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Developmental steps in acquiring competence for shoot development in Arabidopsis tissue culture

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Abstract

Arabidopsis shoots regenerate from root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium (CIM) followed by incubation on a cytokinin-rich shoot induction medium (SIM). During CIM preincubation, root explants acquire competence to respond to shoot induction signals. During CIM preincubation, pericycle cells in root explants undergo cell divisions and dedifferentiate, losing the expression of a pericycle cell-specific marker. These cells acquire competence to form green callus only after one day CIM preincubation and to form shoots after 2–3 days CIM preincubation. Reversible DNA synthesis inhibitors interfered with the acquisition of competence to form shoots. Genes requiring CIM preincubation for upregulation on SIM were identified by microarray analysis and included RESPONSE REGULATOR 15 (ARR15), POLYGALACTURONASE INHIBITING PROTEIN 2 (PGIP2) and WUSCHEL (WUS). These genes served as developmental markers for the acquisition of competence because the CIM preincubation requirements for ARR15 and PGIP2 upregulation correlated well with the acquisition of competence to form green callus, and the CIM preincubation requirements for WUS upregulation matched those for shoot formation. Unlike ARR15, another cytokinin inducible, A-type ARR gene, ARR5, was upregulated on SIM, but the induction did not require CIM preincubation. These findings indicate that competencies for various events associated with shoot regeneration are acquired progressively during CIM preincubation, and that a set of genes, normally upregulated on SIM, are repressed by a process that can be relieved by CIM preincubation.

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Abbreviations

APH:

Aphidicolin

ARR:

Arabidopsis response regulator

CIM:

Callus induction medium

DTA:

Diphtheria toxin chain A

FDR:

False discovery rate

GUS:

β-glucuronidase

HU:

Hydroxyurea

LRP:

Lateral root primordium

MUG:

4-Methylumbelliferyl β-glucuronide

NAA:

1-Naphthalene acetic acid

NPA:

N-1-naphthylphthalamic acid

PI:

Propidium iodide

RT-PCR:

Reverse transcriptase polymerase chain reaction

SIM:

Shoot induction medium

YFP:

Yellow fluorescent protein

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Acknowledgments

This study was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (2003-35304-13363), by the National Science Foundation (IBN-0236060), and by the Plant Sciences Institute at Iowa State University. We acknowledge the important contributions of Rhonda DeCook for her help with the bioinformatics analysis.

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  1. Plant Sciences Institute, Iowa State University, Ames, IA, 50011, USA

    Ping Che, Sonia Lall & Stephen H. Howell

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  1. Ping Che
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  2. Sonia Lall
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  3. Stephen H. Howell
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Correspondence to Stephen H. Howell.

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Che, P., Lall, S. & Howell, S.H. Developmental steps in acquiring competence for shoot development in Arabidopsis tissue culture. Planta 226, 1183–1194 (2007). https://doi.org/10.1007/s00425-007-0565-4

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