Journal Article

Composite <i>Cucurbita pepo</i> plants with transgenic roots as a tool to study root development

Elena L. Ilina, Anton A. Logachov, Laurent Laplaze, Nikolay P. Demchenko, Katharina Pawlowski and Kirill N. Demchenko

in Annals of Botany

Published on behalf of The Annals of Botany Company

Volume 110, issue 2, pages 479-489
Published in print July 2012 | ISSN: 0305-7364
Published online May 2012 | e-ISSN: 1095-8290 | DOI: http://dx.doi.org/10.1093/aob/mcs086
Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

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  • Ecology and Conservation
  • Evolutionary Biology
  • Plant Sciences and Forestry

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Background and Aims

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash.

Methods

The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively.

Key Results

Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots.

Conclusions

The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.

Keywords: Agrobacterium rhizogenes; auxin; CaMV 35S enhancer; composite plants; Cucurbita pepo; Cucurbitaceae; DR5; hairy roots; root; squash

Journal Article.  7343 words.  Illustrated.

Subjects: Ecology and Conservation ; Evolutionary Biology ; Plant Sciences and Forestry

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