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Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel

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Abstract

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05–0.1 mg dm−3 TDZ. TDZ at 0.1 mg dm−3 produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

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Abbreviations

ANOVA:

Analysis of variance

AtHSP101:

Arabidopsis thaliana heat shock protein 101

BAP:

Benzyl amino purine

CIM:

Callus induction medium

CRD:

Completely randomized design

DNMRT:

Duncan new multiple range test

h:

Hour

LB medium:

Luria–Bertani broth medium

MCS:

Multiple cloning site

min:

Minute

MS medium:

Murashige and Skoog medium

OD:

Optical density

PCR:

Polymerase chain reaction

RLU:

Relative light unit

RT-PCR:

Reverse transcription polymerase chain reaction

SRM:

Shoot regeneration medium

TDZ:

Thidiazuron

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Acknowledgments

The authors are thankful to the Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, for the laboratory facilities provided and the financial support in the form of a research Grant No. 01/01/07/0300RU.

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Authors and Affiliations

  1. Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia

    Seyed Ali Ravanfar, Maheran Abdul Aziz & Halimi Mohd Saud

  2. Laboratory of Plantation Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia

    Seyed Ali Ravanfar & Maheran Abdul Aziz

  3. Department of Microbiology, Faculty of Biotechnology and Bimolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia

    Janna Ong Abdullah

  4. Department of Agronomy, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611-0300, USA

    Seyed Ali Ravanfar

Authors
  1. Seyed Ali Ravanfar
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  2. Maheran Abdul Aziz
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  3. Halimi Mohd Saud
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  4. Janna Ong Abdullah
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Corresponding authors

Correspondence to Seyed Ali Ravanfar or Maheran Abdul Aziz.

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Communicated by M. Kupiec.

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Ravanfar, S.A., Aziz, M.A., Saud, H.M. et al. Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel. Curr Genet 61, 653–663 (2015). https://doi.org/10.1007/s00294-015-0494-x

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  • DOI: https://doi.org/10.1007/s00294-015-0494-x

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