Transformation of Kalanchoe pinnata by Agrobacterium tumefaciens with ZsGreen1

Screening tests in which 30 species of succulent plants were inoculated with A136 or C58 strains of Agrobacterium tumefaciens led to selecting genus Kalanchoe for further study of Agrobacterium -mediated transformation. Regeneration studies showed that 4.55 µM Thidiazuron (TDZ) combined with 2.15 µM...

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Published in:	Plant cell, tissue and organ culture Vol. 146; no. 2; pp. 401 - 407
Main Authors:	Cho, Keun H., Vieira, Annelise E., Kim, Joo Young, Clark, David G., Colquhoun, Thomas A.
Format:	Journal Article
Language:	English
Published:	Dordrecht Springer Netherlands 01-08-2021 Springer Nature B.V
Subjects:	Agrobacterium tumefaciens Biomedical and Life Sciences Callus Disease resistance Emission analysis Emissions Excitation Explants Flowers Fluorescence Gene expression Genomes Green fluorescent protein Kalanchoe pinnata Kanamycin Leaves Life Sciences Plant bacterial diseases Plant Genetics and Genomics Plant growth Plant Pathology Plant Physiology Plant Sciences Plasmids Regeneration Research Note Roots Scrophularia Stems Thidiazuron Transcription Transformations Transgenic plants Viruses Regeneration Transcription Fluorescent protein Figwort mosaic virus 35S promoter TDZ Succulents NAA
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Summary:	Screening tests in which 30 species of succulent plants were inoculated with A136 or C58 strains of Agrobacterium tumefaciens led to selecting genus Kalanchoe for further study of Agrobacterium -mediated transformation. Regeneration studies showed that 4.55 µM Thidiazuron (TDZ) combined with 2.15 µM α-naphtaleneacetic acid (NAA) significantly proliferated callus development from Kalanchoe pinnata leaf explants in Murashige and Skoog (MS) medium. Subsequently, 4.55 µM TDZ significantly enhanced shoot formation from the green and compact callus. NAA (5.37 µM) accelerated root growth leading to establishment of transgenic plants in soil. A gene encoding the fluorescent protein, ZsGreen1, was cloned into a plasmid vector harboring a constitutive figwort mosaic virus (FMV) 35S promoter, a kanamycin resistance gene, and a NOS-terminator. The plasmids were delivered into the genome of K. pinnata by A. tumefaciens strain C58 (ABI). The ZsGreen1 -transgenic plants exhibited very strong, green fluorescence from the callus, leaves, stems, roots, and flowers visible through a green emission filter (GFP, 480/510 nm, excitation/emission) under excitation by ultra-violet light. Transcription analysis by sqRT-PCR and qPCR confirmed the stable integration of the ZsGreen1 in the genome of K. pinnata . The FMV 35S promoter conferred uniform expression throughout whole plant organ. ZsGreen1 was proven to be a stable marker for gene expression in genetic transformation of K. pinnata . Key message The ZsGreen1 -transgenic Kalanchoe pinnata mediated by Agrobacterium tumefaciens exhibited strong, green fluorescence from the callus, leaves, stems, roots and flowers under the control of the figwort mosaic virus (FMV) 35S promoter.
ISSN:	0167-6857 1573-5044
DOI:	10.1007/s11240-021-02061-y