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Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants

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Abstract

Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple.

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References

  • Bolar JP, Brown SK, Norelli JL, Aldwinckle HS (1999) Factors affecting the transformation of ‘Marshall McIntosh’ apple by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 55:31–38

    Article  Google Scholar 

  • Chang L, Zhang Z, Yang H, Li H, Dai H (2007) Detection of strawberry RNA and DNA viruses by RT-PCR using total nucleic acid as a template. J Phytopathol 155:431–436

    Article  CAS  Google Scholar 

  • Dai H, Zhang Z, Gao X, Wu L (2004) Establishment and optimization of micropropagation system of sweet cherry cultivars. J Fruit Sci 21:216–219

    Google Scholar 

  • Dai H, Zhang Z, Guo X (2007) Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.). In Vitro Cell Dev Biol Plant 43:2–8

    Google Scholar 

  • Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1–42

    Article  Google Scholar 

  • Ellul P, Garcia-Sogo B, Pineda B, Ríos G, Roig LA, Moreno V (2003) The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculentum Mill.) is genotype and procedure dependent. Theor Appl Genet 106:231–238

    CAS  PubMed  Google Scholar 

  • Flachowsky H, Peil A, Sopanen T, Elo A, Hanke V (2007) Overexpression of BpMADS4 from silver birch (Betula pendula Roth.) induces early-flowering in apple (Malus × domestica Borkh). Plant Breed 126:137–145

    Article  CAS  Google Scholar 

  • Guo D, Zhu Z, Hu X, Zheng S (2005) Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (Brassica juncea var. tsatsai). Plant Cell Tissue Organ Cult 83:123–127

    Article  CAS  Google Scholar 

  • James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector. Plant Cell Rep 7:658–661

    CAS  PubMed  Google Scholar 

  • Janick J, Cummins JN, Brown SK, Hemmat M (1996) Apples. In: Janick J, Moore JN (eds) Fruit breeding: trees and tropical fruits. Wiley, New York

    Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan NW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed Central  PubMed  Google Scholar 

  • Jesen PJ, Makalowska I, Altman N, Fazio G, Praul C, Maximova SN, Crassweller RM, Travis JW, McNellis TW (2010) Rootstock-regulated gene expression patterns in apple tree scions. Tree Genet Genome 6:57–72

    Article  Google Scholar 

  • Korban SS (1986) Interspecific hybridization in Malus. Hortscience 21:41–48

    Google Scholar 

  • Lambert C, Tepfer D (1992) Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones. Theor Appl Genet 85:105–109

    Article  CAS  PubMed  Google Scholar 

  • Luth D, Moore G (1999) Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tissue Organ Cult 57:219–222

    Article  CAS  Google Scholar 

  • Maximova SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting. Plant Mol Biol 37:549–559

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EH, Allan AC, Beuning LL, Bowen JH, Gera E, Jamieson KR, Janssen BJ, Laing WA, McArtney S, Nain B, Ross GS, Snowden KC, Souleyre EJ, Walton EF, Yauk YK (2006) Analyses of expressed sequence tags from apple. Plant Physiol 141:147–166

    Article  PubMed Central  PubMed  Google Scholar 

  • Puite KJ, Schaart JG (1996) Genetic modification of the commercial apple cultivars Gala, Golden Delicious and Elstar via an Agrobacterium tumefaciens-mediated transformation method. Plant Sci 119:125–133

    Article  CAS  Google Scholar 

  • Roach FA (1985) History and evolution of fruit crops. Hortscience 23:51–55

    Google Scholar 

  • Santarem ER, Trick HN, Essig JS, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Rep 17:752–759

    Article  CAS  Google Scholar 

  • Schmidt R, Willmitzer L (1988) High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants. Plant Cell Rep 7:583–586

    Article  CAS  PubMed  Google Scholar 

  • Seong ES, Song KJ (2008) Factors affecting the early gene transfer step in the development of transgenic ‘Fuji’ apple plants. Plant Growth Regul 54:89–95

    Article  CAS  Google Scholar 

  • Seong ES, Song KJ, Jeong S, Yu CY, Chung IM (2005) Silver nitrate and aminoethoxyvinylglycine affect Agrobacterium-mediated apple transformation. Plant Growth Regul 45:75–82

    Article  CAS  Google Scholar 

  • Sriskandarajah S, Goodwin PB, Speirs J (1994) Genetic transformation of the apple scion cultivar ‘Delicious’ via Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 36:317–329

    Article  Google Scholar 

  • Sujatha M, Vijay S, Vasavi S, Veera RP, Chander RS (2012) Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.). Plant Cell Tissue Organ Cult 110:275–287

    Article  Google Scholar 

  • Szankowski I, Briviba K, Fleschhut J, Schönherr J, Jacobsen HJ, Kiesecker H (2003) Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep 22:141–149

    Article  CAS  PubMed  Google Scholar 

  • Tian J, Shen H, Zhang J, Song T, Yao Y (2011) Characteristics of chalcone synthase promoters from different leaf-color Malus crabapple cultivars. Sci Hortic 129:449–458

    Article  CAS  Google Scholar 

  • Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagné D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R (2010) The genome of the domesticated apple (Malus × domestica Borkh.). Nat Genet 42:833–839

    Article  CAS  PubMed  Google Scholar 

  • Yang Y, Zhang Z, Liu Y, He P, Zhou Y, Dai H, Zhao G (2010) Establishment of Agrobacterium tumefaciens-mediated transformation system for Hanfu apple. J Fruit Sci 27:174–178

    CAS  Google Scholar 

  • Yao JL, Cohen D, Atkinson R, Richardson K, Morris B (1995) Regeneration of transgenic plants from the commercial apple cultivar Royal Gala. Plant Cell Rep 14:407–412

    Article  CAS  PubMed  Google Scholar 

  • Zhang Z, Jing S, Wang G, Fang H, Wu L (1997) Genetic transformation of the commercial apple cultivar New Jonagold and regeneration of its transgenic plants. Acta Hortic Sin 24:378–380

    Google Scholar 

  • Zhang Z, Sun A, Cong Y, Sheng B, Yao Q, Cheng Z (2006) Agrobacterium-mediated transformation of the apple rootstock Malus micromalus Makino with the RolC gene. In Vitro Cell Dev Biol Plant 42:491–497

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Acknowledgment

This work was supported by the National Natural Science Foundation of China (grant no. 31171927).

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Authors and Affiliations

  1. College of Horticulture, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning, 110866, China

    Hongyan Dai, Wenran Li, Wenjuan Mao, Guofen Han, Kai Zhao, Yuexue Liu & Zhihong Zhang

  2. College of Bioscience and Biotechnology, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning, 110866, China

    Lei Zhang

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  1. Hongyan Dai
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  2. Wenran Li
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  3. Wenjuan Mao
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  4. Lei Zhang
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  5. Guofen Han
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  6. Kai Zhao
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  7. Yuexue Liu
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  8. Zhihong Zhang
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Correspondence to Zhihong Zhang.

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Editor: J. Forster

Hongyan Dai and Wenran Li contributed equally to this work.

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Dai, H., Li, W., Mao, W. et al. Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants. In Vitro Cell.Dev.Biol.-Plant 50, 1–8 (2014). https://doi.org/10.1007/s11627-013-9544-6

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  • DOI: https://doi.org/10.1007/s11627-013-9544-6

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