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High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics

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Abstract

Fragaria vesca L., a diploid (2n=2x=14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6–7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l−1 hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14–15 weeks to complete its life cycle in different seasons in the greenhouse.

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Abbreviations

BA:

Benzyladenine

GFP:

Green fluorescent protein

IBA:

Indole-3-butyric acid

hpt :

Hygromycin phosphotransferase

Kn:

Kanamycin

References

  • Akiyama Y, Yamamoto Y, Ohmido N, Ohshima M, Fukui K (2001) Estimation of the nuclear DNA content of strawberries (Fragaria spp.) compared with Arabidopsis thaliana by using dual-step flow cytometry. Cytologia 66:431–436

    Google Scholar 

  • Alsheikh MK, Suso H-P, Robson M, Battey NH, Wetten A (2002) Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F.v. semperflorens. Plant Cell Rep 20:1173–1180

    Article  CAS  Google Scholar 

  • Barceló M, El-Mansouri I, Mercado JA, Quesada MA, Pliego-Alfaro F (1998) Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler. Plant Cell Tissue Organ Cult 54:29–36

    Article  Google Scholar 

  • Battey NH, Le Mière P, Tehranifar A, Cekic C, Taylor S, Shrives KJ, Hadley P, Greenland AJ, Darby J, Wilkinson MJ (1998) Genetic and environmental control of flowering in strawberry. In: Cockshull KE, Gray D, Seymour GB, Thomas B (eds) Genetic and environmental manipulation of horticultural crops. CAB Int., Wallingford, pp 111–131

    Google Scholar 

  • Brown T, Wareing PF (1965) The genetical control of the everbearing habit and three other characters in varieties of Fragaria vesca. Euphytica 14:97–112

    Google Scholar 

  • Christou P, Ford TL (1995) Recovery of chimeric rice plants from dry seed using electric discharge particle acceleration. Ann Bot 75:449–454

    Article  Google Scholar 

  • Davis TM, Yu H (1997) A linkage map of the diploid strawberry, Fragaria vesca. J Hered 88:215–221

    CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • El Mansouri I, Mercado JA, Valpuesta V, López-Aranda JM, Pliego-Alfaro F, Quesada MA (1996) Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L. Plant Cell Rep 15:642–646

    Article  CAS  Google Scholar 

  • Elliott AR, Campbell JA, Brettell RIS, Grof CPL (1998) Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker. Aust J Plant Physiol 25:739–743

    Article  CAS  Google Scholar 

  • Elliott AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL (1999) Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep 18:707–714

    Article  CAS  Google Scholar 

  • Firoozabady E, Moy Y, Tucker W, Robinson K, Gutterson N (1995) Efficient transformation and regeneration of carnation cultivars using Agrobacterium. Mol Breed 1:283–293

    Article  CAS  Google Scholar 

  • van der Fits L, Deakin EA, Hoge JHC, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol, 43:495–502

    Article  PubMed  Google Scholar 

  • Galletta GJ, Maas JL (1990) Strawberry genetics. HortScience 25:871–879

    Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  PubMed  CAS  Google Scholar 

  • Ghorbel R, Juárez J, Navarro L, Peña L (1999) Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. Theor Appl Genet 99:350–358

    Article  Google Scholar 

  • Given NK, Venis MA, Grierson D (1988) Hormonal regulation of ripening in the strawberry, a non-climacteric fruit. Planta 174:402–406

    Article  CAS  Google Scholar 

  • Halfhill MD, Richards HA, Mabon SA, Stewart CN Jr (2001) Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa. Theor Appl Genet 103:659–667

    Article  CAS  Google Scholar 

  • Haymes KM, Davis TM (1998) Agrobacterium-mediated transformation of ‘Alpine’ Fragaria vesca, and transmission of transgenes to R1 progeny. Plant Cell Rep 17:279–283

    Article  CAS  Google Scholar 

  • Hisano H, Kimoto Y, Hayakawa H, Takeichi J, Domae T, Hashimoto R, Abe J, Asano S, Kanazawa A, Shimamoto Y (2004) High frequency Agrobacterium-mediated transformation and plant regeneration via direct shoot formation from leaf explants in Beta vulgaris and Beta maritima. Plant Cell Rep 22:910–918

    Article  PubMed  CAS  Google Scholar 

  • Irish VF (1991) Cell lineage in plant development. Curr Opin Genet Dev 1:169–173

    Article  PubMed  CAS  Google Scholar 

  • James DJ, Passey AJ, Barbara DJ (1990) Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria × ananassa Duch) using disarmed binary vectors. Plant Sci 69:79–94

    Article  CAS  Google Scholar 

  • Jelenkovic G, Chin CK, Billings S (1986) Transformation studies of Fragaria × ananassa Duch. by Ti plasmid of Agrobacterium tumefaciens. HortScience 21:695

    Google Scholar 

  • Jordan MC (2000) Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Rep 19:1069–1075

    Article  CAS  Google Scholar 

  • Kaeppler HF, Menon GK, Skadsen RW, Nuutila AM, Carlson AR (2000a) Transgenic oat plants via visual selection of cells expressing green fluorescent protein. Plant Cell Rep 19:661–666

    Article  CAS  Google Scholar 

  • Kaeppler SM, Kaeppler HF, Rhee Y (2000b) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188

    Article  PubMed  CAS  Google Scholar 

  • Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396

    Article  CAS  Google Scholar 

  • Lodhi MA, Ye G-N, Weeden NF, Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Amelopsis. Plant Mol Biol Rep 12:6–13

    Article  CAS  Google Scholar 

  • Manning K (1993) Soft fruit. In: Seymour GB, Taylor JE, Tucher GA (eds) Biochemistry of fruit ripening. Chapman and Hall, London, pp 347–377

    Google Scholar 

  • Manning K (1998) Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits. Planta 205:622–631

    Article  PubMed  CAS  Google Scholar 

  • Mathews H, Dewey V, Wagoner W, Bestwick RK (1998) Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols. Transgenic Res 7:123–129

    Article  CAS  Google Scholar 

  • Mathews H, Wagoner W, Kellogg J, Bestwick R (1995) Genetic transformation of strawberry: stable integration of a gene to control biosynthesis of ethylene. In Vitro Cell Dev Biol 31:36–43

    Article  CAS  Google Scholar 

  • Mathur J, Szabados L, Schaefer S, Grunenberg B, Lossow A, Jonas-Straube E, Schell J, Koncz C, Koncz-Kálmán Z (1998) Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension. Plant J 13:707–716

    Article  PubMed  CAS  Google Scholar 

  • Molinier J, Himber C, Hahne G (2000) Use of green fluorescent protein for detection of transformed shoots and homozygous offspring. Plant Cell Rep 19:219–223

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murthy HN, Jeong JH, Choi YE, Paek KY (2003) Agrobacterium-mediated transformation of niger [Guizotia abyssinica (L. f.) Cass.] using seedling explants. Plant Cell Rep 21:1183–1187

    Article  PubMed  CAS  Google Scholar 

  • Nam Y-W, Tichit L, Leperlier M, Cuerq B, Marty I, Lelièvre J-M (1999) Isolation and characterization of mRNAs differentially expressed during ripening of wild strawberry (Fragaria vesca L.) fruits. Plant Mol Biol 39:629–636

    Article  PubMed  CAS  Google Scholar 

  • Nehra NS, Chibbar RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C (1990a) Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants. Plant Cell Rep 9:10–13

    CAS  Google Scholar 

  • Nehra NS, Chibbar RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C (1990b) Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Rep 9:293–298

    CAS  Google Scholar 

  • Nehra NS, Stushnoff C, Kartha KK (1989) Direct shoot regeneration from strawberry leaf disks. J Am Soc Hort Sci 114:1014–1018

    Google Scholar 

  • Olhoft PM, Phillips RL (1999) Genetic and epigenetic instability in tissue culture and regenerated progenies. In: Lerner HR (ed) Plant responses to environmental stresses: from phytohormones to genome reorganization. Marcel Dekker Inc., New York, pp 111–148

    Google Scholar 

  • Ontivero M, Arias M, Ricci JD, Babot J, Albornoz P, Castagnaro A (2000) Analysis of genetic similarities among species of Fragaria, Potentilla, and Duchesnea found in northwest Argentina by using morphological, anatomical, and molecular characters. Can J Bot 78:547–556

    Article  CAS  Google Scholar 

  • Passey AJ, Barrett KJ, James DJ (2003) Adventitious shoot regeneration from seven commercial strawberry cultivars (Fragaria × ananassa Duch.) using a range of explant types. Plant Cell Rep 21:397–401

    PubMed  CAS  Google Scholar 

  • Poethig S (1989) Genetic mosaics and cell lineage analysis in plants. Trends Genet 5:273–277

    Article  PubMed  CAS  Google Scholar 

  • Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15:8–15

    Article  CAS  Google Scholar 

  • Potter D, Luby JJ, Harrison RE (2000) Phylogenetic relationships among species of Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast DNA sequences. Syst Bot 25:337–348

    Article  Google Scholar 

  • Ricardo VG, Coll Y, Castagnaro A, Ricci JCD (2003) Transformation of a strawberry cultivar using a modified regeneration medium. HortScience 38:277–280

    Google Scholar 

  • Rugini E, Orlando R (1992) High efficiency shoot regeneration from calluses of strawberry (Fragaria × ananassa Duch.) stipules of in vitro shoot cultures. J Hort Sci 67:577–582

    Google Scholar 

  • Sargent DJ, Davis TM, Tobutt KR, Wilkinson MJ, Battey NH, Simpson DW (2004) A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria. Theor Appl Genet 109:1385–1391

    Article  PubMed  CAS  Google Scholar 

  • Sorvari S, Ulvinen S, Hietaranta T, Hiirsalmi H (1993) Preculture medium promotes direct shoot regeneration from micropropagated strawberry leaf disks. HortScience 28:55–57

    Google Scholar 

  • Stewart CN Jr (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep 20:376–382

    Article  PubMed  CAS  Google Scholar 

  • Tian L, Levée V, Mentag R, Charest PJ, Séguin A (1999) Green fluorescent protein as a tool for monitoring transgene expression in forest tree species. Tree Physiol 19:541–546

    PubMed  CAS  Google Scholar 

  • Wang Z-Y, Bell J, Lehmann D (2004) Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells. Plant Cell Rep 22:903–909

    PubMed  CAS  Google Scholar 

  • Zhao Y, Liu Q, Davis RE (2004) Transgene expression in strawberries driven by a heterologous phloem-specific promoter. Plant Cell Rep 23:224–230

    Article  PubMed  CAS  Google Scholar 

  • Zhang CL, Chen DF, McCormac AC, Scott NW, Elliott MC, Slater A (2001) Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.). Mol Biotechnol 17:109–117

    Article  PubMed  Google Scholar 

  • Zhu YJ, Agbayani R, Moore PH (2004) Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation. Plant Cell Rep 22:660–667

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This project was funded by a Virginia Tech ASPIRES grant. We thank Dr. Kim Hummer, Dr. Nalha Bassil, and Bruce Bartlett at the NCGR for providing seed and in vitro cultured material of F. vesca. We thank Scott Rapier for help with plant maintenance in the greenhouse. We thank Dr. Jerzy Nowak, Dr. Ron Mittler, and Jim Walke for critically reading the manuscript.

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Authors and Affiliations

  1. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Washington St, Blacksburg, VA, 24061, USA

    Teruko Oosumi, Hope A. Gruszewski, Leslie A. Blischak, Aaron J. Baxter, Joel L. Shuman & Vladimir Shulaev

  2. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

    Leslie A. Blischak, Aaron J. Baxter, Phillip A. Wadl, Joel L. Shuman, Richard E. Veilleux & Vladimir Shulaev

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  1. Teruko Oosumi
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  2. Hope A. Gruszewski
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  3. Leslie A. Blischak
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  4. Aaron J. Baxter
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  5. Phillip A. Wadl
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  7. Richard E. Veilleux
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  8. Vladimir Shulaev
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Correspondence to Vladimir Shulaev.

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Oosumi, T., Gruszewski, H.A., Blischak, L.A. et al. High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics. Planta 223, 1219–1230 (2006). https://doi.org/10.1007/s00425-005-0170-3

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  • DOI: https://doi.org/10.1007/s00425-005-0170-3

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