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Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death

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Abstract

Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

ABA:

Abscisic acid

BA:

N6-benzyladenine

DCR:

Basal culture medium (Gupta and Durzan 1985)

DMSO:

Dimethyl sulfoxide

IAA:

Indoleacetic acid

NPA:

1-N-napthylphthalamic acid

PAT:

Polar auxin transport

PCD:

Programmed cell death

PCIB:

2-(4-chlorophenoxy)-2-methylpropionic acid

PEG:

Polyethylene glycol

PEM:

Proembryogenic mass

PGR:

Plant growth regulator (auxin and cytokinin)

TUNEL:

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling

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Acknowledgments

This work was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning. Silvia Valladares was supported by an Angeles Alvariño postdoctoral fellowship from Xunta de Galicia (Spain).

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Author notes

  1. Silvia Valladares

    Present address: Department of Plant Physiology, Instituto de Investigaciones Agrobiologicas de Galicia (CSIC), Apartado 122, 15780, Santiago de Compostela, Spain

Authors and Affiliations

  1. Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7080, 750 07, Uppsala, Sweden

    Malin Abrahamsson, Silvia Valladares, Emma Larsson, David Clapham & Sara von Arnold

Authors
  1. Malin Abrahamsson
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  2. Silvia Valladares
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  3. Emma Larsson
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  4. David Clapham
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  5. Sara von Arnold
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Corresponding author

Correspondence to Malin Abrahamsson.

Additional information

Malin Abrahamsson and Silvia Valladares contributed equally to this work.

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11240_2011_103_MOESM1_ESM.tif

Mature somatic embryos after NPA-treatment. (a) Embryo showing normal morphology with 4 separated cotyledons. (b) Embryo showing partially fused cotyledons. (c) Embryos showing severely fused cotyledons. (d) Frequency of fully mature embryos with partially or severely fused cotyledons in cell line 12:12 (grey bars) and 3:10 (black bars). Values are mean percentages (± SE) of the total number of embryos formed per treatment. The data are based on 10 replicates. Within each cell line an asterisk denotes significant differences between the control and NPA treatment at the p≤ 0.05 level according to Duncan´s multiple range test. Bars 250 μm (TIFF 5184 kb)

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Abrahamsson, M., Valladares, S., Larsson, E. et al. Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death. Plant Cell Tiss Organ Cult 109, 391–400 (2012). https://doi.org/10.1007/s11240-011-0103-8

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  • DOI: https://doi.org/10.1007/s11240-011-0103-8

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