RESEARCH PAPER
The influence of isolation stress on the (re)organization of cell walls in protoplasts of in vitro recalcitrant plants

A study of cell-wall regeneration was conducted on protoplast cultures of four recalcitrant plant species: yellow

lupin and grasspea (grain legumes), and also hyacinth and asparagus (ornamental monocots). The rate of cell wall

resynthesis was investigated together with the arrangement of cellulosic fibers on the surface of protoplast-derived

cells. Localization of arabinogalactan proteins in cell walls was also performed. Quick cell wall renewal occurred

in grasspea and hyacinth cultures, where the percentage of protoplast-derived cells after 24h of culture accounted

for 45-50%. In contrast, the presence of cell walls in half of the population was not observed in asparagus

and lupin cultures until 5 and 7 days later, respectively. Grasspea, hyacinth and asparagus cells budded intensively.

Moreover, the cellulosic material of the cell wall was disorganized and unevenly distributed in lupin, asparagus

and hyacinth. In all the tested species arabinogalactan proteins (AGPs) were detected mainly in spherical cells

with condensed cytoplasm, but were absent in the budding cells. Notwithstanding the observed differences among

species, we concluded that incorrect cell wall structure/composition, together with a deregulated cell cycle might

contribute to protoplast recalcitrance in the examined plants. Abnormalities could be a result of the isolation

process itself and defective stress-recovering mechanisms. Subsequently, these could arrest regeneration competences

and direct cells at apoptotic pathways.