Transformation of oat and inheritance of bar gene expression

Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus, with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected uidA gene were detected. Stable transmission and expression of the bar gene in the T₁ inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T₂ lines tested from this transformant. This revised version was published online in June 2006 with corrections to the Cover Date.     

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.     

In situ localization of faba bean and oat legumin-type proteins in transgenic tobacco seeds by a highly sensitive immunological tissue print technique

We have used a highly sensitive immunological tissue print technique to study cell- and tissue-specific expression of heterologous genes in transgenic plants. Primary polyclonal antibodies, raised against legumin of faba bean (Vicia faba L.) and 12S globulin of oat (Avena sativa L.) were used to localize these proteins in transgenic tobacco seeds in a streptavidin-alkaline phosphatase assay in combination with biotinylated secondary antibodies producing a higher sensitivity (by several amplification steps) of the assay. Both storage protein genes were found to be expressed in a specific pattern. While legumin is preferentially accumulated in certain parts of the embryo, the oat legumin-type globulin is restricted to the endosperm. The applied technique is highly sensitive with a resolution power down to the singlecell level and allows rapid screening of large numbers of samples.     

Purification and characterization of a 60-kDa protein from oat, formerly known as a TCP1-related chaperone

Recently, Mummertet al. [Nature 363, 644–648 (1993)] isolated a proposed TCP1-related chaperone. Here we report several findings concerning the protein which they sequenced. Two similar N-terminal sequences were obtained from this abundant 60-kDa protein. Internal sequences were also acquired by protease digestion. Initially it was believed the protein was able to completely inhibit citrate synthase aggregation, but later purifications demonstrated that the 60-kDa polypeptide lacked both chaperone activity and the previously reported kinase activity [Grimmet al., Planta 178, 199–206 (1989)]. It is now our belief that this protein is neither a chaperone nor a kinase.     

Anoxia tolerance and ethanol sensitivity of rice and oat coleoptiles

Anoxia tolerance and ethanol sensitivity of rice (Oryza sativa L.) and oat (Avena sativa L.) seedlings were evaluated to clarify their growth habit in anoxia. Anoxic stress inhibited elongation and dry weight gain of coleoptiles of the oat and rice seedlings; however, the inhibition of the oat coleoptiles was much greater than that of the rice coleoptiles. Anoxic stress increased endogenous ethanol concentration and alcohol dehydrogenase activity in oat and rice coleoptiles and their increases in the rice coleoptiles were much greater than those in the oat coleoptiles. At concentrations greater than 30 mM and 300 mM, exogenously applied ethanol inhibited the elongation and weight gain for the oat and the rice coleoptiles, respectively, and the inhibition was increased with increasing ethanol concentrations with marked inhibition being achieved on the oat coleoptiles. These results suggest that anoxia tolerance and induction of ethanolic fermentation in anoxia may be greater in rice than oat, and ethanol sensitivity of rice may be lower than that of oat.     

Evidence for translational control of storage protein biosynthesis during embryogenesis ofAvena sativa L. (oat endosperm)

Oat polysomes direct the synthesisin vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A⁺ RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased amount of the other products synthesizedin vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments using oat poly A⁺ mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is partially responsible for the levels of globulin in the mature grain.     

Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

In vivo and in vitro effects of cadmium on H+ ATPase activity of plasma membrane vesicles from oat (Avena sativa L.) roots

The effect of an in vivo and in vitro treatment with cadmium on transport activities of root plasma membrane enriched vesicles was studied in oat (Avena sativa L. cv. Argentina) plants. Addition of 100 mumol/L CdSO4 to nutrient solution decreases both proton transport activity and ATPase activity to the same level. In vitro experiments show that cadmium seems to have a differential inhibiting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H(+)-accumulation, suggesting that cadmium would interfere with membrane permeability properties. This is indeed the case. The results demonstrate that cadmium decreases passive permeability to protons.     

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.     

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.     

Mapping of beta-tubulin genomic sequences in hexaploid oat (Arena sativa L.)

Summary The allohexaploid nature of Avena sativa L. (2n=6x=42) and the availability of aneuploid lines was exploited in designing a strategy for mapping beta-tubulin sequences in the oat genome. Evidence for a minimum of eight beta-tubulin genes was obtained by Southern-blot analysis. Three betatubulin sequences were localized to chromosomes using DNA from monosomic and nullisomic lines in the variety Sun II. One sequence was localized to the chromosome missing in nullisome I. Two other sequences were mapped to satellite chromosome 2, the chromosome that is missing in nullisome VIII and to which one ribosomal RNA gene cluster had previously been mapped. Restriction fragments carrying these two beta-tubulin genomic sequences and the cluster of ribosomal RNA sequences were missing in DNA from nullisomics VIII, IX and X, suggesting that all three nullisome classes are deficient for an identical chromosomal segment that includes these three loci. This study demonstrates how molecular analyses can be used to characterize aneuploid stocks and to better define their genetic constitution.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Minnesota and does not imply its approval to the exclusion of other products or vendors that may be suitable     

Cytological and molecular characterization of oat x maize partial hybrids

In cereals, interspecific and intergeneric hybridizations (wide crosses) which yield karyotypically stable hybrid plants have been used as starting points to widen the genetic base of a crop and to construct stocks for genetic analysis. Also, uniparental genome elimination in karyotypically unstable hybrids has been utilized for cereal haploid production. We have crossed hexaploid oat (2n=6x=42, Avena sativa L.) and maize (2n=2x=20, Zea mays L.) and recovered 90 progenies through embryo rescue. Fifty-two plants (58%) produced from oatxmaize hybridization were oat haploids (2n=3x=21) following maize chromosome elimination. Twenty-eight plants (31%) were found to be stable partial hybrids with 1–4 maize chromosomes in addition to a haploid set of 21 oat chromosomes (2n=21+1 to 2n=21+4). Ten of the ninety plants produced were found to be apparent chromosomal chimeras, where some tissues in a given plant contained maize chromosomes while other tissues did not, or else different tissues contained a different number of maize chromosomes. DNA restriction fragment length polymorphisms (RFLPs) were used to identify the maize chromosome(s) present in the various oat-maize progenies. Maize chromosomes 2, 3, 4, 5, 6, 7, 8, and 9 were detected in partial hybrids and chromosomal chimeras. Maize chromosomes 1 and 10 were not detected in the plants analyzed to-date. Furthermore, partial self-fertility, which is common in oat haploids, was also observed in some oat-maize hybrids. Upon selfing, partial hybrids with one or two maize chromosomes showed nearly complete transmission of the maize chromosome to give self-fertile maize-chromosome-addition oat plants. Fertile lines were recovered that contained an added maize chromosome or chromosome pair representing six of the ten maize chromosomes. Four independently derived disomic maize chromosome addition lines contained chromosome 4, one line carried chromosome 7, two lines had chromosome 9, one had chromosome 2, and one had chromosome 3. One maize chromosome-8 monosomic addition line was also identified. We also identified a double disomic addition line containing both maize chromosomes 4 and 7. This constitutes the first report of the production of karyotypically stable partial hybrids involving highly unrelated species from two subfamilies of the Gramineae (Pooideae — oat, and Panicoideae — maize) and the subsequent recovery of fertile oat-maize chromosome addition lines. These represent novel material for gene/ marker mapping, maize chromosome manipulation, the study of maize gene expression in oat, and the transfer of maize DNA, genes, or active transposons to oat.Joint contribution of the Minnesota Agricultural Experiment Station and USDA-ARS. Scientific journal series paper No. 21 859 of the Minnesota Agricultural Experiment Station. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the USDA-ARS or the University of Minnesota and does not imply approval over other products that also may be suitable     

Red/far-red light modulates phospholipase D activity in oat seedlings: relation of enzyme photosensitivity to photosynthesis

Phospholipase D (PLD) activity was found to be higher in etiolated oat seedlings than in green seedlings. White and red (R) light exposure inhibited PLD activity in etiolated seedlings. Far-red light eliminated R-light-induced decrease in PLD activity, indicating phytochrome participation in observed photomodulation. Inhibitor of electron transport in chloroplast 3-(3,4-dichlorophenyl)-1,1-dimethylurea stimulated and glucose suppressed PLD activity in green and etiolated oat seedlings, respectively. These results suggest that PLD activity in oat seedling is regulated by light with involvement of phytochrome photoreceptor, and associated with photosynthesis process.     

Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5-untranslated region and the coding region, but the 3-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)⁺ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5- and 3-untranslated regions that might be important for PHYA mRNA degradation.     

Effect of anaerobiosis on alcohol dehydrogenase in oat seedlings

In order to clarify the induction of alcohol dehydrogenase (ADH) by anaerobiosis in oat (Avena sativa L.), the seedlings were exposed to anaerobiosis and activity of ADH and ADH isozyme profiles were determined. The anaerobiosis increased ADH activities in shoots and roots of the seedlings. By day 2, the activity increased 5 and 4 times in the roots and the shoots, respectively, compared with those under aerobic condition. Based on nondenaturing electrophoresis, ADH isozyme composition analysis revealed six bands consisting of a dimmer enzyme with submits encoded by three different Adh genes. Changes in staining intensity of the isozymes indicated that the increase in ADH activity in oat under anaerobiosis resulted from increased enzyme synthesis.     

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages – regenerating tissue and differentiated shoots. We observed significant (P<0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P<0.05), and lower levels of Spm (P<0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P<0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P<0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.     

Soil humic substances affect transport properties of tonoplast vesicles isolated from oat roots

The effect of a low molecular size (<5 KDa) humic fraction, essentially fulvic acids, on microsomal and tonoplast ion-stimulated ATPase activity was studied. After 20 min of pre-incubation with microsomal vesicles from oat roots, humic substances at organic C concentration of up to 0.5 μg cm^-3 increased KCl-stimulated ATPase activity, while they inhibited enzyme activity at higher concentrations. Cl^--stimulated ATPase activity of tightly sealed tonoplast-enriched vesicles was similarly affected by <5 KDa humic substances. This behaviour was not observed when gramicidin D was added to the assay medium. Proton transport by vesicles incubated up to 5 min with <5 KDa humic molecules was affected in a concentration-dependent manner, strongly resembling that observed for ATP hydrolysis, whereas it was severely reduced when the assay conditions were close to those used for measuring ATP hydrolysis (20 min pre-incubation of vesicles with humic substances). The transmembrane electrical potential was negatively affected, irrespective of the concentration of humic molecules. Furthermore, a 15-min pre-incubation strongly reduced the formation of a potential gradient. The size and concentrations of humic substances employed make an interaction with the vacuolar membrane of root cells plausible. The results show that the main target of humic molecules is the electrical membrane potential and suggest a possible way of interference of these naturally occurring substances with the biochemical mechanisms involved in plant mineral nutrition.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.