Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts

Summary Protoplasts isolated from a barley cell suspension (cv Dissa) were transformed with plasmid DNA containing the neomycinphosphotransferase II (NPT) and -glucuronidase (GUS) genes, using polyethyleneglycol (PEG) to induce DNA uptake. Transformed microcalli were selected in media containing G418 sulphate. NPT activity was detected in all antibiotic-resistant cell lines, but not all NPT-positive cell lines had GUS activity. Southern analysis confirmed the presence of sequences homologous to the APT and GUS genes in DNA of G418-resistant callus.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

Delivery of foreign genes to intact barley cells by high-velocity microprojectiles

Summary Foreign DNA was introduced through the cell walls of intact suspension culture cells of barley (Hordeum vulgare L.) by utilizing the particle acceleration approach. DNA-coated microscopic tungsten particles were accelerated to velocities that permitted their penetration of intact cells. Chimaeric constructs of -glucuronidase and neomycin phosphotransferase II under the control of the dual Agrobacterium T_R 12 promoter or the cauliflower mosaic virus 35S promoter served as reporter genes. Three days after particle delivery, high-level expression of both reporter genes was observed. That plasmid size could be critical for stabilizing DNA in the course of particle delivery will be discussed.     

Rapid production of fertile transgenic barley (Hordeum vulgare L.) by direct gene transfer to primary callus-derived protoplasts

Protoplasts were isolated from primary calli of barley (Hordeum vulgare L.), and an antibiotic (G418) resistance gene was introduced into these protoplasts using a polyethylene glycol (PEG) DNA uptake method. Sixty-four G418 resistant calli were obtained in nine experiments, and two plants were regenerated from these calli. NPTII ELISA and Southern analysis indicated that the G418 resistance gene was introduced and expressed in two T₀ plants. These plants set seed and the introduced gene was transmitted to T₁ plants. These results suggest that our transformation system using primary callus-derived protoplasts is a useful method for the generation of transgenic barley. Received: 14 November 1997 / Revision received: 12 March 1998 / Accepted: 24 April 1998     

Isolation and culture of barley megasporocyte protoplasts

An efficient technique has been developed for the isolation of barley megasporocyte protoplasts at early meiotic prophase. Ovules were dissected out of ovaries under aseptic conditions, subjected to a brief enzymatic digestion, and then transferred to a modified Kao medium with 90 g/l sucrose and 20 mM CaCl₂. A small incision was made with a scalpel through the softened epidermal cell layer of the nucellus and the megasporocyte could then be liberated into the medium by applying gentle pressure on the nucellus. The megasporocyte appeared to be completely devoid of a wall and changed its in situ pyriform shape to completely spherical when extruded into the medium. Four to nine protoplasts could typically be isolated per spike. Protoplasts cultured in medium degenerated after a few days. Viability was dramatically improved if protoplasts were co-cultivated with barley microspores undergoing microspore embryogenesis. More than half of the protoplasts were still alive after 6 days of culture, and in some cases they survived more than 12 days of culture. Fluorescence microscopy of the cultured protoplasts stained with 4,6-diamidino-2-phenylindole (DAPI) or aniline blue revealed that the protoplasts remained uninuclear and reformed their callose wall.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

Antioxidative protection in the leaves of dark-senescing intact barley seedlings

In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H₂O₂ and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H₂O₂ was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.     

Correlation of gibberellin-induced growth, polyamine levels and amine oxidases in epicotyl, root and leaf blade of barley seedlings

The potential role of diamine oxidase (DAO) and polyamine oxidase (PAO) in relation to polyamines was investigated in epicotyls, roots and leaf blades at 3 and 6 days after gibberellic acid (GA) application in barley (Hordeum vulgare L.) seedlings of cvs. Maythorpe (non-mutant parent) and Golden Promise (semi-dwarf mutant). There was a significant increase in epicotyl and leaf-blade elongation rates in GA-treated seedlings of cv. Maythorpe as compared to cv. Golden Promise. DAO and PAO were detectable in all segments of the leaf blade, but the highest activities were present in basal segments. These enzymes, which are thought to have a role in the elimination of cellular polyamines, increased in activity following GA application compared to controls. Application of 10⁻⁶ M GA to the first leaf, significantly increased endogenous bound putrescine (Put) levels in both the epicotyl and leaf blade of cv. Maythorpe. In contrast, there was only a slight increase in cv. Golden Promise. Levels of soluble Put increased in roots and leaf blades of both cultivars following GA treatment but the effect was greatest in leaves of cv. Maythorpe. It is suggested that polyamines may play a role in GA-induced epicotyl and leaf-blade elongation in barley.     

Transfection of germinating barley seed electrophoretically with exogenous DNA

Summary A method is described for transfection (genetic transformation) of barley caryopsis electrophoretically with DNA. -Glucuronidase activity was detected after the electrophoretic transfection with plasmid pBI221 DNA carrying the cauliflower mosaic virus promotor and bacterial -glucuronidase coding sequence. Electrophoretic transfection is evidently effective with pieces of callus and seeds of many plants.     

The effects of DNA hypomethylating drugs on androgenesis in barley (Hordeum vulgare L.)

Summary The effects of DNA hypomethylating drugs (azacytidine and ethionine) on induction of microspore-derived calluses and embryos were studied in barley (Hordeum vulgare L.) ev. Igri. The results were as follows: (1) Yield of calluses and embryos pretreated with the different concentrations of azacytidine for 3 d was several-fold higher than that of the control. The highest yield of calluses and embryos in all treatments appeared at a concentration of 3 mg l⁻¹, which reached 11.03 per anther. It was 110-fold higher than the control. (2) There was a significant difference in yield of calluses and embryos between the different days of pretreatment. The highest yield was obtained at a 3-d pretreatment. If the period of pretreatment was shorter or longer than 3 d, yield of calluses and embryos was reduced sharply, and was similar to that of the control. (3) The data obtained with ethionine pretreatment were very similar to those obtained with azacytidine. (4) Tests on the different methods of pretreatment showed that yield of calluses and embryos pretreated with distilled H₂O, mannitol, azacytidine, and ethionine was much higher than other pretreatments and the control, and reached 6.53–11.39 per anther. The yield of calluses and embryos pretreated with DNA hypomethylating drugs was higher than with mannitol. However, pretreatment with hypomethylation drugs supplemented with induction medium was not effective.     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Effects of aluminum and cadmium toxicity on growth and antioxidant enzyme activities of two barley genotypes with different Al resistance

A hydroponic experiment was carried out to study genotypic differences in effect of Al and Cd on growth and antioxidant enzyme activities by using 2 two-row winter barley genotypes (Hordeum vulgare L.) with different Al resistance, the relatively resistant Gebeina and the sensitive Shang 70–119. The seedling growth, presented as shoot height, root length and dry weight of root and shoot, and tillers per plant were inhibited by all stress treatments, including low pH, 100 M Al (pH 4.0) and 1.0 M Cd+100 M Al (pH 4.0), while 1.0 M Cd showed a slight stimulation of growth. The inhibition was more severe in 1.0 M Cd +100 M Al (pH 4.0) than in 100 M Al (pH 4.0), indicating that the effect of Cd and Al is synergistic. Al-sensitive genotype Shang 70–119 was more inhibited than Al-resistant genotype Gebeina. Proline concentration in leaves was significantly increased when plants were exposed to all stress treatments, being more pronounced in Shang 70–119 than in Gebeina. A highly significant increase in malonaldehyde (MDA) concentration, and a stimulation of superoxide dismutase (SOD) and peroxidase (POD) activities were recorded in the plants subjected to low pH, 100 M Al (pH 4.0) and 1.0 M Cd +100 M Al(pH 4.0) treatments, and the extent of the increase varied greatly depending on concentration and time of exposure. Shang 70–119 had a higher MDA concentration, and less increase in SOD activity when first exposed than Gebeina had.     

Response of barley seedlings to UV-B radiation as affected by NaCl

The response of barley seedlings, subjected to 150 mmol/L NaCl for 4 days at different light regimes (4 d in the light, 4 d in darkness and a 12 h light/dark cycle) before UV-B radiation was investigated. NaCl treatment resulted in a decrease of total chlorophyll content and an increase in H2O2, free proline and lipid peroxidation, as quantified by measurement of malondialdehyde. Significantly more proline was accumulated in the light than in darkness. The combination of UV-B and NaCl treatment produced an additive effect on most of the parameters studied. UV-B radiation reduced the chlorophyll/carotenoids ratio and photochemical efficiency of PSII as estimated by chlorophyll fluorescence. NaCl pre-exposure decreased H2O2 generation and lipid peroxidation and alleviated the inhibitory effect of UV-B on PSII activity. Proline accumulated under salt stress conditions might be one of the reasons for the observed tolerance of barley seedlings to UV-B radiation.     

Chromosomal variation in dividing protoplasts derived from cell suspensions of barley (Hordeum vulgare L.)

Summary Numerical and structural chromosome variation was analysed in dividing protoplasts isolated from suspension cells of barley. Five cell lines exhibited distribution patterns in chromosome number with different peaks and ranges. Embryogenic/morphogenic cell lines showed a peak at 2n = 14 (ca. 50%) after 6–7 months in culture, while older non-embryogenic cell lines had peaks at aneuploid or polyploid chromosome numbers. Culture duration had a clear effect on numerical and structural chromosome variation in embryogenic cell lines. With ageing of the cultures chromosome variation accumulated and the proportion of 2n = 14 cells decreased. The effect of protoplast isolation and culture on chromosome variation was examined; more cells with normal chromosome sets (12%) were maintained in protoplast-derived colonies than in source suspension cells (4%) of the same culture age.Abbreviations DC Dicentric - F fragment - T telocentric     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Summary A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant Nia30 protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad -irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10^-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.     

A comparison of Hordeum bulbosum-mediated haploid production efficiency in barley using in vitro floret and tiller culture

Summary A high efficiency of Hordeum bulbosum-mediated haploid production in barley has been achieved using a floret culture technique in which florets pollinated with Hordeum bulbosum are cultured on modified N₆ media containing 0.5 mg/l kinetin and 1.2 mg/l2,4-D. Cultures were maintained at 25 °C with a 16 h photoperiod for 9 days before embryo rescue. In a comparison of haploid production efficiency using five F₁ hybrids from winter x winter and winter x spring barley crosses, 41.6 haploid plants/100 florets pollinated were produced using floret culture. Using detached tiller culture, 13.5 haploid plants/100 florets pollinated were produced. Higher efficiencies achieved with floret culture are attributed to the formation of larger, differentiated embryos. Such embryos lead to higher frequencies of plant regeneration. The F₁ from a winter x winter cross was inferior in haploid production compared to F₁s from winter x spring crosses. No genotype x technique interaction was observed.Oregon Agricultural Experiment Station Technical Paper No. 8653     

Cadmium translocation and accumulation in developing barley grains

Soil cadmium (Cd) contamination has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide. In order to study the transport of Cd into the developing grains, detached ears of two-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing the markers for phloem (rubidium) and xylem (strontium) transport. Cd concentration in each part of detached spikes increased with external Cd levels, and Cd concentration in various organs over the three Cd levels of 0.5, 2, 8 μM Cd on 15-day Cd exposure was in the order: awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grains with the proportion of 51.0% relative to the total Cd amount in the five parts of detached spikes. Cd accumulation in grains increased not only with external Cd levels but the time of exposure contrast to stem, awn, rachis and glume. Those four parts of detached spike showed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 and increase again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cd concentration in grains, and sucrose or zinc (Zn) addition to the medium and higher relative humidity (RH) also induced dramatic reduction in Cd transport to developing grains. The results indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as an important physiological process influencing the quality of harvested grains. Our results suggested that increasing RH to 90% and Zn addition in the medium at grain filling stage would be beneficial to decrease Cd accumulation in grains.     

A barley RFLP map: alignment of three barley maps and comparisons to Gramineae species

Several gene linkage maps have been produced for cultivated barley. We have produced a new linkage map for barley, based on a cross between Hordeum vulgare subsp. spontaneum and Hordeum vulgare subsp. vulgare (Hvs x Hvv), having a higher level of polymorphism than most of the previous barley crosses used for RFLP mapping. Of 133 markers mapped in the Hvs x Hvv F₂ population, 69 were previously mapped on other barley maps, and 26 were mapped in rice, maize, or wheat. Two known gene clones were mapped as well as two morphological markers. The distributions of previously mapped markers were compared with their respective barley maps to align the different maps into one consensus map. The distributions of common markers among barley, wheat, rice and maize were also compared, indicating colinear linkage groups among these species.To be considered dual first authorsPublished with the approval of the Director of the Colorado State University/Agricultural Experiment Station.     

The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms

Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses. Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes. In grains, the expression of HVA22 gene appears to be correlated with the dormancy status. The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains. In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition. Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA. The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm. The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought. Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabitis elegans, man, mouse and yeast, but not in any prokaryotes. Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible. These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.