35–44
Jones SW, Christison R, Bundell K, Voyce CJ, Brockbank SMV, Newham P, Lindsay MA (2005) Characterisation of cell-penetrating peptide-mediated peptide delivery. Brit J Pharmacol 145:1093–1102
Kumlehn J, Serazetdinova L, Hensel G, Becker D, Loerz H (2006) Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotech J 4:251–261
Lanzov VA (2007) RecA homologous DNA transferase: functional activities and a search for homology by recombining DNA molecules. Mol Biol 41:467–477
Leipe DD, Aravind L, Grishin NV, Koonin EV (2000) The bacterial replicate helicase DNAB evolved from a RecA duplication. Genome Res 10:5–16
Li W, Sun G, Liu J, Masilamany P, Taylor JH, Yan W, Kasha KJ, Pauls KP (2004) Inheritance of plant regeneration from maize (Zea mays L.) shoot meristem cultures derived from germinated seeds and the identification of associated RAPD and SSR markers. Theoret Appl Genet 108:681–687
Ogawa T, Yu X, Shinohara A, Engelman EH (1993) Similarity of the yeast Rad51 filaments to the bacterial RecA filament. Science 259:1896–1899
Rauth S, Song KY, Ayares D, Wallace L, Moore PD, Kucherlapati R (1986) Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts. Proc Natl Acad Sci 83:5587–5591
Reiss B, Klemm M, Kosak H, Schell J (1996) RecA protein stimulates homologous recombination in plants. Proc Natl Acad Sci 93:3094–3098
Reiss B, Schubert I, Köpchen K, Wendeler E, Schell J, Puchta H (2000) RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium. Proc Natl Acad Sci 97:3358–3363
Shim Y-S, Pauls KP, Kasha KJ (2009) Transformation of isolated barley (Hordeum vulgare L.) microspores: II. Timing of pretreatment and temperatures relative to results of bombardment. Genome 53:175–190
Tinland B (1996) The integration of T-DNA into plant genomes. Trends Plant Sci 1:178–184
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
Wender PA, Mitchell DJ, Pattabiraman ET, Pelkey ET, Steinman L, Rothbard JB (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 97:13003–13008
Weng H, Pan A, Yang L, Zhang C, Liu Z, Zhang D (2004) Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol Bio Rep 22:289–300
Zheng MY, Liu W, Weng Y, Polle E, Konzak CF (2001) Culture of freshly isolated wheat (Triticum aestivum L.) microspores treated with inducer chemicals. Plant Cell Rep 20:685–690
Ziemienowicz A, Shim YS, Matsuoka A, Eudes F, Kovalchuk I (2012) A novel method of transgene delivery into triticale plants using the Agrobacterium T-DNA-derived nano-complex. Plant Physiol. doi:10.1104/pp.111.192856
Acknowledgments
The authors thank Denise Nilsson and Eric Amundsen for their technical support and Alicja Ziemienowicz for the scientific discussions during the course of these experiments. The funding support from Alberta Agriculture Research Institute and Agriculture and Agri-Food Canada was instrumental, and both organizations are thanked.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: J. Forster
Rights and permissions
About this article
Cite this article
Shim, YS., Eudes, F. & Kovalchuk, I. dsDNA and protein co-delivery in triticale microspores. In Vitro Cell.Dev.Biol.-Plant 49, 156–165 (2013). https://doi.org/10.1007/s11627-012-9471-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11627-012-9471-y