Morten Joersbo

Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.

A novel principle for selection of transgenic plant cells: positive selection

SummaryA novel principle for selection of transgenic plant cells is presented. In contrast to traditional selection where the transgenic cells acquire the ability to survive on selective media while the non-transgenic cells are killed (negative selection), this selection method actively favours regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed. Therefore, this selection strategy is termed ‘positive selection’. TheE. coli β-glucuronidase gene was used as selectable (as well as screenable) gene and a glucuronide derivative of the cytokinin benzyladenine as selective agent which is inactive as cytokinin but, upon hydrolysis by GUS, active cytokinin is released stimulating the transformed cells to regenerate. Selection ofAgrobacterium tumefaciens inoculated of tobacco leaf discs on benzyladenine N-3-glucuronide (7.5–15 mg/l) resulted in 1.7–2.9 fold higher transformation frequencies compared to kanamycin selection. A significant advantage of this selection procedure is the elimination of the need for herbicide and antibiotic resistance genes.

Direct gene transfer to plant protoplasts by mild sonication.

SummaryA novel procedure employing mild sonication for transformation of plant protoplasts is described. Transient expression of a chloramphenicol acetyltransferase (CAT) gene in protoplasts of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) was obtained by a brief exposure of the protoplasts to 20 kHz ultrasound in the presence of plasmid DNA. Maximum levels of CAT activity were achieved by sonication for 500–900 ms at 30–70 W electric power (0.65–1.6 W/cm2 acoustic power). This reduced the viability to 15–20 % and 60 % for sugar beet and tobacco protoplasts, respectively. Up to 12 % (sugar beet) and 81 % (tobacco) of maximum transient expression could be achieved with no significant loss of viability. Protoplasts surviving exposure to ultrasound were found to have a similar long-term viability and to regenerate to micro-calli as untreated protoplasts. Plasmid DNA concentrations of 80–110 μg/ml and sucrose concentrations of 21–28 % in the sonication medium were found to be optimal for transient expression.

A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene

A new selection system based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene is presented. A broad range of plant species, including agronomically important crops such as maize and rice, is sensitive to low dosages of galactose. The toxicity of galactose is believed to be due to accumulation of galactose-1-phosphate, generated by endogenous galactokinase after uptake. Here, it is demonstrated that this toxicity can be sufficiently alleviated by the Agrobacterium tumefaciens-mediated introduction of the E. coli UDP-glucose:galactose-1-phosphate uridyltransferase (galT) gene, driven by a 35S-promoter, to allow transgenic shoots of potato and oil seed rape to regenerate on galactose containing selection media, resulting in high transformation frequencies (up to 35% for potato). Analysis of genomic DNA and UDP-glucose:galactose-1-phosphate uridyltransferase activity in randomly selected potato transformants confirmed the presence and active expression of the galT gene. The agricultural performance of transgenic potatoes was evaluated by monitoring the phenotype and tuber yield for two generations and these characters were found to be indistinguishable from non-transgenic controls. Thus, the galactose selection system provides a new alternative being distinct from conventional antibiotic and herbicide selection systems as well as so-called positive selection systems where the selective agent has a beneficial effect.

PROTEIN SYNTHESIS STIMULATED IN SONICATED SUGAR BEET CELLS AND PROTOPLASTS

Sugar beet suspension cells and protoplasts were exposed to 20 kHz ultrasound and the amount of 35S-methionine incorporated into cellular protein was determined after 2 days of culture. The incorporation of 35S-methionine in cells was enhanced up to 90% after sonication for 220-770 ms at 1.2-3.5 W/cm2 which reduced viability 5-11% and decreased the average size of cell aggregates. In protoplasts, the 35S-methionine incorporation was increased up to 31% after sonication for 220-550 ms at 0.3-1.0 W/cm2 with a concomitant reduction in viability of 10-16%. At higher ultrasonic energies, 35S-methionine incorporation declined proportionally to the reduction in protoplast viability. At concentrations up to 30 mM, cystamine and cysteamine did not affect 35S-methionine incorporation or viability of the sonicated protoplasts. These results show that mild ultrasonic irradiation can stimulate protein synthesis in plant cells and protoplasts significantly.

Isolation and expression of two cDNA clones encoding UDP-galactose epimerase expressed in developing seeds of the endospermous legume guar

Abstract UDP-galactose 4′-epimerase (UDPG epimerase) catalyses the reversible conversion of UDP- d -glucose to UDP- d -galactose. This compound is a precursor for the biosynthesis of various galactosides and cell wall polymers, including galactomannan which is the main storage polysaccharide in endospermous legumes. Using functional complementation of a UDPG epimerase deficient Escherichia coli mutant (PL-2) by a cDNA expression library from immature guar ( Cyamopsis tetragonoloba ) seeds, galactose metabolising colonies with UDPG epimerase activities comparable to wild type level were obtained. Two cDNA clones (GEPI42 and GEPI48) encoding two different UDPG epimerases were isolated. Re-transformation of PL-2 by plasmid DNA, isolated from either of the two clones, resulted in numerous galactose-metabolising colonies, all with high UDPG epimerase activities. GEPI42 and GEPI48 encoded proteins with 354 and 350 amino acid residues, respectively, corresponding to deduced molecular weights of 39 286 and 38 373 Dalton, respectively. The amino acid sequence identity was 66.9%. Southern analysis of genomic guar DNA confirmed the origin and distinctness of the two UDPG epimerase genes. Analysis by immunohistochemistry showed the presence of significant levels of UDPG epimerase antigen in the endosperm of immature seeds with rapid galactomannan biosynthesis. In the endosperm of seeds close to maturity where galactomannan deposition has ceased, no antigen was detected. These data indicate that one or both of the two cloned UDPG epimerase genes are expressed in guar endosperm at a developmental stage where galactomannan biosynthesis occurs, suggesting that one or both may be involved in this process.

Direct gene transfer to plant protoplasts by electroporation by alternating, rectangular and exponentially decaying pulses.

SummaryThe efficiency of electroporation of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) protoplasts by alternating, rectangular and exponentially decaying pulses was studied by assaying transient expression of an introduced gene for chloramphenicol acetyltransferase. A simple device for electroporation by alternating current was constructed. The mains (220 V) were used as power supply and the pulse duration was controlled by the blow-out of a small fuse. Electroporation of sugar beet protoplasts by alternating current and exponentially decaying pulses resulted in 3–4 fold higher transient expression compared to rectangular pulses. Transient expression in tobacco protoplasts electroporated by exponentially decaying pulses was 30 % and 85 % higher than when electroporated by rectangular and alternating current pulses, respectively.

Quantitative Relationship between Parameters of Electroporation

Abstract The efficiency of electroporation was investigated by observations on the percentage of stained protoplasts of carrot ( Daucus carota L.) and sugar beet ( Beta vulgaris L.) suspension cells and mesophyll protoplasts of wall-rocket ( Diplotaxis muralis L.) electroporated in the presence of the dye, phenosafranine, at various electrical conditions. By computer analysis of the data, an empirical equation was derived correlating the electric field strength, E, and the pulse duration, t, to the efficiency of electroporation, ∈: aaaaaaaaaaaaa where the constants a, b and c were found to be 1.0-1.4, 2.7-2.9 and 345-1653 · 106, respectively. The relatively small variation in the exponents of t and E suggests that they may be generally applicable for many species of protoplasts while the magnitude of c is probably species dependent. The equation suggests that the efficiency of electroporation is closely related to the electric energy delivered to the protoplasts. Analysis of the equation with respect to half value and critical voltage was performed and these parameters are discussed.

Transient electropermeabilization of barley (Hordeum vulgare L.) microspores to propidium iodide

Microspores of barley (Hordeum vulgare L.) cultivar ‘Igri’ were isolated in the late uninuclear stage and the viability of the microspore samples was improved threefold through Percoll gradient centrifugation. The fraction on top of 20% Percoll contained 70% viable grains. These grains were electroporated in the presence or absence of propidium iodide. Five to 17% of the grains became transiently permeabilized to propidium iodide, the percentage depending on the electroporetic conditions. Optimal electric conditions were five pulses of 100–400 μs at 120 V/mm. Microspore samples subjected to electroporation were able to develop microcalli/proembryos and subsequently plants.

Inoculation of sugar beet protoplasts with beet necrotic yellow vein virus particles by mild sonication.

A novel procedure employing mild sonication for in vitro inoculation of plant protoplasts with virus particles has been established. Sugar beet (Beta vulgaris L.) protoplasts were briefly exposed to 20 kHz ultrasound in the presence of beet necrotic yellow vein virus (BNYVV) particles. The accumulation of BNYVV coat protein was analyzed by an enzyme-linked immunosorbent assay. The infection was detectable after 16 h and reached maximum 3-3 1/2 days after inoculation. Maximum levels of BNYVV coat protein in inoculated protoplasts were obtained by sonication for 500-600 ms at 45-60 W. This reduced the viability to 15-30%. The efficiency of the inoculation increased with the concentration of BNYVV particles up to 28 micrograms/ml. Infection was found to be optimal when the BNYVV particles were added just before sonication but low levels of infection could be obtained by addition of virus particles up to 60 min after sonication.