Transgenic Oat Research Articles

Metabolic engineering-induced transcriptome reprogramming of lipid biosynthesis enhances oil composition in oat.

The endeavour to elevate the nutritional value of oat (Avena sativa) by altering the oil composition and content positions it as an optimal crop for fostering human health and animal feed. However, optimization of oil traits on oat through conventional breeding is challenging due to its quantitative nature and complexity of the oat genome. We introduced two constructs containing three key genes integral to lipid biosynthesis and/or regulatory pathways from Arabidopsis (AtWRI1 and AtDGAT1) and Sesame (SiOLEOSIN) into the oat cultivar 'Park' to modify the fatty acid composition. Four homozygous transgenic lines were generated with a transformation frequency of 7%. The expression of these introduced genes initiated a comprehensive transcriptional reprogramming in oat grains and leaves. Notably, endogenous DGAT, WRI1 and OLEOSIN genes experienced upregulation, while genes associated with fatty acid biosynthesis, such as KASII, SACPD and FAD2, displayed antagonistic expression patterns between oat grains and leaves. Transcriptomic analyses highlighted significant differential gene expression, particularly enriched in lipid metabolism. Comparing the transgenic oat plants with the wild type, we observed a remarkable increase of up to 34% in oleic acid content in oat grains. Furthermore, there were marked improvements in the total oil content in oat leaves, as well as primary metabolites changes in both oat grains and leaves, while maintaining homeostasis in the transgenic oat plants. These findings underscore the effectiveness of genetic engineering in manipulating oat oil composition and content, offering promising implications for human consumption and animal feeding through oat crop improvement programmes.

A stress-responsive transcription factor PeNAC1 regulating beta-D-glucan biosynthetic genes enhances salt tolerance in oat.

A Populus euphratica NAC gene regulates (1,3; 1,4)-β-D-glucan content in oat developing seed and improves the spikelet number and grain number per spike in transgenic oat under salinity conditions Salinity is the major factor affecting the production and quality of oat, and improving oat salt tolerance to increase yield and quality is vital. (1,3;1,4)-β-D-glucan in Gramineae is the key component in response to various environmental signals, and it is the most important functional ingredient in oat grain. The NAC transcription factors are important candidate genes used in genetic engineering to improve plant abiotic stress tolerance. In this study, we introduced Populus euphratica PeNAC1, controlled by its own promoter, into hexaploid cultivated oat and produced six transgenic lines. Compared to the non-transgenic control, the expression of PeNAC1 significantly improved the seed germination rate, seedling survival rate, and leaf chlorophyll content in the transgenic plants under salt stress. These physiological changes increased the spikelet number and grain number per spike in the transgenic oat under salinity conditions and reduced the yield loss per plant. The results indicated that the heterologous expression of PeNAC1 plays an effective role in improving the salt tolerance in transgenic oat. In addition, overexpressing PeNAC1 significantly increased the (1,3;1,4)-β-D-glucan content as well as the expression level of the (1,3;1,4)-β-D-glucan biosynthetic genes AsCslF3, AsCslF6, and AsCslF9 in the transgenic lines under salt stress, which suggested that PeNAC1 regulates the synthesis of (1,3;1,4)-β-D-glucan. Our research should assist in the discovery of the diverse action modes of NAC proteins, while PeNAC1 will be useful for improving the salt tolerance and quality of oat through molecular breeding.

Changes in the concentration of avenanthramides in response to salinity stress in CBF3 transgenic oat

Oat avenanthramides have long been known to possess potential nutraceutical and therapeutical properties. The change in avenanthramides 2p, 2c and 2f concentration in four salinity tolerant transgenic oat plants containing CBF3 gene and non-transgenic control exposed to different levels of salinity stress was investigated. Determination of oat avenanthramides at the nano-scale level was performed using a well-optimized and highly sensitive sequential injection chemiluminescence (SIA-CL) method enhanced by eco-friendly gold nanoparticles biosynthesized from oat biomass extract. Under the conditions of this study, the predominant avenanthramide, which also exhibited the strongest scavenging capacity, was 2c followed by 2p and 2f. At no stress, there was no significant (p ≤ 0.05) difference between the transgenic lines and control regarding the concentrations of the three determined avenanthramides. After exposure to 250 mmol L−1 NaCl, avenanthramide 2c dramatically increased by 170.9%, 580%, 353.6%, 457.6% and 229.1% in the control and the four transgenic lines, respectively. Among the transgenic lines, Agrogle-1 maintained the highest avenanthramides concentration under all salinity levels with maximum values of 71.5 mg kg−1 for 2p, 221.0 mg kg−1 for 2c and 62.0 mg kg−1 for 2f detected at 250 mmol L−1NaCl. The results of this study demonstrated that oat avenanthramides might have a potential role in enhancing abiotic stress tolerance in oats.

Agrobacterium-Mediated Transformation of Leaf Base Segments.

Agrobacterium-mediated transformation has become a routine method of genetic engineering of cereals, gradually replacing the biolistic protocols. Simple integration patterns of transgenic loci, decent transformation efficiency, and technical simplicity are the main advantages offered by this method. Here we present a detailed protocol for the production of transgenic oat plants by Agrobacterium-mediated transformation of leaf base segments. The use of leaf explants as target tissues for transformation and in vitro regeneration of transgenic plants may be a good alternative for genotypes which are not susceptible to regeneration from immature or mature embryos. We also describe the biochemical and molecular analysis procedures of the transgenic plants including a GUS histochemical assay, and Southern blot, both of which are optimized for application in oat.

Physiological and biochemical changes of CBF3 transgenic oat in response to salinity stress

Salinity is a major abiotic constraint affecting oat productivity. Several physiological and biochemical traits have been found to be related to yield maintenance under salinity. The impact of introducing the Arabidopsis CBF3 gene controlled by the rd29A stress-inducible promoter in T2 transgenic oat on salinity tolerance and associated physiological changes were studied. Compared with the non-transgenic control, transgenic T2 plants exhibited greater growth and showed significant maintenance of leaf area, relative water content, chlorophyll content, photosynthetic and transpiration rates as well as increased levels of proline and soluble sugars under high salt stress. These physiological changes delayed leaf-wilting symptoms, increased tolerance and reduced yield loss. At a salinity stress level of 100mM, the CBF3-overexpressing transgenic oat showed a yield loss of 4–11% compared with >56% for the non-transgenic control. These results demonstrate that stress-inducible over-expression of CBF3 may have the potential to enhance abiotic stress tolerance in oat.

Aluminum resistance mechanisms in oat (Avena sativa L.)

Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known. A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24 h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat. Oat seedlings were highly resistant to Al, as a concentration of 325 μM AlK(SO4)2 was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion. Release of malate from oat roots was associated with Al resistance, which suggests that malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement.

Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties in vitro

Transgenic anti-fungal gene expression in heterologous species provides a means to test resistance protein combinations across species barriers. This is the first report of transgenic anti-fungal seed storage protein accumulation in oat seed. An anti-fungal barley (Hordeum vulgare L.) hordothionin (Hthl) gene was genetically engineered into oat (Avena sativa L.) to determine the effect of hordothionin on pathogen resistance. The transgene was expressed in both leaf and seed tissue, with transgenic protein accumulation occurring only in the seed. Transgenic oat line HTH-Av5 expressed c. 94 μg HTH/g seed, 19% of native barley seed levels. The anti-fungal activities of HTH fractions from barley cv. Morex and oat (transgenic and control) were tested in an in vitro growth assay against an important small grain pathogen. Fusarium graminearum. The partially purified HTH fractions from control oat seeds did not inhibit fungal growth, while HPLC-purified HTH positive control, as well as partially purified barley and transgenic oat HTH inhibited growth similarly over a range of concentrations. These results indicate hordothionin can be expressed in a heterologous cereal species and still maintain its anti-fungal properties. Future studies with HTH targeted to additional tissues are planned to test for increased fungal resistance.

Barley HVA1 Gene Confers Salt Tolerance in R3 Transgenic Oat

A major cause of oat crop yield loss worldwide is osmotic stress due to drought and/or salinity. This study investigated the third generation of transgenic oat (Avena sativa L.) expressing barley HVA1 stress tolerance, β‐glucuronidase (uidA; gus) and bar herbicide resistance genes. Transgenic plants showed normal 9:7 third generation inheritance for glufosinate ammonium herbicide resistance. Molecular and histochemical studies confirmed the presence and stable expression of all three genes. Compared with the nontransgenic control plants, transgenic R3 plants exhibited greater growth and showed a significant (P < 0.05) increase in tolerance to salt stress conditions (200 mM NaCl) for traits including number of days to heading, plant height, flag leaf area, root length, panicle length, number of spikelets/panicle, number of tillers/plant, number of kernels/panicle, 1000‐kernel weight, and kernel yield/plant.

Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures

Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures

Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment.

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.

Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures.

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic oat ( Avena sativa L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by a rice actin promoter. In vitro shoot meristematic cultures (SMCs) induced from shoot apices of germinating mature seeds of a commercial oat cultivar, Garry, were used as a transformation target. Proliferating SMCs were bombarded with a mixture of plasmids containing the sgfp(S65T) gene and one of three selectable marker genes, phosphinothricin acetyltransferase (bar), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII). Cultures were selected with bialaphos, hygromycin B and geneticin (G418), respectively, to identify transgenic tissues. From 289 individual explants bombarded with the sgfp(S65T) gene and one of the three selectable marker genes, 23 independent transgenic events were obtained, giving a 8.0% transformation frequency. All 23 transgenic events were regenerable, and 64% produced fertile plants. Strong GFP expression driven by the rice actin promoter was observed in a variety of tissues of the T(0) plants and their progeny in 13 out of 23 independent transgenic lines. Stable GFP expression was observed in T(2) progeny from five independent GFP-expressing lines tested, and homozygous plants from two lines were obtained. Transgene silencing was observed in T(0) plants and their progeny of some transgenic lines.

Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement.

To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of the delivered DNA, and inverted and imperfect direct repeats were detected in the same transgene locus indicating that homologous recombination and synthesis-dependent mechanism(s), respectively, were also involved in transgene locus rearrangement. The most unexpected result was the small size of the fragments of delivered and genomic DNA incorporated into the transgene loci via illegitimate recombination; 50 of the 82 delivered DNA fragments were shorter than 200 bp. Eleven transgene and genomic fragments were shorter than the DNA lengths required for Ku-mediated non-homologous end joining. Detection of these small fragments provided evidence that illegitimate recombination was most likely mediated by a synthesis-dependent strand-annealing mechanism that resulted in transgene scrambling. Taken together, these results indicate that transgene locus formation involves the concerted action of several DNA break-repair mechanisms.

Genomic interspersions determine the size and complexity of transgene loci in transgenic plants produced by microprojectile bombardment

The structure of transgene loci in six transgenic allohexaploid oat (Avena sativa L.) lines produced using microprojectile bombardment was characterized using fluorescence in situ hybridization (FISH) on extended DNA fibers (fiber-FISH). The transgene loci in five lines were composed of multiple copies of delivered DNA interspersed with genomic DNA fragments ranging in size from ca. 3 kb to at least several hundred kilobases, and in greater numbers than detected using Southern blot analysis. Although Southern analysis predicted that the transgene locus in one line consisted of long tandem repeats of the delivered DNA, fiber-FISH revealed that the locus actually contained multiple genomic interspersions. These observations indicated that transgene locus size and structure were determined by the number of transgene copies and, possibly to a greater extent, the number and the length of interspersing genomic DNA sequences within the locus. Large genomic interspersions detected in several lines were most likely the products of chromosomal breakage induced either by tissue culture conditions or, more likely, by DNA delivery into the nucleus using microprojectile bombardment. We propose that copies of transgene along with other extrachromosomal DNA fragments are used as patches to repair double-strand breaks (DSBs) in the plant genome resulting in the formation of transgene loci.

Erratum to: “High frequency of cytogenetic aberration in transgenic oat ( Avena sativa L.) plants” : [Plant Science 156 (2000) 85–94

Erratum to: “High frequency of cytogenetic aberration in transgenic oat ( Avena sativa L.) plants” : [Plant Science 156 (2000) 85–94

Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from 55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expression of gfp, demonstrating efficiency and repeatability of the selection system.

High frequency of cytogenetic aberration in transgenic oat ( Avena sativa L.) plants

Cytological abnormalities were observed in transgenic oat ( Avena sativa L. cv. GAF/Park-1) produced by microprojectile bombardment of mature seed-derived highly regenerative tissues. Of the plants from 48 independent transgenic lines examined, plants from only 20 lines (42%) were karyotypically normal (2n=6x=42) without detectable chromosomal aberrations; plants from 28 lines (58%) had chromosomal variation, i.e. aneuploids and structural changes. No significant difference in cytological aberration was observed between the two different culturing systems used for transformation: 57% chromosomal abnormalities in plants derived from D′BC2 medium (2.0 mg/l 2,4-D, 0. 1 mg/l BAP and 5.0 μM cupric sulfate) used for tissue initiation and maintenance and 60% in plants from tissue initiated on D′BC2 and maintained on DBC3 (1.0 mg/l 2,4-D, 0.5 mg/l BAP and 5.0 μM cupric sulfate). Comparative differences in chromosomal status frequently occurred among plants regenerated from the same T 0 line. The most common cytological aberration in transgenic plants was aneuploidy, followed by deletion of chromosomal segments; no change in ploidy level was observed. In contrast, nontransgenic plants, regenerated from tissues comparable in age and culture media to that used for transgenic tissues, had a much lower percentage of karyotypic abnormality (0–14%). Our data indicate that some stress(es) imposed by the transformation process, e.g. osmotic treatment, bombardment and selection, leads to cytological variation in transgenic oat plants, an observation similar to that observed in our recent studies with transgenic barley plants.

Transgenic oat plants via visual selection of cells expressing green fluorescent protein.

New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines.

Association of transgene integration sites with chromosome rearrangements in hexaploid oat

Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombardment were characterized using phenotypic and genotypic segregation, Southern blot analysis, and fluorescence in situ hybridization (FISH). Twenty-five transgene loci were detected; 8 lines exhibited single transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A/D-genome-specific dispersed and clustered repeats showed no preferences in the distribution of transgene loci among the highly heterochromatic C genome and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes. Transgene integration sites were detected at different locations along individual chromosomes, although the majority of transformants had transgenes integrated into subtelomeric and telomeric regions. Transgene integration sites exhibited different levels of structural complexity, ranging from simple integration structures of two apparently contiguous transgene copies to tightly linked clusters of multiple copies of transgenes interspersed with oat DNA. The size of the genomic interspersions observed in these transgene clusters was estimated from FISH results on prometaphase chromosomes to be megabases long, indicating that some transgene loci were significantly larger than previously determined by Southern blot analysis. Overall, 6 of the 25 transgene loci were associated with rearranged chromosomes. These results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal breakage and rearrangements.

Genetic transformation of commercial cultivars of oat ( Avena sativa L.) and barley ( Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T0 plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T1 seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T1 and T2 progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T1 and T2 progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T1 and T2 progeny tested.

Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA.

Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35-280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.