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     

Plant regeneration from protoplasts of wild barley (Hordeum murinum L.)

Summary We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - L1, L2 medium according to Lazzeri et al. 1991 - L3 medium medium according to Jähne et al. 1991a     

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (flat). When anthers were plated with only one loculus in contact with the medium (up), a high proportion of the anthers produced calluses. The top loculus of the up anthers was most productive. Flat anthers, when compared with up anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in flat anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.     

Chromosomal variation in immature embryo derived calluses of barley (Hordeum vulgare L.)

Summary Chromosome counts of ten morphogenic and seven non-morphogenic immature embryo derived calluses of barley,Hordeum vulgare L. cv. Himalaya, were determined. Morphogenic calluses carried the normal chromosome complement (2n=2x=14) in a majority of the cells. A low frequency of haploid (2n=x=7), triploid (2n=3x=21), tetraploid (2n=4x=28) and octoploid (2n=8x=56) cells were also observed. In contrast, non-regenerability of a callus was attributed to the cells having numerical and structural chromosomal changes. In these calluses, aneuploid cells around diploid, triploid, and tetraploid chromosome numbers predominated. It has been demonstrated that chromosomal changes were induced during the culture and that they did not pre-exist in the cultured barley embryos. Based on this study, it is suggested that chromosome analysis of a non-regenerable callus should be conducted before altering the media composition.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Triple hybridization with cultivated barley (Hordeum vulgare L.)

Summary A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. Gull, Golden Promise and Vada. Generally, cv Gull had high crossability in crosses with related species in the primary hybrid. It is suggested that Gull has a genetic factor for crossability not present in cv Vada and cv Golden Promise. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv Vada but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x Gull (2n=7–30) and (H. jubatum x H. lechleri) x Gull (2n=20–22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19–22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Superoxide dismutase and peroxidase activities in drought sensitive and resistant barley (Hordeum vulgare L.) varieties

The effects of water deficit on relative water content (RWC), on the activity superoxide dismutase (SOD) and peroxidase (POX) from leaves of two drought-resistant barley strains (Hordeum vulgare L.) varieties (TOKAK-157/37 and 56000/MISC-233) and one sensitive (ERGINEL-90) were studied. In 21 day old seedlings, drought stress was initiated by withholding water and lasted for 12 days. Activity of SOD increased by the effect of drought treatments in the leaves of drought-resistant varieties TOKAK-157/37 and 56000/MISC-233 as compared to sensitive variety ERGINEL-90. The drought treatment resulted in a 418 % and 59 % increase in SOD activity in resistant varieties at the end of the 12^th day of experimental period. However, an increase in activity of SOD was not accompanied by an increase in activity of POX in drought-resistant TOKAK-157/37 and 56000/MISC-233 except on the 6^th day of drought treatment in 56000/MISC-233. In drought-sensitive variety, ERGINEL-90, POX activity did not change throughout drought period.     

Production of gynogenic haploids of Hordeum vulgare L.

Haploid plants were regenerated from cultured unfertilized ovaries of Hordeum vulgare L. (barley). Optimal response was obtained by the addition of 0.6 M 4-chloro-2-methylphenoxyacetic acid (MCPA), 2.8 M indole-3-acetic acid (IAA) and 4.4 M 6-benzyladenine (BA) in the N₆ medium. Further increase in the rate of callus formation and the number of green plants produced was possible with the addition of 90 g/l sucrose and 100 g/l coconut water. The stage of development of the ovaries at the time of culture was critical; the largest number of plants being produced by ovaries from flowers at the trinucleate stage of pollen.Abbreviations (BA) 6-benzyladenine - (MCPA) 4-chloro-2-methylphenoxyaceticacid - (2,4-D) 2,4-dichlorophenoxyaceticacid - (GA₃) gibberellic acid - (IAA) indole-3-acetic acid     

The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.)

The influence of temperature stress pre-treatment on anther culture response has been examined in eight commercially desirable barley cultivars. Spikes were pre-treated in darkness at 4°C for periods of 0, 7, 14, 21 and 28 days. Overall, the optimum pre-treatment period was 21 days, although there were large genotype by pre-treatment interactions. The most responsive cultivar was Igri, with a mean of 38% anthers responding, and relatively little effect of pre-treatment. The greatest effect of pre-treatment was in cv. Heriot, which had 3% response with no pre-treatment and 52% response from 14 days pre-treatment.     

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.     

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     

Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS

We have applied a refined microdissection procedure to create a plasmid library of the barley (Hordeum vulgare L.) chromosome arm 1HS. The technical improvements involved include synchronization of meristematic root tissue, a metaphase drop-spread technique, paraffin protection of the collection drop to avoid evaporation, and a motorized and programmable microscope stage. Thirteen readily-discernible telocentric chromosomes have been excised from metaphases of synchronized root-tip mitoses. After lysis in a collection drop (2 nl), the DNA was purified, restricted withRsaI, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. Finally, the amplified DNA was cloned into a standard plasmid vector. The size of the library was estimated to be approximately 44,000 recombinant plasmids, of which approximately 13% can be utilized for RFLP analysis. Tandem repetitive probes could be rapidly excluded from further analysis after colony hybridization with labelled total barley DNA. Analysis of 552 recombinant plasmids established that: (1) the insert sizes ranged between 70 and 1150 bp with a mean of 250 bp, (2) approximately 60% of the clones contained highly repetitive sequences, and (3) all single- or low-copy probes tested originate from chromosome 1HS. Four probes were genetically mapped, using an interspecificH. vulgare xH. spontaneum F₂ population. One of these probes was found to be closely linked to theMla locus conferring mildew resistance.     

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

Summary With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.     

Production and identification of new structural chromosome mutations in barley (Hordeum vulgare L.)

A total of 52 reciprocal translocations and 9 pericentric inversions were induced and identified in both standard and cytologically marked barley karyotypes using gamma-rays as the clastogenic agent. An analysis based upon Giemsa N-banding patterns and arm length measurements of the reconstructed chromosomes enabled a rather precise cytological localization of intra- and interchange breakpoints. This analysis was significantly facilitated and improved, especially for the identification of pericentric inversions, when the reconstructed karyotype T-1586 was used as starting material. The majority, if not all, of the aberration breakpoints proved to be localized in interband regions or in medial and terminal parts of the chromosomes, i.e., in regions which are deficient in constitutive heterochromatin. A great number of the structural mutations produced in this study contain specific cytological markers covering nearly all of the chromosomes of barley karyotype. This material might be of considerable interest in solving various problems of barley cytogenetics and chromosome engineering and especially in constructing a physical map of barley genome.     

Influence of Fe concentration in the medium on multicellular pollen grains and haploid plants induced by mannitol pretreatment in barley (Hordeum vulgare L.)

Summary. This study aims to clarify the short- and long-term effects of the iron concentration in the medium on androgenesis induced in barley by isolated microspore culture. The ultrastructural features and pectin composition of the intine wall were studied in the initial stages of androgenesis. The evolution of electron-dense iron deposits on the intine was analysed in multicellular pollen grains obtained by isolated microspore culture performed for 3, 6, and 9 days using various concentrations of FeNa₂ EDTA. Finally, the number of embryo-like structures and green plants obtained by microspore culture using different Fe concentrations was evaluated in order to estimate the optimum concentration for isolated microspore culture. Correspondence and reprints: Departamento de Bioquimica, Biologia Celular y Molecular de Plantas, Estación Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, Profesor Albareda 1, 18008 Granada, Spain.     

Characterization of a gibberellin sensitive dwarf mutant of barley (Hordeum vulgare L.,Mut. Dornburg 576)

The radiation (³²P) induced dwarf mutant of spring barley,Hordeum vulgare L., Mut. Dornburg 576, was genetically studied by crosses with the mother variety and characterized by seedling assays and investigations on development and yield formation. In comparison to the normal mother variety (Hordeum vulgare L., cv. Saale) mutant plants exhibit drastically reduced culm length, intensive tillering, a prolonged life cycle and a smaller biomass and grain yield formation. These characters are controlled by one gene in a pleiotropic way. The mutant responds with normal growth and development to the application of gibberellins.     

Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.)

Tocotrienols are lipophilic antioxidants belonging to the tocochromanols, better known as vitamin E. Although present in cereal grains in high quantities not much is known about their function in plants. In a detailed study the temporal and spatial accumulation of tocotrienols and tocopherols during grain development in two barley cultivars was analyzed. Tocochromanols and lipids accumulated in parallel until 80% of the final dry weight of the kernels was reached. Later on the tocochromanol content did not change while the lipid content decreased. Generally, only about 13% of the tocochromanols were found in the germ fraction, whereas the pericarp fraction contained about 50% and the endosperm fraction about 37% of the tocochromanols. Altogether, about 85% of the tocochromanols were tocotrienols in both cultivars. In case of the tocopherols about 80% were found in the germ fraction and the remaining 20% in the pericarp fraction. Tocotrienols were almost equally present in the pericarp and the endosperm fraction. Individual forms of tocopherols and tocotrienols accumulated with different kinetics during barley grain development. The differences in distribution and accumulation indicate different functions of the individual tocochromanols during grain development.     

The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli -glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.NRCC No. 36482