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Superoxide dismutase (SOD) plays an important role in cellular defense against oxidative stress in aerobic organisms. To generate cucumber (Cucumis sativus L.) fruits producing high yields of SOD for an anti-aging cosmetic material as a plant bioreactor, the CuZnSOD cDNA (mSOD1) from cassava was introduced into cucumber fruits by Agrobacterium-mediated transformation using the ascorbate oxidase promoter with high expression in fruits. The bialaphos-resistant shoots were selected on medium containing MS basal salts, 2 mg l-1 BA, 0.1 mg l-1 IAA, 300 mg l-1 claforan, and 2 mg l-1 bialaphos. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 1 mg l-1 IAA, 300 mg l-1 claforan, 2 mg l-1 bialaphos to induce roots. Southern blot analysis confirmed that the mSOD1 gene was properly integrated into the nuclear genomes of three cucumber plants tested. The mSOD1 gene was highly expressed in the transgenic cucumber fruits, whereas it was expressed at a low level in the transgenic leaves. The SOD specific activity (units/mg protein) in transgenic fruits was approximately 3 times higher than in those of non-transgenic plants. [ABSTRACT FROM AUTHOR]

Weed control is a substantial economic input for production of mint oils, the most commercially important of which are obtained from peppermint. The objective of this research is to obtain peppermint plants resistant to the broad-spectrum herbicide glufosinate, which can be used for development of economically efficacious weed control strategies and, perhaps, serve as a paradigm in perennial crops. The bar gene, which encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate-ammonium or phosphinothricin (PPT), was constructed into Agrobacterium tumefaciens binary vectors under the nopaline synthase (NOS) or a chimeric promoter containing a trimer of the OCS-upstream-activating sequence (UAS) to a MAS promoter/activator region[ (OCS)3 MAS]. A total of 142 independent transgenic peppermint (cv. Black Mitcham) plants were obtained (107 and 35 were obtained with pGPTV (and pCAS1) and pATC940 vectors, respectively) and evaluated for herbicide resistance in the greenhouse after foliar application of glufosinate herbicide Liberty as the commercial product. All transgenic plants exhibited substantially less herbicide symptom development than non-transgenic Black Mitcham or untransformed tissue cultured-derived plants, albeit variation for herbicide resistance occurred amongst the transformed lines. Plants from 35 of the 142 lines were selected at random and all were PCR-positive for the presence of bar. Five lines, that were least affected, exhibited no injury symptoms to Liberty concentrations that are 4 times the standard level for control of weeds in peppermint fields. The most resistant transgenic plants had the greatest steady-state PAT mRNA levels and PAT activities. No experimental difference in herbicide resistance was evident between plant populations obtained with pGPTV (pCAS1)-bar or pATC940-bar vector. However, 4 of 35 lines transformed with (ocs)3 MAS-bar exhibited maximal resistance while only 1 of 107 NOS-bar lines has comparable resistance. These herbicide resistant peppermint plants will facilitate development of post-emergent herbicide control strategies that use newer generation herbicides, like glufosinate, which have reduced environmental and product residual because of metabolism by microbes and the transgenic plants. [ABSTRACT FROM AUTHOR]

The Aspergillus niger phytase-encoding gene ( phyA) has been constitutively expressed in wheat. Transgenic wheat lines were generated by microprojectile bombardment of immature embryos, using the bar-Bialaphos selection system. The bar and the phyA gene expression were controlled by the maize ubiquitin-1 promoter. To ensure secretion and glycosylation of the microbial phytase, an expression cassette was designed (Ubi-SP-Phy) where an α-amylase signal peptide sequence was inserted between the promoter and the phytase coding region. A similar cassette was constructed without the signal peptide sequence (Ubi-Phy). Five lines of fertile wheat transformed with the Ubi-SP-Phy were generated and two lines with the Ubi-Phy construct. The inheritance of the phyA gene was monitored through three generations. Western blotting of leaf and seed derived protein revealed the presence of an immunoreacting polypeptide of the size expected for the Aspergillus phytase. Up to 25 days after pollination, the heterologous phytase was exclusively present in the pericarp-seed coat-aleurone fraction. Thereafter, it accumulated in the endosperm in amounts exceeding that found in the seed coat and aleurone. The phyA mRNA and derived protein could at no stage be detected in the embryo. The Ubi-SP-Phy transgenic seeds exhibited up to 4-fold increase of phytase activity while up to 56% increase was found in Ubi-Phy plants. It is concluded that a functional Aspergillus phytase can be produced in significant amounts in wheat grains. This may be of relevance for improving the phytate-phosphorus digestibility when wheat grains are used for non-ruminant animal feed. [ABSTRACT FROM AUTHOR]

Durum wheat ( Triticum turgidum L. var. durum) is traditionally used for the production of numerous types of pasta, and significant amounts are also used for bread-making, particularly in southern Italy. The research reported here centres on the glutenin subunits 1Dx5 and 1Dy10 encoded by chromosome 1D, and whose presence in hexaploid wheats is positively correlated with higher dough strength. In order to study the effects of stable expression of the 1Dx5 and 1Dy10 glutenin subunits in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area (‘Svevo’, ‘Creso’, ‘Varano’ and ‘Latino’) were co-transformed, via particle bombardment of cultured immature embryos, with the two wheat genes Glu-D1-1d and Glu-D1-2b encoding the glutenin subunits, and a third plasmid containing the bar gene as a selectable marker. Protein gel analyses of T1 generation seed extracts showed expression of one or both glutenin genes in four different transformed durum wheat plants. One of these transgenic lines, DC2-65, showed co-suppression of all HMW-GS, including the endogenous ones. Transgene stability in the transgenic lines has been studied over four generations (T1–T4). Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes from T4 plants showed that the integration of transgenes occurred in both telomeric and centromeric regions. The three plasmids were found inserted at a single locus in two lines and in two loci on the same chromosome arm in one line. The fourth line had two transgenic loci on different chromosomes: one with both glutenin plasmids and a different one containing only the construct with the gene encoding the 1Dy10 glutenin subunit. Segregation of these two loci in subsequent generations allowed establishment of two sublines, one containing both 1Dx5 and 1Dy10 and the other containing only 1Dy10. Small-scale quality tests showed that accumulation of Dx5, Dy10 or both in transgenic durum wheat seeds resulted in doughs with stronger mixing characteristics. [ABSTRACT FROM AUTHOR]

Maize ( Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat ( Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase ( pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T1 progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T2 plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T2 plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T2 wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation. [ABSTRACT FROM AUTHOR]

Rice inflorescences were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pJD4 with application of vacuum infiltration. After co-cultivation, callus was initiated and subjected to hygromycin selection, and plants were regenerated from resistant callus lines. Based on the total number of co-cultivated inflorescences bearing flowers 1 to 3 mm in length, the average frequency for recovering independent transgenic rice plants was at least 30%. Seeds from selfed R0 plants were harvested within 6 months after initiation of the experiments. Genomic DNA blot analysis showed that genes in the T-DNA of the binary plasmid were stably integrated into the rice genome, typically at low copy number. In most, but not all, cases the transgene was transmitted to R1 progeny at a frequency characteristic for Mendelian inheritance of a single dominant trait. For selfed progeny of one two-locus insertion line, reactivation of GUS expression was observed for a single copy locus that segregated from a silenced multicopy locus. For this line and some additional plants, fluorescence in situ hybridization was used to visualize the chromosomal location of the transgene insert. [ABSTRACT FROM AUTHOR]

Wheat ( Triticum aestivum L. cv. Hi-Line) immature embryos were transformed with the replicase gene (NIb) of wheat streak mosaic virus (WSMV) by the biolistic method. Six independent transgenic plant lines were analyzed for transgene expression and for resistance to mechanical inoculation of WSMV at R3 or R4 generation. Four out of the six lines showed various degree of resistance to WSMV. These lines had initially milder symptoms than controls, and the new growth ranged from milder symptoms, a substantial delay in symptom development, or asymptomatic. Two lines displayed higher resistance with very mild virus symptoms after inoculation and the new growth of 72% and 32% plants from these lines were asymptomatic and had no detectable virus through the plant life cycle. Interestingly, five out of the six transgenic lines had no detectable transgene mRNA expression by RNA gel blot hybridization. The only line that had detectable transgene mRNA did not show delay in the symptom development but had overall milder symptom to the virus. [ABSTRACT FROM AUTHOR]

The basic helix-loop-helix (bHLH) transcription factor has been shown to play an important role in various physiological processes. However, its functions and mechanisms in drought tolerance still remain poorly understood. Here, we reported a bHLH transcription factor — CgbHLH001 — from Chenopodium glaucum, which was able to confer drought tolerance in maize. CgbHLH001-overexpressed maize lines exhibited drought-tolerant phenotype and improved ear traits by accumulating the contents of soluble sugar and proline and elevating the activities of antioxidant enzymes (SOD, POD, and CAT) under drought stress, accompanying with the upregulation of some stress-related genes, which may balance the redox and osmotic homeostasis compared with the non-transgenic and CgbHLH001-RNAi plants. These findings suggest that CgbHLH001 can confer drought tolerance and has the potential for utilization in improving drought resistance in maize breeding. [ABSTRACT FROM AUTHOR]

A bioassay was developed for identifying transgenic wheat plants based on De Block et al.'s [8] ammonium-multiwell assay which allows qualitative and quantitative evaluation of the expression of the enzyme phosphinothricin acetyl transferase (PAT). Important parameters in the assay are the use of young leaf tissues, short incubation period (6 h) and a high light intensity during incubation (250 μmol s−1m−2). The assay is quick and results are obtained within a day. Ammonium measurements based on a colourimetric (modified Berthelot) reaction are conducted using sodium salicylate to avoid the use of phenol. Results of the assay show high correlation with Basta leaf painting tests and polymerase chain reaction (PCR) results. Thus, the assay may be used as a facile screen for bar-expressing transgenic cereals. [ABSTRACT FROM AUTHOR]

Maize functional genomics research and genetic improvement strategies have been greatly accelerated and refined through the development and utilization of genetic transformation systems. Maize transformation is a composite technology based on decades’ efforts in optimizing multiple factors involving microbiology and physical/biochemical DNA delivery, as well as cellular and molecular biology. This review provides a historical reflection on the development of maize transformation technology including the early failures and successful milestones. It also provides a current perspective on the understanding of tissue culture responses and their impact on plant regeneration, the pros and cons of different DNA delivery methods, the identification of a palette of selectable/screenable markers, and most recently the development of growth-stimulating or morphogenic genes to improve efficiencies and extend the range of transformable genotypes. Steady research progress in these interdependent components has been punctuated by benchmark reports celebrating the progress in maize transformation, which invariably relied on a large volume of supporting research that contributed to each step and to the current state of the art. The recent explosive use of CRISPR/Cas9-mediated genome editing has heightened the demand for higher transformation efficiencies, especially for important inbreds, to support increasingly sophisticated and complicated genomic modifications, in a manner that is widely accessible. These trends place an urgent demand on taking maize transformation to the next level, presaging a new generation of improvements on the horizon. Once realized, we anticipate a near-future where readily accessible, genotype-independent maize transformation, together with advanced genomics, genome editing, and accelerated breeding, will contribute to world agriculture and global food security. [ABSTRACT FROM AUTHOR]

The bioscavenger butyrylcholinesterase (BChE) can detoxify organophosphates used as chemical weapons or pesticides. It has potential use as a therapeutic or prophylactic treatment for exposure to these compounds as well. Current methods of obtaining BChE rely on extracting out-of-date blood supplies but this is not sufficient to meet potential needs in the event of large-scale population exposure. Therefore, in an effort to create a new low-cost source of the enzyme, we have stably expressed BChE in transgenic maize grain using constructs with a maize-optimized BChE coding region under control of either an endosperm-preferred or embryo-preferred promoter. Expression was also monitored after targeting to different subcellular locations. The highest level of enzyme activity was achieved with constructs under control of an endosperm-preferred promoter and targeted to the endoplasmic reticulum with accumulation of up to 50 mg BChE/kg seed. [ABSTRACT FROM AUTHOR]

Efforts in hybrid breeding have made this technology one of the main factors contributing to the substantial global rise in agricultural output over the last few decades. For hybrid breeding, an efficient pollination control system is necessary to avoid the unwanted self-pollination or sib-pollination of the female parental line. This review will provide a historical overview of pollination control systems and their use in hybrid crop breeding. We outline the prerequisites for commercial hybrid breeding and summarize the most important non-biological and biological technologies. Our main focus is on hybrid systems that are based on genetically engineered plants. We describe their suitability for pollination control, propagation of the male-sterile crossing partner, fertility restoration and mixed planting. Additionally, we report on the latest findings in the development of inducible sterility systems and various technologies that enable pollination control via metabolic engineering. We discuss the pros and cons of the different pollination control strategies. [ABSTRACT FROM AUTHOR]

We generated and characterized a transgenic wheat ( Triticum aestivum L.) population expressing constitutively an oat arginine decarboxylase ( ADC) cDNA. Transgenic wheat plants expressing the transgene had a significant increase in ADC activity with no concomitant changes in ODC activity. We measured 2-fold increases in putrescine, spermidine and spermine content in leaves of transgenic plants compared to wild-type. These plants also exhibited significant increases in the three polyamines in seeds (7-fold putrescine, 2.5-fold spermidine and 1.8-fold spermine). This significant polyamine accumulation was heritable in subsequent generations. In order to characterize molecularly this population we cloned a partial TaADC cDNA. This sequence was used as a probe to ascertain the presence of two distinct ADC transcripts in wheat. Further DNA analyses confirmed the presence of at least two distinct ADC genes in the wheat genome. Our results demonstrate that polyamine levels can be altered in transgenic wheat plants resulting in phenotypically normal and fertile plants. This transgenic wheat population will serve as useful germplasm to embark on further in depth studies to ascertain the role of polyamines in abiotic stress responses. [ABSTRACT FROM AUTHOR]

Organophosphate hydrolase (OPH, E.C. 3.1.8.1; encoded by the bacterial opd gene) provides a new scoreable and selectable genetic marker system for use in plant cell culture and regenerated plant tissue. OPH hydrolyzes a wide range of substrates that produce visually detectable products, which can be readily quantified in biological tissues. A variety of different OP compounds, both herbicides and pesticides, have been identified as acceptable enzymatic substrates, which can be used to generate transgenic markers for various types of plant tissues. For example, transgenic leaf tissue was easily differentiated from non-transgenic tissue by a simple fluorescent assay utilizing the OP insecticide coroxon. Transformed callus and intact whole seed could be easily distinguished from non-transformed tissue using novel non-destructive methods which allowed callus or seeds to grow and/or to germinate after phenotypic scoring with non-herbicidal OP insecticides such as paraoxon. In addition to being used as a scoreable phenotypic markers with various OP pesticides, the OP compounds Haloxon and Bensulide (Bensumec-4LF) were effective as positive selection agents for callus and germinating seeds. [ABSTRACT FROM AUTHOR]

To develop a salt-tolerant upland rice cultivar ( Oryza sativa L.), OsNHX1, a vacuolar-type Na+/H+ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T2 generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na+ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil. [ABSTRACT FROM AUTHOR]

Corn lines with improved culturability and transformability were produced using Marker Assisted Breeding (MAB) to introgress specific regions from the highly transformable hybrid, Hi-II, into the elite line, FBLL that responds very poorly in culture. FBLL is a female inbred parental stiff-stalk line that has been used to produce a series of some of DEKALB’s historically best selling hybrids. Five unlinked regions important for culturability and transformability were identified by segregation distortion analysis and introgressed into FBLL to produce the highly transformable FBLL-MAB lines. Agrobacterium mediated transformation was used to screen the FBLL-MAB lines and select the most efficient lines for transformation using immature embryo explants. Two highly efficient transformation systems were developed using kanamycin and glyphosate as selective agents. To evaluate agronomics, two testcross hybrids were produced for each of the three lead FBLL-MAB lines. A 25-location, 3-replication yield trial was used to evaluate grain yield, yield stability, and agronomic characteristics of the hybrids. Yields were found to be 2–5% lower and more stable (across a diverse set of environments) among hybrids produced with the FBLL-MAB lines as compared to the same hybrids produced with FBLL. [ABSTRACT FROM AUTHOR]

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric β-glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station. [ABSTRACT FROM AUTHOR]

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R1 and R2 generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system. [ABSTRACT FROM AUTHOR]

Extension of the vegetative growth phase through delay of flowering is an important goal in today’s breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species. [ABSTRACT FROM AUTHOR]

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer. [ABSTRACT FROM AUTHOR]

So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean (Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial aspartate kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals. [ABSTRACT FROM AUTHOR]

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens. [ABSTRACT FROM AUTHOR]

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the β-glucuronidase reporter gene ( gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T1 progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome. [ABSTRACT FROM AUTHOR]

Improved gene transfer techniques are necessary to obtain adequatenumbers of stable transgenic wheat plants needed for practical purposes.Considering that wheat transformation is genotype-dependent, we used cv. Combiin all experiments, which had been selected from agronomically important Germanspring wheat cultivars because of its high transformation ability. In mostwheatgene transfer attempts, immature embryos or embryogenic scutellar calli weremicrobombarded. We compared both methods under optimised conditions, using bar, uidA, and gfp as markers in co-transformation attempts. Integrationof the genes mentioned above was proven by Southern blotting, expression levelswere measured by assays on phosphinothricin acetyltransferase andβ-glucuronidase activities, and by monitoring for green fluorescentproteinin most developmental stages. Following bombardment of scutellar calli, anaverage transformation frequency of 0.13% was attained. Using immature embryos,mean transformation frequency (1.06%) was 8-fold higher. In addition, embryotechniques were over 2 weeks faster than scutellar callus procedures.Introducing gfp as a vital marker led to an improvement ofembryo-based techniques. In a first screening, transient gfp-expressing embryos were transferred tophosphinothricincontaining callus medium. Only gfp-expressing calli whichdeveloped on it were cultured further on phosphinothricin containingregeneration medium. Shoots obtained from gfp-expressingcalli were rooted on phosphinothricin-free medium, and cultured exvitro. Average transformation frequency (4.93%) was 38-fold higherthan with scutellar callus techniques. Differences between the transformationstrategies used were of high statistical significance. Combining greenfluorescent protein screening with phosphinothricin selection in embryo-basedtechniques offers a promising system to obtain high wheat transformationfrequencies. [ABSTRACT FROM AUTHOR]

Drought and salt stresses are two major factors that lower plant productivity. Transgenic approaches offer powerful means to better understand and then minimize loss of yield due to these abiotic stresses. In this study, we have generated transgenic rice plants expressing a wheat LEA group 2 protein (PMA80) gene, and separately the wheat LEA group 1 protein (PMA1959) gene. Molecular analysis of the transgenic plants revealed the stable integration of the transgenes. Immunoblot analysis showed the presence of the LEA group 2 protein (39 kDa) and the LEA group 1 protein (25 kDa) in most of the plant lines. Second-generation transgenic plants were subjected to dehydration or salt stress. The results showed that accumulation of either PMA80 or PMA1959 correlates with increased tolerance of transgenic rice plants to these stresses. [ABSTRACT FROM AUTHOR]

In this study, six plasmids were constructed to study the effects of the tobacco Rb7 matrix attachment region (MAR) sequence on rice transgene expression. Among them, each of four plasmids contained two identical copies of the MAR sequence flanking two different reporter genes, which encode the green fluorescent protein and β-glucuronidase. Two control plasmids contained no MAR sequences. Microprojectile bombardment was used to separately introduce these six plasmids into rice calli. Transgenic rice plants were regenerated, and gene expression was measured in rice leaf extracts of two-month-old transgenic plants. By comparing transgenic plants with the corresponding control plants, transgenic plants harboring each of four plasmids that included the MAR sequence resulted in average gene expression levels enhanced by 3.3-fold, 18-fold, 376-fold and 650-fold. These results suggest that the same MAR sequence can affect the expression of different genes to different extents. One goal of plant scientists and breeders is to maximize expression levels of transgenes, especially in the production of transgene-encoded protein. Therefore, inclusion of the Rb7 MAR sequence would be beneficial. [ABSTRACT FROM AUTHOR]

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes. [ABSTRACT FROM AUTHOR]

Transgenic plants were obtained after particle bombardment of embryogenic callus derived from stem segments of two tetraploid Alstroemeria genotypes with plasmids containing different selection/reporter genes. Firstly, a plasmid containing a firefly luciferase reporter gene driven by the maize ubiquitin promoter ( Ubi1), was bombarded into both friable embryogenic callus and proembryos. Transient and stable expression of luciferase was visually detected by a luminometer. This selection method is non-destructive and can be applied over the whole developmental process from callus to embryo and plantlet. Molecular proof of transformation was obtained both by PCR analysis and Southern hybridization. Secondly, a plasmid containing the bar gene together with an uidA gene coding for β-glucuronidase both driven by the Ubi1 promoter was bombarded into proembryos. The transgenic callus was effectively selected from the callus clumps four months after bombardment on a medium containing 5 mg/l phosphinotricin (PPT). Selection by PPT was efficient and labour-saving. Stable expression of GUS was confirmed by the histochemical staining assay and molecular proof was obtained by PCR analysis. [ABSTRACT FROM AUTHOR]

The feasibility of producing plant cell wall polysaccharide-hydrolysing feed enzymes in the endosperm of barley grain was investigated. The coding region of a modified xylanase gene ( xynA) from the rumen fungus, Neocallimastix patriciarum, linked with an endosperm-specific promoter from cereal storage protein genes was introduced into barley by Agrobacterium-mediated transformation. Twenty-four independently transformed barley lines with the xylanase gene were produced and analysed. The fungal xylanase was produced in the developing endosperm under the control of either the rice glutelin B-1 ( GluB-1) or barley B1 hordein ( Hor2-4) promoter. The rice GluB-1 promoter provided an apparently higher expression level of recombinant proteins in barley grain than the barley Hor2-4 promoter in both transient and stable expression experiments. In particular, the mean value for the fungal xylanase activity driven by the GluB-1 promoter in the mature grains of transgenic barley was more than twice that with the Hor2-4 promoter. Expression of the xylanase transgene under these endosperm-specific promoters was not observed in the leaf, stem and root tissues. Accumulation of the fungal xylanase in the developing grains of transgenic barley followed the pattern of storage protein deposition. The xylanase was stably maintained in the grain during grain maturation and desiccation and post-harvest storage. These results indicate that the cereal grain expression system may provide an economic means for large scale production of feed enzymes in the future. [ABSTRACT FROM AUTHOR]

The development of genetic transformation technology for plants has stimulated an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutical value. In this regard, we report the generation and characterization of transgenic maize lines producing recombinant aprotinin. The transgenic aprotinin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced into immature maize embryos via particle bombardment. Aprotinin gene expression was driven by the maize ubiquitin promoter and protein accumulation was targeted to the extracellular matrix. One line that showed a high level of aprotinin expression was characterized in detail. The protein accumulates primarily in the embryo of the seed. Southern blot analysis showed that the line had at least 20 copies of the bar and aprotinin genes. Further genetic analysis revealed that numerous plants derived from this transgenic line had a large range of levels of expression of the aprotinin gene (0–0.069%) of water-soluble protein in T2 seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental transgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T2 lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of the purified aprotinin from seeds revealed that the recombinant aprotinin had the same molecular weight, N-terminal amino acid sequence, isoelectric point, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seeds has biochemical and functional properties identical to its native counterpart provides a proof-of-concept example for producing new generation products for the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects. [ABSTRACT FROM AUTHOR]

The microbial bar and pat genes confer tolerance to the non-selective herbicide phosphinothricin (PPT; sold as Basta or Finale). This tolerance in plants could provide an environmental gain compared to current-day herbicide cocktails, but the safety of such a transgene approach is questioned by many. The biosafety of the presence of these herbicide tolerance genes in plants is evaluated in a ‘transgene-centered approach’. Potentially, the introduction of transgenic PPT-tolerant crops could result in acquired PPT tolerance in weedy relatives of these crops. Assuming responsible use of this trait in agronomy, the ecological consequences with respect to weediness or spread of the transgenic PPT tolerance are concluded to be negligible. The key issue for the toxicological evaluation is whether or not the plant has actually been sprayed with PPT. Consumption of the gene and/or gene product from unsprayed transgenic plant material will not have adverse effects. In case of PPT-sprayed material, PPT or its derivatives could be present in food and feed and crop-specific metabolites might be formed. To date, the toxicological impact of such a putative exposure is not sufficiently clear, and further premarket testing is recommended. [ABSTRACT FROM AUTHOR]

We have generated transgenic maize seed containing β-glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant β-glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant β-glucuronidase (GUS) was compared to native β-glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a Km of 0.20 mM and a Vmax of 0.29 mM MUG per hour, and the native enzyme had a Km and Vmax of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of β-glucuronidase, the Ki of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor. [ABSTRACT FROM AUTHOR]

In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits. [ABSTRACT FROM AUTHOR]

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces. An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at −29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods. [ABSTRACT FROM AUTHOR]

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs. Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation. [ABSTRACT FROM AUTHOR]

Transient expression of the maize anthocyanin regulatory elements, R and C1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5-8×10 cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression of R and C1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression of luc, aphA or bar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. When luc and aphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67-79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane. [ABSTRACT FROM AUTHOR]