Suspensions of plant cells in microgravity.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana spec. was performed as part of the German Sounding Rocket Program (TEXUS). The results indicate a significant increase not only in the yield of 1:1 fusion products, but also in homo- and multifusion products. Heterokaryons obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent oxygen evolution (independent of other substrates than bicarbonate). Furthermore we were able to regenerate hybrid plants from suspensions of vacuolated and evacuolated protoplasts which were pulse-treated under microgravity. These expressed characteristics intermediate to those of the parental plants (Nicotiana tabacum (cv. Samsun), evacuolated; N. rustica, vacuolated).

Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability.

By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 µs/1.5 kV*cm(-1) and 200 µs/0.5 kV*cm(-1), hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths.

Electric pulse induced membrane permeabilization. Spatial orientation and kinetics of solute efflux in freely suspended and dielectrophoretically aligned plant mesophyll protoplasts.

Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.

Effects of microgravitation on electrofusion of plant cell protoplasts.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana tabacum was performed as part of the German Sounding Rocket Program (TEXUS 17, 1988). The results indicate a significant increase not only in the yield of 1:1 hybrids, but also in homo- and multifusion products. Hybrids obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent O2-evolution (independent of other substrates than bicarbonate). This finding is of interest for fusion experiments where only limited numbers of fusion partners are available (e.g. protoplasts from embryogenic tissues) or where fusion yields are extremely low under 1 x gravity (e.g. protoplasts of different specific density).

Assay of photosynthetic oxygen evolution from single protoplasts.

A semiquantitative assay for light-dependent O(2) evolution by a single mesophyll protoplast is described. The assay indicator is the density of aerotactic bacteria (Pseudomonas aeruginosa, ATCC 10145; ;Engelmann experiment') attracted to the protoplast. Quantification is by dark field microphotometry. The sensitivity is about 50 femtomoles O(2) per protoplast per minute. The results demonstrate the biphasic nature of O(2) evolution of a single protoplast during photosynthetic induction. Computerized data acquisition yields traces which, until a steady state of photosynthetic O(2) evolution is reached, are identical to ordinary O(2) electrode traces.

Mass electrofusion and mass selection of functional hybrids from vacuolate × evacuolate protoplasts.

Vacuolate mesophyll protoplasts of Nicotiana tabacum L. (cv. Samsun) were electrically fused with evacuolate protoplasts of the same species. For this purpose a mass fusion chamber was constructed. Due to distinct differences in the specific density of the respective protoplast populations, interspecific hybrids could be separated from intraspecific ones as well as from unfused parental protoplasts on an isoosmotic density gradient. The interspecific hybrids appeared to be viable to about 60% as assayed by a bacterial test for photosynthetic oxygen evolution.