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Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen

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Abstract

A successful in vitro Agrobacterium-mediated transformation protocol was developed for Mimulus aurantiacus, a model species for ecological and evolutionary genetics and a promising ornamental plant. Three binary vectors were tested, each containing the hptII selectable marker gene and one of the reporter genes: gusA, EGFP or ZsGreen, all of them under CaMV 35S promoter. Genetic transformation was achieved through 4 days of co-cultivation of leaf, petiole and hypocotyl explants with Agrobacterium tumefaciens strain LBA 4404. Explants produced transformed callus tissue on solid modified Murashige and Skoog medium supplemented with 1 mg L−1 6-benzylaminopurine, 0.5 mg L−1 1-naphthaleneacetic acid, 30 g L−1 sucrose and 20 or 50 mg L−1 hygromycin B. All three reporter genes were expressed in callus tissue but the intensity of expression gradually decreased during further plant development. The new reporter gene ZsGreen proved suitable for plant transformation experiments since very intense and bright fluorescence was detected. Out of 1,760 co-cultured explants, 110 plants were regenerated and all of them were found to be PCR positive for the selection and/or reporter genes. Chemiluminescent Southern blot analysis revealed that 91 % of the regenerated plants (100 T0 plants) contained T-DNA integrated in their genome. Transformation efficiency varied from 1.4 to 23.3 % for hypocotyl and petiole explants, respectively. Integration of some backbone sequences in plant genomes was confirmed in 75.3 % of T0 plants. Using this protocol, stable transformants expressing selectable marker gene hptII and one of the reporter genes (gusA, ZsGreen or EGFP) were obtained in 4–5 months.

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Abbreviations

AS:

Acetosyringone

BAP:

6-Benzylaminopurine

CaMV 35S:

Cauliflower mosaic virus 35S promoter

CTAB:

Cetyl trimethylammonium bromide

EGFP:

Enhanced green fluorescent protein

EM:

Shoot elongation medium

GUS:

b-Glucuronidase

hptII:

Hygromycin phosphotransferase gene

IAA:

Indole-3-acetic acid

MS:

Murashige and Skoog medium (Murashige and Skoog 1962)

NAA:

1-Naphthaleneacetic acid

RM:

Regeneration medium

TDZ:

Thidiazuron

X-Gluc:

5-Bromo-4-chloro-3-indolyl-β-glucuronic acid

ZsGreen:

Green fluorescent protein derived from Zoanthus sp

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Acknowledgments

This work was supported by research grant P4-0077 from the Slovenian Research Agency.

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  1. Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia

    Nik Susič, Borut Bohanec & Jana Murovec

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  1. Nik Susič
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  2. Borut Bohanec
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  3. Jana Murovec
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Correspondence to Jana Murovec.

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Susič, N., Bohanec, B. & Murovec, J. Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen . Plant Cell Tiss Organ Cult 116, 243–251 (2014). https://doi.org/10.1007/s11240-013-0402-3

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