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Establishment of an efficient Agrobacterium-mediated genetic transformation system in Pelargonium graveolens: an important aromatic plant

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Abstract

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56 ± 1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94 ± 2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5 ± 2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.

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Abbreviations

MS:

Murashige and skoog

BAP:

Benzylaminopurine

NAA:

Naphthalene acetic acid

IBA:

Indole-3-butyric acid

ADS:

Adenine di-sulphate

gusA:

β-Glucuronidase

nptII :

Neomycin phosphotransferase

X-Gluc:

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

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Acknowledgements

The authors are thankful to the Director CSIR-CIMAP for providing necessary facilities to carrying out the research work and Dr. Ajay Kohli for editing the Manuscript. The first author also gratefully acknowledges CSIR Network Project ChemBio (BSC 203) for providing financial support.

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Authors and Affiliations

  1. Plant Biotechnology Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Picnic Spot Road, Lucknow, U.P, 226015, India

    Pooja Singh, Sana Khan & Laiq ur Rahman

  2. Plant Molecular Virology Laboratory, CPMB Division, National Botanical Research Institute, Lucknow, U.P, 226001, India

    Susheel Kumar

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  1. Pooja Singh
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  2. Sana Khan
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  3. Susheel Kumar
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  4. Laiq ur Rahman
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Correspondence to Laiq ur Rahman.

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Singh, P., Khan, S., Kumar, S. et al. Establishment of an efficient Agrobacterium-mediated genetic transformation system in Pelargonium graveolens: an important aromatic plant. Plant Cell Tiss Organ Cult 129, 35–44 (2017). https://doi.org/10.1007/s11240-016-1153-8

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