Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Enhanced GUS gene expression in cereal/grass cell suspensions and immature embryos using the maize uhiquitin-based plasmid pAHC25

Transient GUS (-glucuronidase) expression was visualized in cell suspensions of Triticum aestivum, Zea mays, Pennisetum glaucum, Saccharum officinarum, Pennisetum purpureum and Panicum maximum after microprojectile bombardment with pBARGUS and pAHC25 plasmid DNAs. pBARGUS contains the GUS (UidA) gene coding region driven by the Adh1 promoter and the Adh1 intron 1, as well as the BAR gene coding region driven by the CaMV 35S promoter and the Adh1 intron 1. pAHC25 contains the GUS and BAR gene coding regions driven by the maize ubiquitin promoter, first exon and first intron (Ubi1). The effectiveness of the constructs was first compared in cell suspension cultures by counting blue expression units (b.e.u.). The expression of construct pAHC25 ranged from 3 to 50 fold greater than pBARGUS in different species. In addition, the two plasmids were quantitatively compared in Triticum aestivum and Zea mays by using the more sensitive GUS fluorometric assay to determine the amount of methylumbellyferride (MU) produced. There was more than a 30 fold increase in MU production with pAHC25 than with pBARGUS in the wheat suspension, while the maize suspension showed only a 2.5 fold increase with the pAHC25 construct. Transient GUS expression was also visualized in immature embryos of Pennisetum glaucum following bombardment with pBARGUS and pAHC25 DNA. Expression of plasmid pAHC25 was twice as high as pBARGUS. A comparison of two DNA/gold preparation methods, as well as repeated sonications of the DNA/gold mixture, had no effect on the number of b.e.u.     

Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment

The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.     

Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content in grain starch

The waxy gene encodes a granule-bound starch synthase. A 1.0-kb portion of the sequence of the rice waxy gene, which includes the region between exon 4 and exon 9, was inserted in an antisense orientation between the 35 S promoter and the GUS gene of pBI221. The resultant plasmid, pWXA23, was introduced into rice protoplasts by electroporation. GUS activity was clearly detected in derived callus lines, suggesting that the antisense component of the fusion gene was also expressed. Transgenic rice plants were regenerated from these callus lines and their GUS activity was confirmed. Some of the rice seeds from these transformants showed a significant reduction in the amylose content of grain starch, even though they had become polyploid. These results suggest that even when intron sequences are included, antisense constructs can bring about a reduced level of expression of a target gene. The utility of GUS, included as a reporter gene, for the simple detection of expression of an antisense gene, was apparent from these results.     

Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.)

A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15–21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.Abbreviations DMSO dimethyl sulfoxide - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtylacetic acid - FDA fluorescein diacetate     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.)

Embryogenic callus initiated from basal segments of micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable of yielding large numbers of protoplasts which underwent sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast-derived callus and are currently being grown to maturity. This is the first report of plant regeneration from protoplasts of a wild species of Oryza.Abbreviations BAP 6-benzyalamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2[N-morpholino] ethanesulphonic acid - NAA -naphthalene acetic acid - PAR photosynthetically active radiation - SCV settled-cell volume     

Transient expression of chimaeric genes in dividing and non-dividing cereal protoplasts after PEG-induced DNA uptake

Transient expression of chimaeric genes (neomycin phosphotransferase fused to four different promoters) was detected in suspension culture derived protoplasts of maize, barley and rice, in mesophyll protoplasts of maize, rice, rye, and root protoplasts of maize. The introduction and expression of foreign genes could be performed with both dividing and non-dividing protoplasts by applying the PEG transformation method. The significance of this method for the functional analysis of genes was demonstrated by the differential expression of a regulated gene in protoplasts of different tissues in agreement with its expression in the donor tissue.Abreviations PEG polyethylene glycol - NPT neomycin phosphotransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - caseinh. caseinhydrolysate     

Intron-mediated enhancement of heterologous gene expression in maize

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5 translated region but not when it was located upstream of the promoter or within the 3 untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity of mRNA.     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

Intron position affects expression from thetpi promoter in rice

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the firsttpi intron, and the gene encoding bacterial -glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the ricetpi gene enhanced expression of the GUS gene from thetpi promoter when it was placed 5 of the GUS gene. When thetpi intron was placed in the 3-untranslated region no enhancement of GUS gene expression was observed, indicating the importance of position in intron-mediated enhancement of gene expression.     

Evaluation of five promoters for use in transformation of oil palm (Elaeis guineensis Jacq.)

The efficiency of GUS (-Glucuronidase) gene expression in embryogenic callus and young leaflets of mature and seedling palm after microprojectile bombardment with five constructs (pEmuGN, pAHC25, pAct1-F4, pGH24 and pBARGUS) was evaluated to identify the most suitable promoter(s) to use in transformation attempts in oil palm. Expression of the GUS gene driven by theEmu, Ubi1, Act1 35S orAdh1 was assayed, both histochemically and fluorometrically, from a total of 200 plates of tissues in eight independent experiments two days after bombardment. A completely randomized experimental design was used for each experiment, and the data analysed by ANOVA and Duncan's Multiple Range Test. The expression level of GUS driven by theEmu orUbi1 promoters was significantly higher than that of the Act], 35S and Adhl promoters in many experiments, and that of theAdhl was significantly lower than those of the other four promoters. Both histochemical and fluorometric data indicate that in embryogenic callus, the expression of theEmu promoter was higher than that of theUbi1 whereas in young leaflets from mature palm the Ubi1 expression was stronger. The performances of the five promoters were also tested in tobacco callus using a fluorometric GUS assay. The activity of the 35S promoter was highest, and significantly different from that of all the other promoters except theEmu, and that of theAct1 promoter was lowest. These results indicate that either theUbil orEmu promoter should facilitate the expression of desired genes in oil palm and aid in development of an efficient stable transformation system.Abbreviations GUS -Glucuronidase - EC embryogenic callus - YLMP young leaflet from mature palm - YLSP young leaflet from seedling palm - MU methyl umbelliferone - MUG 4-methyl--D-glucuronide - X-glue 5-bromo-4-chloro-3-indoyl-glucuronide - Ubil maize ubiquitin 1 - Actl rice actin 1 - Adh1 maize alcohol dehydrogenase 1 - Emu a recombinant truncated maize alcohol dehydrogenase 1 - ANOVA Analysis of variance - DMRT Duncan's Multiple Range Test Communicated by W A. Parrott     

Fertile Transgenic Indica Rice Produced by Expression of Maize Ubiquitin Promoter-bar Chimaeric Gene in the Protoplasts

We report production of fertile transgenic Indica rice plants by transferring a chimaeric construct consisting of promoter, first exon and intron of maize ubiquitin gene (Ubi-1) and the coding sequences of the bar gene from Streptomyces hygroscopicus to the rice protoplasts through electroporation. In total, 11 plants were regenerated. All of them were fertile and set seeds on maturity. These plants were resistant to high concentration of PPT (400 mg l^?1) which was otherwise toxic to the untransformed controls. The gene was inherited to the progenies of the five plants in Mendelian ratio.     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.)

Protoplasts isolated from embryogenic (Mustang and Chinese Spring) and non-embryogenic (Mit) calli of wheat (Triticum aestivum L.) genotypes transiently expressed -glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm^-1 and 250 F for 2 s using 50 g/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.     

Stably transformed herbicide resistant callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts

Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.Abbreviations GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - PMSF phenylmethylsulfonyl fluoride - MES 2[N-Morpholino]ethanesulfonic acid - HEPES [N-2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] - PAT Phosphinothricin acetyltransferase - CTAB cetyltrimethylammonium bromide     

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects.The 1.9 kb 5-upstream fragment (–1559 to +342) of the CatA gene was fused with the Escherichia coli -glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between –1564 to –699. Abscisic acid (ABA) at a final concentration of 10^–6 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at –266 to –254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light.The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.author for corresondenceThe nucleotide sequence data reported will appear in the DDBJ EMBL and GenBank Nucleotide Sequence Databases under the accession number D29966.