Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Abstract

Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

AS:

Acetosyringone

BAP:

6-Benzylaminopurine

CaMV35S:

Cauliflower mosaic virus 35S

Cx:

Cefotaxime

IAA:

Indole-3-acetic acid

Kin:

Kinetin

Km:

Kanamycin

MS:

Murashige and Skoog

NAA:

1-Naphthaleneacetic acid

X-Gluc:

5-Bromo-4-chloro-3-indolyl β-D-glucuronide

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Acknowledgements

This paper is part of the PhD dissertation of the first author. These experiments were conducted at the Molecular Biology Laboratory and Tissue Culture Laboratory of Faculty of Agricultural Sciences and Natural Resources, Imam Khomeini International University, Qazvin, Iran. The authors would like to thank PhD Students; Reza Heidari Japelaghi and Meysam Bastami for their technical assistance.

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BS: Performance of experiments, collection and analysis of data and preparation of manuscript; RH: Corresponding author, chief scientist, assistance to data collection and analysis; financial supports, experimental design. MB: Project academic advisor, preparation of plant materials and assistance to experimental design.

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Correspondence to Raheem Haddad.

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Communicated by Danny Geelen.

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Sedaghati, B., Haddad, R. & Bandehpour, M. Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant. Plant Cell Tiss Organ Cult 136, 231–245 (2019). https://doi.org/10.1007/s11240-018-1509-3

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Keywords

  • Purslane
  • Cytokinin
  • Somatic embryogenesis
  • Agrobacterium tumefaciens
  • uidA gene
  • Southern hybridization