Veli Kauppinen

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T0), and four of the 90 T0 spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T1). The four transgenic shoots of Toivo (the T0 plant) produced 25 plantlets as T1 progeny. Altogether fifteen of these T1 plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T2 progeny.

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

SummaryProduction and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. β-Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.

Secretion of a heat-stable fungalβ-glucanase from transgenic, suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostableβ-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of solubleβ-glucans during mashing.

Regulation of α-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation

SummaryTransient gene expression was studied in isolated protoplasts of barley (Hordeum vulgare L. cv Himalaya) transformed by electroporation. Two plasmid constructions were used, both of which contained the gene coding for neomycin phosphotransferase II (nptII) as a reporter gene. In one plasmid the reporter gene was under the control of an α-amylase group 1 gene promoter of barley and in the other, used as a control, under the CaMV 35S transcript promoter. Protoplasts were isolated from three different types of tissue: the aleurone layer, the scutellar epithelium and the mesophyll. All three types of protoplasts electroporated with 35S -nptII plasmid construction showed strong NPTII activity on which GA3 and ABA had no effect. In protoplasts isolated from the aleurone layer and scutellum the expression of amy-nptII was low when compared with the expression of 35S -nptII. In aleurone protoplasts GA3 enhanced the expression of amy-nptII about tenfold and ABA prevented the action of GA3. In protoplasts isolated from the scutellar epithelium GA3 did not affect the low level of expression of amy-nptII. In mesophyll protoplasts the amy-nptII was not expressed at all.

Efficient regeneration of fertile plants from protoplasts isolated from microspore cultures of barley (Hordeum vulgare L.).

SummaryCultures of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar of malting barley) were used for isolation of protoplasts. The protoplasts were cultured embedded in agarose. The plating efficiency varied from 0.002% to 0.015%. Several hundred green plants were regenerated from the cultures. Plantlets regenerated from protoplasts were potted in soil within 4–5 months of collecting the spikes for microspore culture and the first plants are now setting seed.

Characterization of a birch (Betula pendula Roth.) embryogenic gene, BP8

We have isolated the birch homologue (BP8) for the carrot embryogenic gene DC8 by heterologous hybridization. The birch BP8 gene encodes a putative protein of 53 kDa, showing 52% sequence identity with the DC8 gene at the amino acid level. The putative BP8 protein contains 20 repeats of 11 amino acids and thus belongs to the group of LEA proteins isolated from such plants as carrot, cotton and wheat. Northern hybridization of mRNA isolated from birch cells representing different stages of somatic embryogenesis and non-embryogenetic material with a PB8 probe gave no signals, suggesting a low expression level of the BP8 gene.

The effect of salinity, nitrate concentration, pH and temperature on eicosapentaenoic acid (EPA) production by the red unicellular alga Porphyridium purpureum

Abstract The effects of salinity, nitrate concentration, pH and temperature on eicosapentaenoic acid (EPA) production by Porphyridium purpureum were studied. Both systematic and stepwise optimization were used in order to determine the best conditions for production of EPA. An analysis method for fatty acids was also developed. The highest EPA production per cell was achieved at pH 7.6 with 41.8 g l −1 NaCl and 12.1 mM nitrate in the production medium and using a growth temperature of 8°C.

Measurement of α-amylase and glucoamylase activities produced during fermentation

Abstract A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α- d -glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.

Culture conditions for efficient induction of green plants from isolated microspores of barley

Subculture regime and carbohydrate concentration of the medium had a marked effect on the regeneration of green plantlets from mechanically isolated microspores of Hordeum vulgare L. cv. Kymppi. A sevenfold increase in the yield of green plants was obtained by shortening the suspension culture time of the developing proembryo mass from 4 to 3 weeks. A further twofold increase was obtained by increasing the maltose concentration of the microspore isolation medium and of the culture medium. Under optimal conditions, a mean of 169±97 green plants per spike were regenerated.

Automatic monitoring of protease activity during fermentation

Abstract An automatic method for the determination of bacterial protease activity was coupled to a laboratory fermenter. The assay procedure was based on the solubilization of an insoluble dye-protein complex. Monitoring of protease production by Bacillus subtilis was successfully carried out.