Desiccation tolerance of protoplasts isolated from pea embryos

To facilitate studies of desiccation tolerance at the cellular level, a technique to isolate protoplasts from desiccation-tolerant pea (Pisum sativum L. cv. Alaska) embryos has been developed. Using FDA (fluorescein diacetate) as a probe, viability of the protoplasts was investigated before and after drying to determine whether the protoplasts could survive desiccation in a manner similar to the tissue from which they were isolated. Protoplasts were isolated from 12 h imbibed pea axes, suspended in several different sugar solutions, then dried to water contents less than 0.2 g H(2)O g(-1) DW. Protoplasts only survived drying if the rate was rapid (<2 h), while slow drying (24 h) was lethal. Maximal survival (75%) was obtained after drying protoplasts with a mixture of sucrose and raffinose, while pure sucrose and trehalose were somewhat less effective protectants. Low survival was obtained after drying protoplasts with monosaccharides and pure raffinose. Protoplasts isolated from germinated seedlings did not survive dehydration below 0.2 g H(2)O g(-1) DW. Transmission electron microscopy revealed that dried desiccation-tolerant protoplasts appeared shrunken, with folded membranes, while dried protoplasts from sensitive tissue had disrupted membranes. While isolated protoplasts maintained some of the desiccation tolerance of orthodox seeds, their inability to survive complete drying and their sensitivity to drying rate is similar to the behaviour of recalcitrant embryos.