Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts |
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Authors: | K C Katsirdakis K A Roubelakis-Angelakis |
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Institution: | (1) Department of Biology, University of Crete, P. O. Box 1470, 711 10 Heraklio, Crete, Greece;(2) Institute of Molecular Biology and Biotechnology, P.O. Box 1470, 711 10 Heraklio, Crete, Greece |
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Abstract: | Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition
was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils
were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and
its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study
was undertaken on U-14C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts
of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher U-14C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and
noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the
labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts.
Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl
sulfoxide to the culture medium resulted in a dramatic increase in U-14C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect
on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic
and noncellulosic cell wall fractions. |
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Keywords: | grapevine protoplasts Vitis scanning electron microscopy cellulosic noncellulosic cell wall dimethyl sulfoxide |
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