Genetic transformation of selected mature cork oak (Quercus suber L.) trees

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and -glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.Abbreviations BA N⁶-Benzyladenine - GUS -Glucuronidase - MSSH Expression-proliferation medium - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase gene - uidA -Glucuronidase gene     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Transient expression of uidA constructs in in vitro onion ( Allium cepa L.) cultures following particle bombardment and Agrobacterium‐mediated DNA delivery

Particle bombardment and Agrobacterium-mediated DNA delivery into immature embryos and microbulbs were used to investigate the expression of the uidA gene in in vitro onion cultures. Both methods were successful in delivering DNA and subsequent uidA expression was observed. Optimal transient -glucuronidase activity was observed in immature embryos that had been pre-cultured for three days and bombarded at a distance of 3 cm from the stopping plate, under 25 in Hg vacuum, using 900–1300 psi rupture discs. The CaMV35S-uidA gene construct gave five fold higher transient -glucuronidase activity than the uidA gene construct regulated by any of four other promoters initially chosen for high experession in monocotyledonous tissues.Abbreviations GUS -glucuronidase - IE immature embryo - MUG methylumbelliferyl -D-glucuronide     

High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) containing<Emphasis Type="Italic"> npt-II</Emphasis> and antimicrobial peptide genes

A reliable and efficient system for transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) plants via microprojectile bombardment was developed. Improvements over the previous biolistic transformation system included: (1) the use of gold particles for bombardment; (2) step-wise selection at 10 then 15 mg/l kanamycin; and (3) embryo induction at 27°C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for -glucuronidase (GUS), or were co-bombarded with pSAN237 carrying the npt-II (neomycin phosphotransferase II) selectable marker gene, and a second plasmid with an antimicrobial peptide gene. A large number of transient (7,883±1,928) and stable (46±32) blue spots per plate at 2 and 95 days after bombardment, respectively, were obtained according to GUS expression analyses. A total of 447 putative transgenic embryos was harvested from 84 bombarded plates. From these embryos, 242 (54%) were regenerated into plants within the first year of the experiment. Southern blot analyses confirmed integration of the transgenes into the grape genome. Co-transformation was tested with four separate antimicrobial constructs. The co-transformation frequency of unlinked genes was 48% as measured by polymerase chain reaction (PCR), and 56% as estimated by dot blot hybridization. Expression of the gus gene, and PCR and Southern blot analyses of npt-II and antimicrobial genes from regenerated plants document stable transformation of 'Chardonnay' and establish the parameters for highly-efficient biolistic transformation in V. vinifera.Abbreviations AMP Antimicrobial peptide - DBH Dot blot hybridization - GM+NOA Glycerol and maltose liquid medium with -naphthoxyacetic acid - gus -Glucuronidase gene - GUS -Glucuronidase - Km Kanamycin - Km^R Kanamycin resistant - mag2 Magainin-2 - MS Murashige and Skoog medium - MS/2 Half-strength Murashige and Skoog medium - nos Nopaline synthase - npt-II Neomycin phosphotransferase II gene - PCR Polymerase chain reaction - PGL Peptidyl-glycine-leucine - Pubq3 Arabidopsis ubiquitin-3 promoter - Pubq10-L Arabidopsis ubiquitin-10L promoter - Pubq11 Arabidopsis ubiquitin-11 promoter - SP Signal peptide - Tnos Nopaline synthase terminator - WPM Woody plant mediumCommunicated by E.D. Earle     

Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin

Summary Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to -tubulin. ThebenA gene of three independently isolated rhizoxin-resistant (Rhi^r) mutants ofAspergillus nidulans was cloned, sequenced and compared with that of the wild-type, rhizoxin-sensitive (Rhi^s) strain. In all three Rhi^r mutants, the AAC codon for Asn-100 of thebenA -tubulin gene was altered to ATC, coding for Ile. Sequence displacement experiments confirmed that the substitution of Ile for Asn-100 confers resistance to rhizoxin in this organism. The amino acid sequences of -tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions. The fission yeastSchizosaccharomyces pombe and the budding yeastSaccharomyces cerevisiae are naturally occurring Rhi^r organisms whose -tubulin genes encode Ile and Val respectively at the 100th amino acid residue. The Ile-100 ofS. pombe and the Val-100 ofS. cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques. The resultant haploid strains of these two yeasts uniquely expressing -tubulin (Asn-100) instead of -tubulin (Ile-100 or Val-100) were found to be Rhi^s. Haploid yeast expressing -tubulin (Asn-100) is normal except for its sensitivity to rhizoxin. These results suggest that rhizoxin resistance has a common basis in both naturally occurring species and experimentally selected mutants in the substitution of Ile or Val for Asn-100 in -tubulin.     

Effects of promoter,intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts

Various chimaeric promoter regions coupled to the uidA -glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5 transcribed region decreased expression strength.     

Spurious localizations of diX-indigo microcrystals generated by the histochemical GUS assay

For several models expressing theuidA orgus reporter gene with or without a presequence for mitochondrial targeting, we have demonstrated that the compartmentation of -glucuronidase (E.C. 3.2.1.31) activity was not in agreement within situ localization of the diX-indigo microcrystals generated by the cytoenzymological GUS assay. These crystals were generally associated with the various cytomembranes and lipid inclusions. Experiments with purified -glucuronidase or withgus-expressing bacteria incubated with 5-bromo-4-chloro-3-indolyl--d-glucuronide and maize oil-phosphate buffer emulsion indicated that the intermediate products resulting from the GUS assay actively diffused and crystallized preferentially in association with lipids, sometimes far from the site of enzyme activity. This phenomenon could not be suppressed by the addition of potassium ferricyanide in the incubation medium. These findings are discussed with regard to previously reported biochemical and histochemical data on animal tissues, and focus on the necessity for caution in studies of tissue-specific gene expression using the GUS assay, particularly for lipid-rich plant models.     

TheCaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants

We have evaluated the expression of the reporter -glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity—as estimated by the histochemical GUS assay—in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 mol MU mg protein^–1 h^–1 to 0.26 mol MU mg protein^–1 h^–1. Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.Abbreviations CaMV 35S Cauliflower mosaic virus promoter - GUS -Glucuronidase (EC 3.2.1.31) - MU 4-Methyl umbelliferone - nos Nopaline synthase promoter - nptII Neomycin phosphotransferase - X-Gluc 5-Bromo-4-chloro-3-indolyl--d-glucuronic acid     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

β-1,3-Glucanase is highly-expressed in laticifers of Hevea brasiliensis

Clones encoding -1,3-glucanase have been isolated from a Hevea cDNA library prepared from the latex of Hevea brasiliensis using a probe Nicotiana plumbaginifolia cDNA encoding -1,3-glucanase, gnl. Nucleotide sequence analysis showed that a 1.2 kb Hevea cDNA encoding a basic -1,3-glucanase showed 68% nucleotide homology to gnl cDNA. Northern blot analysis using the Hevea cDNA as probe detected a mRNA of 1.3 kb which was expressed at higher levels in latex than in leaf. In situ hybridization analysis using petiole sections from Hevea localized the -1,3-glucanase mRNA to the laticifer cells. Genomic Southern analysis suggested the presence of a low-copy gene family encoding -1,3-glucanases in H. brasiliensis.     

Anther-specific expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and alters anther development and whole flower growth

Summary The promoter activity of a 2.2-kb DNA fragment from the 5 flanking sequence of the tap1 gene of snapdragon has been characterised in tobacco with the -glucuronidase reporter gene. The tap1 promoter conferred to the reporter gene an expression that was limited to the early stages of anther development. Expression of the rolB gene of Agrobacterium rhizogenes under the control of the tap1 promoter impaired the development of tobacco flowers. Tobacco plants transgenic for the tap1-rolB chimeric gene showed altered anthers and a reduction in whole flower growth. Such growth alterations were correlated with an increase in free IAA content and a decrease in gibberellin activity present in anthers. Since the rolB gene codes for an indole -glucosidase (Estruch et al. 1991), we interpret the phenotypic alterations to be a consequence of the increased content and activity of auxin in anthers. The perturbation of anther development would in turn affect gibberellin production and content, which is reflected in a reduced elongation of flowers.     

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene

Summary A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial -glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of -glucuronidase activity.     

Novel β-carotene ketolases from non-photosynthetic bacteria for canthaxanthin synthesis

We reported previously that the Rhodococcus erythropolis strain AN12 synthesizes the monocyclic carotenoids 4-keto -carotene and -carotene. We also identified a novel lycopene -monocyclase in this strain. Here we report the identification of the rest of the carotenoid synthesis genes in AN12. Two of these showed apparent homology to putative phytoene dehydrogenases. Analysis of Rhodococcus knockout mutants suggested that one of them ( crtI) encodes a phytoene dehydrogenase, whereas the other ( crtO) encodes a -carotene ketolase. Expression of the -carotene ketolase gene in an Escherichia coli strain which accumulates -carotene resulted in the production of canthaxanthin. In vitro assays using a crude extract of the E. coli strain expressing the crtO gene confirmed its ketolase activity. A crtO homologue (DR0093) from Deinococcus radiodurans R1 was also shown to encode a -carotene ketolase, despite its sequence homology to phytoene dehydrogenases. The Rhodococcus and Deinococcus CrtO ketolases both catalyze the symmetric addition of two keto groups to -carotene to produce canthaxanthin. Even though this activity is similar to the CrtW-type of ketolase activity, the CrtO ketolases show no significant sequence homology to CrtW-type ketolases. The presence of six conserved regions may be a signature for the CrtO-type of -carotene ketolases.Communicated by E. Cerdá-Olmedo     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

A partially disarmed<Emphasis Type="Italic"> vir</Emphasis> helper plasmid,pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean

Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T_R) and a portion of T-left (T_L). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T₀ soybean plants derived by transformation using strain KYRT1 contained T_R from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T₀ plants contained T_L DNA. These results suggest that some function coded for by T_R of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.Abbreviations 2,4-D 2,4-Dichlorophenoxyactic acid - GUS -Glucuronidase - hpt Hygromycin phosphotransferase gene - SE Somatic embryo - uidA -Glucuronidase gene     

Fertile,transgenicTriticale ( ×Triticosecale Wittmack)

Fertile transgenicTriticale ( ×Triticosecale Wittmack) plants expressing the-glucuronidase (uidA) and phosphinothricin acetyltransferase (bar) genes were obtained after microprojectile bombardment of scutellar tissue with the plasmid pDB1 containing theuidA gene under the control of the actin-1 promoter (Act1) from rice and the selectable marker genebar under the control of the CaMV 35S promoter. From 465 bombarded scutella about 4000 plantlets were regenerated; 300 plants survived the selection. These regenerants were screened for enzyme activity by the histological GUS assay and by spraying the plants with a herbicide (Basta). Twenty-five regenerants showed GUS activity and survived repeated Basta spraying. Southern blot analysis showed the presence of both marker genes introduced into the genome of analysed plants.All transgenic plants were fertile. They were grown to maturity and set seed. Pollen and progeny analyses provided evidence for inheritance of the introduced genes to the next generation.