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Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh

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Abstract

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.

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Abbreviations

CFDA:

5-(and-6) Carboxyfluorescein diacetate

2,4-D:

2,4-Dichlorophenoxyacetic acid

DSE:

Direct somatic embryogenesis

ISE:

Indirect somatic embryogenesis

IZE:

Immature zygotic embryo

SE:

Somatic embryogenesis

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Acknowledgments

We thank Jane Murfett (Division of Biological Sciences, University of Missouri, Columbia) for providing the Col-0 transgenic line carrying DR5::GUS construct and F. Parcy (Institut des Sciences du Vegetal, Gif-sur-Yvette, France) for providing the LEC2::GUS transgenic line. This work was supported part by grants: N303 092 32/3176 and N301 079 31/2544 from the Ministry of Science and Higher Education of Poland.

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Authors and Affiliations

  1. Laboratory of Cell Biology, University of Silesia, ul. Jagiellońska 28, 40-032, Katowice, Poland

    Ewa U. Kurczyńska & Ewa Mazur

  2. Department of Genetics, University of Silesia, ul. Jagiellońska 28, 40-032, Katowice, Poland

    Małgorzata D. Gaj & Agnieszka Ujczak

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  1. Ewa U. Kurczyńska
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  2. Małgorzata D. Gaj
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Correspondence to Ewa U. Kurczyńska.

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Kurczyńska, E.U., Gaj, M.D., Ujczak, A. et al. Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh. Planta 226, 619–628 (2007). https://doi.org/10.1007/s00425-007-0510-6

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