Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii

Abstract

A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 M sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 M sorbitol layer. Regeneration occurred over a period of 10-24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater.

Nitric Oxide and cGMP Signaling in Calcium-Dependent Development of Cell Polarity in Ceratopteris richardii

“…Spores were surface sterilized by soaking for 90 s in 0.875% sodium hypochlorite and rinsed with sterile water. To enhance the synchronization of spore germination, the spores were resuspended in deionized water and soaked for 4 to 6 d in complete darkness at 29°C (Edwards and Roux, 1998). Petri dishes contained solid growth media made from 1% agarose in half-strength Murashige and Skoog basal salt mixture pH 6.2 (Caisson Labs).…”

Transient and stable transformation of Ceratopteris richardii gametophytes

“…To facilitate Agrobacteria infection, we treated 12-day-old gametophytes with a combination of 1.5% (w/v) cellulase and 0.5% (w/v) macerozyme (containing pectinase, cellulase and hemicellulase). This use of a combination of enzymes is a modification of a previously described protocol for protoplast isolation [9]. After treatment, the gametophytic cells have lost most of their thick cell wall but the prothallus remains largely intact (Figure 1b).…”

“…Under high population density, later-germinating spores develop into smaller male gametophytes, developing antheridia but not archegonia, in response to the antheridiogen produced by earlier-germinated hermaphrodites [6]. Although an array of processes has been investigated using C. richardii , such as gametophytic growth and development [7], sex determination [8], the establishment of polarity during spore germination [9, 10], alternation of generations [11–13], and the evolution of vascular cell walls [14], the full potential of this model organism cannot be realized without a simple and reliable genetic transformation system.…”