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.     

Genetical analysis of microspore derived plants of barley (Hordeum vulgare)

Summary From an F₁ hybrid between the two barley (Hordeum vulgare L.) cultivars Golden Promise and Mazurka a series of doubled haploid (DH) lines were generated both from microspores by anther culture and from immature zygotic embryos after hybridization withH. bulbosum. The DH lines from both sources were used to monitor the segregation of the five major genes, rachilla hair length, DDT susceptibility, height, C hordein polymorphism and mildew resistance. Whereas the microspore-derived samples showed significant departures from the expected 11 ratio for three of the five genes, theH. bulbosum lines showed deviation for only one gene. Analysis of linkage data also showed differences between the two series of DH lines. Cytogenetic analysis revealed a mean chiasma frequency in theH. bulbosum lines which was very similar to the F₁ hybrid. In contrast, four of the ten microspore derived lines examined showed a reduced chiasma frequency. One showed evidence of translocation heterozygosity.     

Nuclear genes affecting percentage of green plants in barley (Hordeum vulgare L.) anther culture

Summary The genetics behind response in barley anther culture was studied with 22 reciprocal and one single: cross between three varieties with high and four varieties with low capacity for green plant formation. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 62 and 76% of total variation, respectively, with almost no genetic effect on the ability to regenerate plants from pollen embryos. Nuclear genes could explain all genotype effects in this plant material, since no reciprocal effects were indicated. The three parents with high and the four parents with low capacity for green plant formation formed two phenotypically homogeneous groups, producing 27–52% and 0–7% green plants, respectively. Genetic variation within hybrids for both embryo and green plant formation could be explained completely by general combining ability (GCA). The results are discussed with respect to a previous similar study in hexaploid wheat and the reported existence of DNA deletions in the plastid genomes in albino plants from anther culture of wheat and barley.     

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.     

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.     

A comparison of several published methods for barley anther culture

A number of methods have been published for barley (Hordeum vulgare L.) anther culture and have gained acceptance in different laboratories. The breeder's requirement is for a compromise method that gives good, repeatable results for a wide range of genotypes. Yet the routine production of spontaneously doubled haploid green regenerants remains difficult. Despite attempts to formulate a widely-applicable anther culture method, the 4 main published methods, compared here with one modified procedure, are quite distinct for a number of important characteristics. The methods interacted strongly with the 3 genotypes, and response ranged from zero to 28 green regenerants per 100 anthers plated. The current methods still require often substantial modification to suit local situations in order that the technology may be exploited by barley breeders.Abbreviations BAP benzylaminopurine - DH doubled haploid - FV final volume - IAA indoleacetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - PABA para-aminobenzoic acid     

Association of two barley yellow mosaic virus (RNA 2) encoded proteins with cytoplasmic inclusion bodies revealed by immunogold localisation

Summary Antisera were raised against the RNA 2-encoded proteins of 28 kDa and 70 kDa of barley yellow mosaic virus (BaYMV) by using the corresponding cDNA sequences of a German isolate for protein overexpression inEscherichia coli BL 21 and subsequent purification. The proposed processing of a 98 kDa precursor polyprotein encoded by the long open reading frame of RNA 2 to two proteins of 28 kDa and 70 kDa could be confirmed by immunoprecipitation of the in vitro transcribed and translated cDNA-clone of RNA 2 and Western blot analysis of fragmentated protein extracts of BaYMV-infected winter barley plants. In situ localisation studies of infected leaf tissue using immunogold labeling techniques for electron microscopy revealed that both viral proteins of BaYMV (RNA 2) were associated with the crystal-like cytoplasmic inclusion bodies. No other parts of the cells and no other inclusions (pinwheelstructures or aggregated virus particles) showed any gold labeling when the 28 kDa and 70 kDa antisera were used. We suppose that both RNA 2-encoded proteins take part in the formation of the crystal-like cytoplasmic inclusion bodies which are the most dominant structures in the cytoplasm of BaYMV-infected tissue. Possible functions of the 28 kDa and 70 kDa protein of BaYMV (RNA 2) are discussed.Abbreviations PBS phosphate-buffered saline - CEA chicken egg albumin - BaYMV barley yellow mosaic virus - BaMMV barley mild mosaic virus     

Enhancement of embryogenesis and plant regeneration from barley anther culture by low doses of gamma irradiation

Summary The effects of 0,5 and 10 Gy doses of gamma irradiation on the enhancement of embryogenesis and plant regeneration efficiency of three barley (Hordeum vulgare L.) genotypes, Igri, Arabi Abiad and AECS 76, were evaluated. Embryo yields at 5 and 10 Gy doses were significantly higher than those of the control (OGy). This effect was genotype-dependent. The most responsive genotype was Igri, with 592.8 embryos 32 anthers exposed to 10 Gy. However, despite a high embryo induction rate, the green plant regeneration rate was low. Arbi Abiad had a higher ability to generate green plants produced from, with 28. 13 plantlets obtained from 32 anthers at 10 Gy; irradiation had no significant effect on regeneration of Igri and AECS 76 genotypes. In general, the 10 Gy dose produced a much higher embryo yield than the 5 Gy dose. The root-tip chromosome number and the fertility of 298 regenerating green plants of cv. Igri revealed that 64% of the tested plants were spontaneously doubled haploids (DHs) and fertile.     

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     

Field performance of lines derived from haploid and diploid tissues of Hordeum vulgare

Summary Plant tissue culture technology is of increasing interest to plant breeders. As part of a continuing investigation into breeding methods with spring barley two studies were conducted to assess the field performance of the progenies of material regenerated in tissue culture. The first study involved two spring barley cultivars, Golden Promise and Mazurka and compared lines produced from immature embryo (IE) derived callus with those from embryos developed by the Hordeum bulbosum (Hb) technique of chromosome elimination. In general the mean values for the seven characters scored were lower for the IE than the Hb material. In the second study F₁ hybrid material (Golden PromisexMazurka) was used and doubled haploid lines produced by the H. bulbosum and microspore culture (M) techniques were compared with single seed descent (SSD) material. Analysis of these F samples indicated that the mean values for the M lines were significantly lower than those of the Hb and SSD lines. Furthermore, data from the M lines showed significant evidence of variation created during the culture phase. The implications of these findings for barley breeding are discussed.     

The inheritance of genetic markers in microspore-derived plants of barley Hordeum vulgare L.

Summary Biochemical, molecular and morphological markers have been used to monitor the segregation of alleles at major gene loci in microspore-derived lines of four spring barley crosses and their parents. Significant deviations from the expected Mendelian ratios were observed for four of the ten markers studied in the cross. Distorted ratios were associated with loci located on chromosomes 4H and 6H. The differential transmission of alleles was in favour of the responsive parent (Blenheim) used in the anther culture studies. For the -Amy-1 locus on chromosome 6H, the preferential transmission of Blenheim alleles was most pronounced in the haploid regenerants that were colchicine treated. These results are discussed in relation to the genetic control of androgenetic response in barley and with respect to the exploitation of another culture in barley improvement.     

Barley anther culture using membrane rafts

When compared to agarose solidified media in small petri dishes, membrane rafts used in conjunction with liquid induction media significantly improved anther culture response in the Australian, malting-quality, spring barley cultivar Clipper. In contrast, the German cultivar Gimpel did not show an increased response on rafts.Abbreviations BA 6-benzylaminopurine - IAA indoleacetic acid - DH doubled haploid     

Chromosomal location of resistance to barley yellow mosaic virus in German winter-barley identified by trisomic analysis

Summary In order to localize a gene for resistance to Barley Yellow Mosaic Virus (BaYMV) of German resistant varieties, cvs. Ogra and Sonate were crossed to a complete trisomic (2n=2x+1=15) set of Shin Ebisu 16. Tests for resistance in F₂ strongly support the conclusion that the German gene for resistance to BaYMV is located on barley chromosome 3.     

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     

Identification of embryogenic microspores of barley (Hordeum vulgare L.) by individual selection and culture and their potential for transformation by microinjection

Summary In order to identify microspores, suitable for transformation via microinjection of DNA, single microspores of barley (Hordeum vulgare L.) were selected after initial preculture of anthers floating on liquid media and analysed for their development in individual culture in microdroplets of culture medium. Conditions for microculture and plant regeneration from single selected embryogenie microspores were established. The technical feasability of intranuclear microinjection was demonstrated by injecting the fluorescent dye Lucifer Yellow. All essential procedures for a transformation system of barley based on microinjection into microspores have thus been performed successfully. Further efforts to increase efficiencies of culture and microinjection procedures are necessary, however, in order to improve the suitability of this approach towards transformation of barley.Abbreviations MES 2 (N-morpholino) ethanesulfonic acid - PEG polyethylene glycol     

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.     

Screening salt-tolerant barley genotypes via F1 anther culture in salt stress media

Summary Anthers of two six-row barley cultivars Diamond (a germination salt sensitive cultivar) and Men Yuan Liang Lan (a germination salt tolerant cultivar), and their F₁ reciprocal crosses were cultured in liquid media containing 0, 0.4, 0.6, and 0.8% Na₂SO₄. A total of 138 green pollen plants were obtained: 7 from Na₂SO₄ media, 128 from Na₂SO₄ free medium. Seeds of two successive generations of 61 pollen plants were germinated in a series of Na₂SO₄ solution (0 to 5.5%). It was found that among 37 progenies from F₁ pollen in Na₂SO₄ free medium, 11 were as sensitive as Diamond, 12 were intermediate to the two parents, 7 were equal to the salt tolerant parent and 7 were more tolerant to Na₂SO₄ than Men Yuan Liang Lan. Whereas, no progeny from F₁ pollen in high salt media was as susceptible as the susceptible parent; 2 were intermediate, 2 were equal to the salt tolerant parent and 2 were more tolerant than the salt tolerant parent. The results indicate that culturing anthers in Na₂SO₄ media effectively eliminated salt susceptible progenies. All 16 microspore-derived lines of Diamond were as susceptible as Diamond to Na₂SO₄. The 5 lines from Men Yuan Liang Lan microspores were as resistant to Na₂SO₄ as Men Yuan Liang Lan. All of the lines breed-true. The results indicate that the lines exhibiting elevated levels of tolerance to salt probably resulted from recombination of genes rather than from spontaneous mutation.     

Plant regeneration by pollen embryogenesis from cultured whole spikes of barley (Hordeum vulgare)

Summary Pollen embryogenesis and subsequent plant regeneration have been established from cultured whole barley spikes in agitated N6 liquid medium (Chu 1978) containing high levels of 2,4-D, Ficoll and potato extract. Microspore division within the anthers and subsequent embryogenic development were obtained in medium containing high amounts of reduced nitrogen with Zeatin, NAA and BAP (all at 0.5 mg/l levels, pH 6.2). Once embryoids were formed in the liquid medium, they produced secondary embryoids from the scutellum and subsequently plants on MS (Murashige and Skoog 1962) agar medium containing BAP and NAA. The ratio of green plants to albino was 18.7.     

Competitive effects in monocultures and mixtures of spring barley (Hordeum vulgare)

Summary The presence of significant levels of intergenotypic competition amongst barley (Hordeum vulgare) genotypes has profound consequences for barley breeding programmes. Breeding programmes based on the pedigree system attempt to identify genotypes in genetically heterogeneous populations but the elite genotypes are grown in monoculture. Thus, to attain varietal status genotypes produced by this breeding strategy must perform well in mixtures as well as in pure stands. The effectiveness of early generation selection may be hampered by intergenotypic competition. To examine this problem in spring barley, a modified substitution experiment (Mather and Caligari 1981, 1983) was used and included genotypes sampled from a random set of inbred lines generated without conscious selection. This approach to the investigation of competitive effects in barley indicated the presence of significant levels of intergenotypic competition for a range of agronomic characters. The analyses allowed a distinction to be made between aggression (a) and response (r) with the component r displaying greater variation than a. The lack of correlation in the distribution of a and r suggested that they were under separate genetic control and hence adjustable by selection. The implications of these results for barley improvement, the use of varietal mixtures and mixed cropping are discussed.