Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.
To develop an effective protocol for observation of dry pea embryonic axes (Pisum sativum L. cv. Alaska), several fixation methods were compared for ease of infiltration and sectioning and for quality of sections. These methods included osmium vapour fixation, freeze-substitution followed by an aqueous wash, and freeze-substitution using anhydrous methanol. Axes fixed with osmium vapour were brittle and difficult to section. Cells fixed using vapour from an aqueous OsO4 solution had cell walls separated from plasma membranes, while cells fixed using osmium crystals exhibited folded walls tightly appressed to plasma membranes. Axes fixed using freeze-substitution followed by an aqueous wash sectioned easily, but appeared plasmolysed, with cell walls completely separated from plasma membranes. Even a brief (10 min) rinse in 90% acetone led to separation of the walls from the membranes. Tissues infiltrated with anhydrous methanol and Spurr’s resin were difficult to section, but showed extensive folding of the cell walls, with plasma membranes appressed to the walls. Additional modifications to the infiltration protocol led to improved sectioning. The highest-quality sections were obtained using freeze-substitution with anhydrous methanol as a solvent, followed by infiltration using methanol and Spurr’s resin, with the catalyst (dimethylaminoethanol) omitted from the infiltration protocol until the final step. Through comparison of several protocols recommended in the literature for the preparation of dry tissues, it is apparent that the presence of water, even in the vapour phase, at any step can cause swelling of the cell walls and the artefactual separation of the walls from the fixed protoplast.
The purpose of this project is to incorporate a community service component into a Biology course at Northern State University (NSU) in Aberdeen, SD. Students in an upper-level botany course (Plant Structure and Function) provide landscaping services to homeowners who have purchased homes through Habitat for Humanity. Homeowner satisfaction with the finished project and student learning are assessed through surveys.
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