The effect of genotype and environment on the fatty acid content of barley (Hordeum vulgare L.) grains

Abstract A comparison was made of the content of total and some individual fatty acids in grains of nine barley varieties grown at six sites in Belgium. The varieties represented six- and two-rowed winter types and two-rowed spring types. The results showed that the winter types contain more linolenic acid (C18 : 3) than spring types and that six-rowed barleys have less total fatty acids than two-rowed barleys, due mainly to a low concentration of palmitic (C16:0), oleic (CI8 : 1) and linoleic (C18 : 2) acids. Analysis of variance showed that fatty acid content is affected by both the genotype and the environment and multiple regression analysis suggested that weather conditions before and after flowering affected lipid composition.     

Light and electron microscope studies on pollen development in barley (Hordeum vulgare L.) grown under copper-sufficient and deficient conditions

Abstract. Pollen development in copper-deficient barley plants is highly irregular resulting in low and variable pollen fertility. The main cause of this sterility was found to be the abnormal development of the tapetum which becomes expansionary and invasive as the pollen develops. The ultrastructure of both tapetum and microspores is different from that of control material with irregularities of exine deposition, endopolyploidy of tapetal nuclei and an alteration of organelle composition being correlated with low fertility.     

Leaf extension in the slender barley mutant: delineation of the zone of cell expansion and changes in translatable mRNA during leaf development

The slender mutant of barley (Hordeum vulgare L.) results from an alteration to a single nuclear gene. Plants homozygous for the mutant allele have long, attenuated leaves as a result of a greatly increased extension rate. Although the growth rate at any one position in the extension zone appears not to differ between slender and normal (wild-type) barley, in slender the length of the zone over which cells extend is approximately 50% greater than that in normal barley. Epidermal cells are both longer and narrower in slender, so the whole-plant phenotype is mirrored at the cell level. Translation in vitro of RNA extracted from successive sections of the young primary leaf, followed by one-dimensional SDS-PAGE separation, facilitated the alignment of equivalent developmental stages in the two genotypes, but failed to demonstrate major differences between the two genotypes. Two-dimensional separation of translation products from total leaf tissue revealed a few small differences between normal and slender. Growth of plants at 8°C compared with 20°C caused changes in some translation products, with one (unknown) product decreasing in abundance in cold-treated normal tissue but not in slender tissue.     

Production and analysis of plants that are somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.)

In order to obtain plants that were somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.), we fused protoplasts that had been isolated from 6-month-old suspension cultures of carrot cells with protoplasts isolated from barley mesophyll by electrofusion. After culture for 1 month at 25°C , the cells were cultured for 5 weeks at 4°C , and were then returned to 25°C for culture on a shoot-inducing medium. Three plants (nos. 1, 2 and 3) were regenerated from the cells. The morphology of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells had about 24 chromosomes, fewer than the sum of the numbers of parent chromosomes which was 32. Southern hybridization analysis with fragments of the rgp1 gene used as probe showed that the regenerated plants contained both barley and carrot genomic DNA. Chloroplast (ct) and mitochondrial (mt) DNAs were also analyzed with several probes. The ctDNA of the regenerated plants yielded hybridization bands specific for both barley and carrot when one fragment of rice ctDNA was used as probe. Furthermore, the regenerated plants yielded a barley specific band and a novel band with another fragment of rice ct DNA as a probe. One of the regenerated plants (no. 1) yielded a novel pattern of hybridized bands of mt DNA (with an atp6 probe) that was not detected with either of the parents. These results indicated that the regenerated plants were somatic hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have occurred. Received: 28 May 1996 / Accepted: 2 August 1996     

A flavonoid mutant of barley (Hordeum vulgare L.) exhibits increased sensitivity to UV-B radiation in the primary leaf

The aim of the present investigation was to define the role of soluble flavonoids as UV-B protectants in the primary leaf of barley (Hordeum vulgare L.). For this purpose we used a mutant line (Ant 287) from the Carlsberg collection of proanthocyanidin-free barley containing only 7% of total extractable flavonoids in the primary leaf as compared to the mother variety (Hiege 550/75). Seven-day-old leaves from plants grown under high visible light with or without supplementary UV-B radiation were used for the determination of UV-B sensitivity. UV-B-induced changes were assessed from parameters of chlorophyll fluorescence of photosystem II, including initial and maximum fluorescence, apparent quantum yield, and photochemical and non-photochemical quenching. A quartz fibre-optic microprobe was used to evaluate the amount of potentially harmful UV-B (310 nm radiation) penetrating into the leaf as a direct consequence of flavonoid deficiency. Our data indicate an essential role of flavonoids in UV-B protection of barley primary leaves. In leaves of the mutant line grown under supplementary UV-B, an increase in 310nm radiation in the mesophyll and a strong decrease in the quantum yield of photosynthesis were observed as compared to the corresponding mother variety. Primary leaves of liege responded to supplementary UV-B radiation with a 30% increase in the major flavonoid saponarin and a 500% increase in the minor compound lutonarin. This is assumed to be an efficient protective response since no changes in variable chlorophyll fluorescence were apparent. In addition, a further reduction in UV-B penetration into the mesophyll was recorded in these leaves.     

Expression of XET-related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.)

Five cDNA clones were isolated from barley (Hordeum vulgare L.) that encoded mRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated that the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA₃), or homozygosity at the ‘slender’ (sln1) locus, resulted in a 2.5-fold (approximately) stimulation of blade elongation rate. Three of the five clones detected mRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GA₃-treated or slender seedlings. The remaining two clones detected mRNAs that were maximally expressed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA₃ treatment revealed similar maximal REGR values despite a 2.5-fold difference in leaf elongation rate. Segments of GA₃-treated leaves attained their maximal REGR values more rapidly, this being associated with enhanced expression of the three ‘basal’ XET-related mRNAs. Highest XET activities were detected in the base of the elongation zone, and in GA₃-treated seedlings a second activity peak was observed near the distal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes with different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.     

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion

An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents. Received: 5 March 1997 / Revision received: 4 September 1997 / Accepted: 13 September 1997     

转TrxS基因啤酒大麦种子中硫氧还蛋白h与淀粉酶活性变化

导入TrxS基因后,转基因大麦籽粒的硫氧还蛋白h活性明显提高;淀粉酶活性也明显提高,其中α-淀粉酶活性在开花后30d提高了3倍以上,随着籽粒的发育,转基因对α-淀粉酶活性影响作用减少,对β-淀粉酶活性的影响有同样的趋势;转基因大麦种子发芽势明显提高。说明TrxS基因有望改善啤酒大麦的制麦特性和品质特性。     

The cytological effects of the gametocides Ethrel and RH-531 on microsporogenesis in barley (Hordeum vulgare L)

Abstract. Barley plants ( Hordeum vulgare L. cv. Midas) raised under controlled environmental conditions were sprayed with either of the gametocides Ethrel and RH-531. At various times after spraying the anthers were fixed for light and electron microscopy. Abortion of sporogenous cells occurred in plants sprayed at both pre- and post-meiotic stages of microsporogenesis. In contrast, cells of the tapetum were insensitive to the immediate effects of gametocides. The cytological effects of the gametocides are similar to those induced by male sterile genes in a variety of plants. These range from the induction of additional mitotic divisions in the pollen mother cells to exine malformations on developing microspores. These observations are discussed in terms of the control mechanisms operating during microsporogenesis.     

Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens

A novel genetic transformation method for barley (Hordeum vulgare L.), based on infection of androgenetic pollen cultures with Agrobacterium tumefaciens, is presented. Winter-type barley cv. 'Igri' was amenable to stable integration of transgenes mediated by A. tumefaciens strain LBA4404 harbouring a vector system that confers hypervirulence, or by the non-hypervirulent strain GV3101 with a standard binary vector. The efficacy of gene transfer was substantially influenced by pollen pre-culture time, choice of Agrobacterium strain and vector system, Agrobacterium population density, medium pH and the concentrations of acetosyringone, CaCl(2) and glutamine. After co-culture, rapid removal of viable agrobacteria was crucial for subsequent development of the pollen culture. To this end, the growth of agrobacteria was suppressed by the concerted effects of appropriate antibiotics, low pH, reduced level of glutamine and high concentrations of CaCl(2) and acetosyringone. Following infection with LBA4404 and GV3101, about 31% and 69%, respectively, of the primary transgenic (T(0)) plants carried a single copy of the sequence integrated. The use of hypervirulent A. tumefaciens and hygromycin resistance as a selectable marker resulted in 3.7 T(0) plants per donor spike. About 60% of the primary transgenic plants set seed, indicating spontaneous genome doubling. An analysis of 20 T(1) populations revealed that four progenies did not segregate for reporter gene expression. This indicates that the approach pursued enables the generation of instantly homozygous primary transgenic plants. The method established will be a valuable tool in functional genomics as well as for the biotechnological improvement of 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.     

Boron toxicity in barley (Hordeum vulgare L.) seedlings in relation to soil temperature

Boron (B) is an essential micronutrient in crop growth but its sufficiency range is narrow. Boron toxicity is a widespread problem in arid and semi-arid areas with cold weather. We investigated the effect of soil temperature (5, 10 and 15°C) on development of symptoms of B toxicity, plant growth and plant development, and on content and concentration of B in tissue of seedlings of four barley lines grown in soil with high level of available B (12 mg kg^–1). Visual symptoms of toxicity were first observed in the high B soil concentration treatment at 5 °C at 12 days after emergence. Concentration of B in tissue decreased with increasing soil-temperatures. There was no effect of soil temperature on B content or B concentration in plant tissue at the final sample (17 days after emergence). High soil B reduced seedling and leaf emergence rates, although the final seedling emergence and number of leaves were unaffected. Barley lines differed in concentration of B in tissues and visual toxicity symptom development. Adaptation to high B was either through maintaining low tissue B concentration or through tolerance to high tissue B concentration. While the investigated range of temperature does influence B toxicity in barley seedlings, it remains to be determined whether it affects crop yield.     

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.     

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.     

XET-related genes and growth kinematics in barley leaves

DV, displacement velocity
GA₃, gibberellic acid
REGR, relative elemental growth rate
XET, xyloglucan-endotransglycosylase

Recently Schünmann et al. (1997 ; Plant, Cell and Environment 20, 1439–1450) investigated the correlation of spatial patterns of xyloglucan-endotransglycosylase (XET) activity, XET-related mRNAs, and growth in elongating barley ( Hordeum vulgare L.) leaves. Here, methodological difficulties in the kinematic growth analysis are discussed, and it is concluded that the role that XET-related gene activity plays in the control of spatial growth patterns remains undetermined.     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves -- the link between jasmonate and abscisic acid

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.     

大麦花药培养中海藻酸钠作用的初步探讨

本试验以普通大麦（Hordeum vulgare L.）品种单核晚期的花药为材料,研究了他们在液体培养过程中海藻酸钠的作用。结果表明：在供试的4个海藻酸钠浓度（1、2、3、6%）中,以浓度为2-3%的效果最佳。当他们代替20-30%的Ficoll时,无论是用BAC3培养基,还是用MS培养基,培养基中加NAA,还是家2,4-D,都能诱导出愈伤组织,但以每升培养基加2mg NAA和1 mg 6-BA的效果最佳,在含这种激素的培养基上,一些基因型的出愈率相当高,供试基因型都在100%以上,而且有些花粉粒能直接发育成能正常发芽出苗的胚状体。Barley anthers at the late uninucleate stage were cultured in order to study the use of sodium alginate in liquidmedium.The results showed that, the effect of 2-3% (W/V) sodium alginate was best in 4 concentration studied (1%,2%,3%and 6%). When the 2-3% sodium alginate was substituted for 20-30% ficoll, the calli could be induced from anthers of all genotypes whether on BAC3 medium or MS medium and whether adding NAA or 2,4-Din medium.2mg/L NAA+1mg/L 6-BA added to medium could give better results and on the medium with these hormones the callus rate of all genotypes were above 100%. Some pollens could grow into embryoids directly.