A comparison of minirhizotron,core and monolith methods for quantifying barley (Hordeum vulgare L.) and fababean (Vicia faba L.) root distribution

Root research has been hampered by a lack of good methods and by the amount of time involved in making measurements. The use of the minirhizotron as a quantitative tool requires comparison with conventional destructive methods. This study was conducted in the greenhouse to compare the minirhizotron technique with core and monolith methods in quantifying barley (Hordeum vulgare L.) and fababean (Vicia faba L.) root distribution. Plants were grown in boxes (80 cm long × 80 cm wide × 75 cm deep) in a hexagonal arrangement to minimize the effects of rooting anistrophy. Minirhizotron observations and destructive sampling to a depth of 70 cm using core and monolith methods were performed at the ripening growth stage. Total root length for the entire depth interval was generally higher in barley (159–309 m) than fababean (110–226 m). Significant correlation coefficients between monolith and core methods for root length density (RLD, cm cm^–3) was observed in both crops (p 0.01). A method and depth interaction showed no significant differences in fababean RLD distribution measured by core and monolith methods. However, the RLD was different for the uppermost 40 cm depth in barley. The relationship for RLD between minirhizotron and core methods was significant only in barley (r=0.77^*). For both crops, estimates of RLD in the top 10-cm layer by the minirhizotron technique were lower than those by core and monolith techniques. In contrast, estimates of RLD were higher in fababean at a depth >30 cm. Destructive sampling still remains the method to quantify root growth in the 0–10 cm soil layer. ei]B E Clothier     

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.     

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.     

Novel approaches for examining the effects of differential soil compaction on xylem sap abscisic acid concentration, stomatal conductance and growth in barley (Hordeum vulgare L.)

Novel techniques were devised to explore the mechanisms mediating the adverse effects of compacted soil on plants. These included growing plants in: (i) profiles containing horizons differing in their degree of compaction and; (ii) split-pots in which the roots were divided between compartments containing moderately (1·4 g cm ^{? 3}) and severely compacted (1·7 g cm ^{? 3}) soil. Wild-type and ABA-deficient genotypes of barley were used to examine the role of abscisic acid (ABA) as a root-to-shoot signal. Shoot dry weight and leaf area were reduced and root : shoot ratio was increased relative to 1·4 g cm ^{? 3} control plants whenever plants of both genotypes encountered severely compacted horizons. In bartey cultivar Steptoe, stomatal conductance decreased within 4 d of the first roots encountering 1·7 g cm ^{? 3} soil and increased over a similar period when roots penetrated from 1·7 g cm ^{? 3} into 1·4 g cm ^{? 3} soil. Conductance was again reduced by a second 1·7 g cm ^{? 3} horizon. These responses were inversely correlated with xylem sap ABA concentration. No equivalent stomatal responses occurred in Az34 (ABA deficient genotype), in which the changes in xylem sap ABA were much smaller. When plants were grown in 1·7 : 1·4 g cm ^{? 3} split-pots, shoot growth was unaffected relative to 1·4 g cm ^{? 3} control plants in Steptoe, but was significantly reduced in Az34. Excision of the roots in compacted soil restored growth to the 1·4 g cm ^{? 3} control level in Az34. Stomatal conductance was reduced in the split-pot treatment of Steptoe, but returned to the 1·4 g cm ^{? 3} control level when the roots in compacted soil were excised. Xylem sap ABA concentration was initially higher than in 1·4 g cm ^{? 3} control plants but subsequently returned to the control level; no recovery occurred if the roots in compacted soil were left intact. Xylem sap ABA concentration in the split-pot treatment of Az34 was initially similar to plants grown in uniform 1·7 g cm ^{? 3} soil, but returned to the 1·4 g cm ^{? 3} control level when the roots in the compacted compartment were excised. These results clearly demonstrate the involvement of a root-sourced signal in mediating responses to compacted soil; the role of ABA in providing this signal and future applications of the compaction procedures reported here are discussed.     

西藏青稞4个B组醇溶蛋白基因的克隆和特征

从两份西藏青稞材料中分离克隆出4个B组醇溶蛋白基因（BH1—BH4）,DNA测序结果表明：它们均包含完整的开放阅读框。推断的氨基酸序列与先前报道的大麦B组醇溶蛋白具有相同的蛋白质基本结构。系统分析表明：它们推断的氨基酸序列与栽培大麦中的B组醇溶蛋白具有较高的相关性,与野生大麦和山羊草属的醇溶谷蛋白相似性较低。并且,在4个基因BH1—BH4中,BH1与先前报道的B组醇溶蛋白基因有较低的序列相似性,因此我们对BH1基因进行了原核表达,含该基因的表达载体在大肠杆菌中表达出相对分子质量为28．15kDa并以包涵体形式存在的蛋白,进一步对其在青稞谷粒品质改良中的潜在价值进行了探讨。     

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     

Pre-germination effects on mechanically impeded root growth of barley (Hordeum vulgare L)

The effects of conditions pre-dating germination on growth rate of impeded barley cv. Harrington roots were measured using an agar-capillary tube technique. Seedling root tips were directed into glass capillary tubes twothirds filled with agar at eight concentrations ranging from 1.6 to 9.6%, equivalent to penetrometer resistances of 25 to 1240 kPa. The rate of unrestricted root elongation (growth in air) of seed stored for 13 months (old seed), and of seed grown for a second generation without subsequent storage (new seed) was compared with growth in agar over a 24-hour interval. Root elongation rate of old and new seed was identical in the absence of resistance. At low to intermediate agar concentrations, elongation was significantly slower in roots from old, compared with new seed. At high agar concentrations root growth of old and new seed was the same. In both old and new seed, root growth through agar was greater in seed that germinated after 24, compared with 48 h. Differences in impeded root growth between old and new seed were lost in progeny of the test seed. Environmental factors that pre-date germination are an important influence on the ability of seedling roots to elongate through soil.LRS Contribution no. 3879349LRS Contribution no. 3879349     

Map construction of sequence-tagged sites (STSs) in barley (Hordeum vulgare L.)

 In order to identify sequence-tagged sites (STSs) appropriate for recombinant inbred lines (RILs) of barley cultivars ‘Azumamugi’ × ‘Kanto Nakate Gold’, a total of 43 STS primer pairs were generated on the basis of the terminal sequences of barley restriction fragment length polymorphism (RFLP) clones. Forty one of the 43 primer pairs amplified PCR products in Azumamugi, Kanto Nakate Gold, or both. Of these, two showed a length polymorphism and two showed the presence or absence of polymorphism between the parents. PCR products of the remaining 37 primers were digested with 46 restriction endonucleases, and polymorphisms were detected for 15 primers. A 383.6-cM linkage map of RILs of Azumamugi×Kanto Nakate Gold was constructed from the 19 polymorphic STS primer pairs (20 loci) developed in this study, 45 previously developed STS primer pairs (47 loci), and two morphological loci. Linkage analysis and analysis of wheat-barley chromosome addition lines showed that with three exceptions, the chromosome locations of the STS markers were identical with those of the RFLP markers. Received: 4 August 1998 / Accepted: 8 October 1998     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin

Triacontanol (TRIA) treatment of plasma membrane-enriched vesicles from barley ( Hordeum vulgare L., cv. Conquest) roots resulted in stimulation of membrane-associated, divalent cation-dependent ATPase activity (EC 3.6.1.3). The stimulation at physiologically active concentrations of TRIA (10⁻¹¹–10⁻⁹ M ) occurred only when the vesicles were treated with TRIA in the presence of calmodulin. Octacosanol, the C₂₈-analogue of TRIA, had no effect on divalent cation-dependent ATPase activity. Consistent with in vivo studies, simultaneous treatment of vesicles with weight equivalents of TRIA and octacosanol reduced the stimulation of ATPase activity. The effect of calmodulin on the stimulation of ATPase activity was diminished by calmidazolium, a specific inhibitor of calmodulin. Circular dichroism studies did not show a change in the α-helix content of calmodulin in the presence of TRIA. TRIA also had no apparent effect on soluble calcium-calmodulin 3',5'-cyclic nucleotide phosphodiesterase activity. Removal of excess TRIA from the medium after treatment still resulted in stimulation of divalent cation-dependent ATPase activity in the presence of calmodulin was comparable to treated vesicles from which excess TRIA had not been removed. These data further support the contention that TRIA affects membrane structure and function.     

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.     

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.     

An RFLP map of diploid Hordeum bulbosum L. and comparison with maps of barley (H. vulgare L.) and wheat (Triticum aestivum L.)

This paper describes the first extensive genetic map of Hordeum bulbosum, the closest wild relative of cultivated barley. H. bulbosum is valuable for haploid production in barley breeding, and because of desirable agronomic characteristics, it also has potential for trait introgression into barley. A H. bulbosum map will assist introgression and provide a basis for the identification of QTLs for crossability with barley and other potentially useful genes. The present study used a population of 111 individuals from a PB1×PB11 cross to develop a genetic linkage map of diploid H. bulbosum (2n=2x=14) based on barley, wheat and other ”anchor” cereal RFLP markers previously mapped in other species. Because of the cross-pollinating and highly polymorphic nature of H. bulbosum, up to four alleles showed segregation at any one locus, and five different segregation types were found. This enabled maps to be developed for the PB1 and PB11 parents, as well as a combined map. In total, 136 RFLP loci were mapped with a marker coverage of 621 cM. The markers were generally colinear with barley but H. bulbosum had less recombination in the centromeric regions and similar or more in the distal regions. Cytological studies on pollen mother cells at metaphase-I showed marked distal localization of chiasmata and a frequency consistent with the genetic map length. This study showed that H. bulbosum was highly polymorphic, making it suitable for trait analysis and supplementing maps of barley. Received: 20 November 2000 / Accepted: 5 January 2001     

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     

Si amelioration of Al toxicity in barley (Hordeum vulgare L.) growing in two Andosols

The influence of silicon on aluminium toxicity in barley (Hordeum vulgare L. cv. Shunrai) was studied in two Andosols. Silicon sources were a solution of sodium metasilicate with pH adjusted to 5.0, silica gel, and an industrial waste, porous hydrated calcium silicate. The waste is produced in large amounts in the manufacturing processes of autoclaved light concrete, and has been used as a silicon source for rice plants. The addition of the waste increased the concentration of Si in the soil solution, soil pH and amelioration of aluminium toxicity was observed. The addition of silica gel and sodium metasilicate solution to both soils increased significantly (p<0.05) the Si concentration of the soil solutions, but no amelioration of aluminium toxicity was observed. An amelioration of aluminium toxicity by the waste porous hydrated calcium silicate was probably due to the increase in soil pH rather than to the increase of silicon concentration in the soil solution.     

The effect of low temperature on patterns of cell division in developing second leaves of wild-type and slender mutant barley (Hordeum vulgare L.)

This study reports on investigations into the effect of long-term growth at reduced temperatures on cell elongation and cell division in the wild type and a temperature-insensitive ( slender ) mutant of barley. Plants were grown under two temperature regimes (20 and 5 °C) and the mitotic index, cell doubling time and cell lengths over the division and elongation zone were monitored at several stages of development in the second leaf. Leaf length and leaf growth rates were characteristically greater in the slender mutant than in the wild type and this was greatly exaggerated by growth at low temperature. Cell length and the length of the division zone were also greater in the slender mutant than in the wild type, and growing the plants at reduced temperature (5 °C) shortened cell lengths only in the wild type. The slender mutant had a higher mitotic index than the wild type, although in neither genotype was change in the mitotic index observed following growth at reduced temperature. Cell doubling time, on the other hand, was reduced by growth at reduced temperature in the wild type but not in the slender mutant. Thus, the data suggest very different growth responses to low temperature in the two genotypes. The results are discussed in terms of the ability of plants to sense their environment and optimize their metabolism for future growth.     

QTL analysis of tolerance to a German strain of BYDV-PAV in barley (Hordeum vulgare L.)

One hundred and forty six barley doubled-haploid lines (DH lines) were tested for variation in grain yield, yield components, plant height, and heading date after artificial infection with a German isolate of barley yellow dwarf virus (BYDV-PAV-Braunschweig). Of these 146 lines 76 were derived from the cross of the barley yellow dwarf virus (BYDV) tolerant cultivar ’Post’ to cv ’Vixen’ (Ryd2) and 70 from the cross of Post to cv ’Nixe’. Phenotypic measurements were gathered on both non-infected plants and plants artificially inoculated with BYDV-PAV by viruliferous aphids in pot and field experiments for three years at two locations. For all traits a continuous variation was observed suggesting a quantitative mode of inheritance for tolerance against BYDV-PAV. Using skeleton maps constructed using SSRs, AFLPs and RAPDs, two QTLs for relative grain yield per plant after BYDV infection, explaining about 47% of the phenotypic variance, were identified in Post × Vixen at the telomeric region of chromosome 2HL and at a region containing the Ryd2 gene on chromosome 3HL. In Post × Nixe, a QTL was found in exactly the same chromosome 2HL marker interval. In this cross, additional QTL were mapped on chromosomes 7H and 4H and together these explained about 40% of the phenotypic variance. QTL for effects of BYDV infection on yield components, plant height, and heading date generally mapped to the same marker intervals, or in the vicinity of the QTL for relative grain yield, on chromosomes 2HL and 3HL, suggesting that these regions are of special importance for tolerance to the Braunschweig isolate of BYDV-PAV. Possible applications of marker-assisted selection for BYDV tolerance based on these results are discussed. Received: 1 December 2000 / Accepted: 9 March 2001     

Isolation of sucrose: sucrose 1-fructosyltransferase (1-SST) from barley (Hordeum vulgare)

The enzyme sucrose: sucrose 1-fructosyltransferase was partially purified from barley leaf growth zones. Four steps (ammonium sulphate precipitation and polyethylene glycol precipitation, followed by chromatography on Concanavalin A-sepharose and hydroxylapatite) yielded a 35-fold purification. The resulting preparation of 1-SST which still contained a number of different activities related to fructan metabolism, was subjected to preparative isoelectric focusing, and sections of the gel were analysed individually for 1-SST and related activities, using sucrose and 1-kestose as substrates. This procedure yielded a 196-fold purification and revealed the presence of two isozymes of 1-SST with pI values of 4.93 and 4.99, as determined by analytical isoelectric focusing of the corresponding fractions. Both isozymes produced glucose and 1-kestose when incubated with sucrose. In addition, small amounts of 6-kestose and tetrasaccharides were formed. In particular, one of the two 1-SST isozymes yielded fructose when incubated with 1-kestose, indicating that it also acts as a fructan exohydrolase. The other isozyme exhibited less fructan exohydrolase activity. Nystose was also degraded by the fructan exohydrolase activity but less than 1-kestose, whereas 6-kestose was not a substrate for the enzyme. Incubation of both 1-SSTs with different concentrations of sucrose showed that the enzyme was not saturated even at 500 mM. As for the barley sucrose: fructan 6-fructosyltransferase, both isozymes of 1-SST yielded two polypeptide bands of molecular weight 50 and 22 kDa upon sodium dodecylsulphate polyacrylamide gel electrophoresis, suggesting their close relationship to invertase (composed of two subunits of similar size), as previously reported for other plants.