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Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N6-(Δ2-isopentenyl)-adenine (iP) and N6-(Δ2-isopentenyl)-adenosine (iPA) levels

  • Cell Biology and Morphogenesis
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Abstract

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N6-(Δ2-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml−1) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5–3.9 pmol g−1 FW of the cytokinins analysed. Seedlings (1.0–1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 μM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g−1 FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g−1 FW) and iPA (63 pmol g−1 FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.

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Abbreviations

BA:

6-Benzyladenine

CW:

Coconut water

DZ:

Dihydrozeatin

DZR:

Dihydrozeatin riboside

DZ9G:

Dihydrozeatin-9-glucoside

DZ5P:

Dihydrozeatin riboside 5′-monophosphate

HPLC:

High performance liquid chromatography

iP:

N6-(Δ2-isopentenyl)-adenine

iPA:

N6-(Δ2-isopentenyl)-adenosine

iPAMP:

N6-(Δ2-isopentenyl)-adenoside-5′-monophosphate

iP9G:

N6-(Δ2-isopentenyl)-adenine-9-glucoside

KC:

Knudson C (1946) medium

PSB:

Phosphate buffer

iP:

N6-(Δ2-isopentenyl)-adenine

RIA:

Radioimmunoassay

TEAA:

Triethylammonium acetate

Z:

Zeatin

ZR:

Zeatin riboside

Z9G:

Zeatin-9-glucoside

Z5P:

Zeatin riboside 5′-monophosphate

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Acknowledgments

Sim GE was supported by a research scholarship from the National University of Singapore (NUS). Loh CS thanks the NUS for a research grant to carry out this project. Loh CS is grateful to Dr David Hanke of the Department of Plant Sciences, University of Cambridge, UK, for an opportunity to learn cytokinin analysis techniques in his lab. We thank Dr. L. H. Jones, University of Cambridge, for providing the tracers used in RIA. We wish to thank Professor Prakash Kumar of NUS for critical reading of the manuscript and Ms Tan Wee Kee and Mr Koh Teng Seah for advice in statistical analyses.

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  1. Guek Eng Sim

    Present address: Plant Biotechnology and Agrotechnology Section, School of Chemical and Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore, Singapore, 139651

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  1. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, Singapore, 117543

    Chong Jin Goh & Chiang Shiong Loh

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  1. Guek Eng Sim
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  2. Chong Jin Goh
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Correspondence to Chiang Shiong Loh.

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Communicated by K. Kamo.

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Sim, G.E., Goh, C.J. & Loh, C.S. Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N6-(Δ2-isopentenyl)-adenine (iP) and N6-(Δ2-isopentenyl)-adenosine (iPA) levels. Plant Cell Rep 27, 1281–1289 (2008). https://doi.org/10.1007/s00299-008-0551-8

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