Fertile transgenic barley to 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.

Stable transformation of barley tissue culture by particle bombardment.

Suspension culture cells of barley (Hordeum vulgare L. cv Pokko) were stably transformed with two separate plasmids containing genes coding for neomycin phosphotransferase II and ß-glucuronidase, respectively. Transformed cultures were selected with the antibiotic Geneticin(R). Enzymatic activity was tested in the Geneticin(R) resistant cultures, and in 96% of them neomycin phosphotransferase could be detected. The non-selected marker, detected as ß-glucuronidase activity, was expressed in 40% of the resistant cultures. Stable transformation was confirmed with Southern blot hybridization.

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

Transient 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.