Agarose embedding affects cell wall regeneration and microtubule organization in sunflower hypocotyl protoplasts

Sunflower hypocotyl protoplasts (Helianthns annuus L, cv. Emil) divide symmetrically to form loosely associated microcolonies when cultured in liquid medium, whereas when embedded in agarose beads they divide asymmetrically to give rise to embryo-like structures. To understand the relationship between protoplast embedding and cell division patterns, we studied the deposition of beta-linked glucan and the dynamics of microtubules during early phases of culture. After one day in culture, under both culture conditions, a small proportion of the protoplasts had already begun to rebuild a beta-glucan cell wall and the process reached completion in all protoplasts after 10 days. Callose deposition was faster in agarose than in liquid medium but it concerned only 30-40% of the protoplasts and was not related to either division type . No marked differences were observed in cortical arrays of microtubules. However, in embedded protoplasts perinuclear microtubules formed a well-defined basket around the nucleus; these microtubules were never observed in liquid-cultured protoplasts. A narrow preprophase band was present only in dividing protoplasts cultured in liquid medium. The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.