Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

Somaclonal variation in the progeny of transgenic barley

 Somaclonal variation (SCV) in transgenic plants may slow the incorporation of introduced genes into commercially competitive cultivars. Somaclonal variation in transgenic barley (Hordeum vulgare L.) was assessed in one experiment by comparing the agronomic characteristics of 44 segregating transgenic lines in the T₂ generation to their non-transformed parent (‘Golden Promise’). A second experiment examined the agronomic characteristics of seven transgenic-derived, null (non-transgenic) segregant lines in the T₂ and T₄ generations. Compared to their uncultured parent, Golden Promise, most of these lines were shorter, lower yielding, and had smaller seed, and the variability among individual plants was higher. The frequency and severity of the observed SCV was unexpectedly high, and the transformation procedure appeared to induce greater SCV than tissue culture in the absence of transformation. Attempts to understand the sources of SCV, and to modify transformation procedures to reduce the generation of SCV, should be made. Received: 26 June 1997 / Accepted: 31 October 1997     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia

The length polymorphism in the waxy gene, which encodes a granule-bound ADP-glucose-glucosyl transferase [granule-bound starch synthase I (GBSS I), E.C. 2.4.1.11] in barley (Hordeum vulgare), was found. The 5′ leader sequence of the waxy gene of barley germplasm from Japan and Korea was analyzed by the polymerase chain reaction (PCR). The waxy gene of these genetic stocks had three types of length polymorphisms, suggesting that there are insertion/deletion mutations at the 5′ leader sequence of the waxy gene. DNA sequence analysis of the polymorphic PCR products showed that: (1) a 403-bp deletion mutation, which included a complete exon I, was found in the wax allele and a 193-bp insertion sequence was located in the intron I, and (2) the insertion sequence was also located in intron I of the Wax allele. The identity of the insertion sequence was completely conserved between the wax allele and the novel Wax allele. These finding s implying that the wax allele, which was found in indigenous waxy barley, originated in non-waxy barley with the novel Wax allele. Received: 12 January 2001 / Accepted: 17 April 2001     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

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     

Histological analysis of somatic embryogenesis in Brazilian cultivars of barley, Hordeum vulgare vulgare, Poaceae

 Histological analysis was performed aimed at elucidating the origin and the developmental process of somatic embryos of two Brazilian cultivars of barley (Hordeum vulgare vulgare), 'MN-599' and 'A-05'. The observed site of somatic embryo origin (SSEO) could originate from a superficial callus cell, possibly indicating a unicellular origin, or from epidermal and subepidermal callus cells, representing a multicellular origin. A fold, the somatic embryo scutellum that subsequently develops into a cotyledonary leaf, indicates the somatic embryo differentiation. The somatic embryos also showed a growth increase of the primary root and, occasionally, a delay in root development. A possible alternative pathway for the origin of somatic embryos is suggested, in which a SSEO forms a clump of somatic embryos. Received: 4 June 1998 / Revision received: 28 August 1998 / Accepted: 7 December 1998     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

Genetic analysis of disease resistance to all strains of BaYMV in a Chinese barley landrace, Mokusekko 3

 A Chinese landrace of barley, Mokusekko 3, is unique in being completely resistant against all strains of barley yellow mosaic virus (BaYMV). The present investigation revealed that the resistance of Mokusekko 3 is governed by two recessive genes. As one of the resistance genes was known to be tightly linked with alleles at the Est complex locus, consisting of the Est1, Est2 and Est4 loci for esterase isozymes, each of the resistance genes could be separated by means of marker-assisted selection using an isozyme allelic combination as a marker. One of the resistance genes, ym1, is linked to K (hooded lemma) and gl3 (glossy leaf 3) with recombination values of 25.3% and 9.7% respectively, and these three genes are located in the order K-gl3-ym1 on chromosome 4. Another newly designated resistance gene, ym5, is linked to alleles at the Est complex locus and cu2 (curly growth 2), with recombination values of 1.9% and 19.5% respectively, in the order cu2-Est-ym5 from proximal to distal on the long arm of chromosome 3. The complete resistance of Mokusekko 3 is caused by combining two resistance genes, ym1 and ym5. However, almost all the “resistant” cultivars derived from crosses with Mokusekko 3 are susceptible to the recently detected strain BaYMV-III in Japan, since they contain only one resistance gene, ym5. Marker-assisted selection to combine resistance genes into a cultivar is discussed for the breeding of stabilizing resistance to BaYMV. Received: 23 September 1996 / Accepted: 8 November 1996     

Transformation of barley scutellum protoplasts: regeneration of fertile transgenic plants

 A system for barley transformation via polyethyleneglycol-mediated DNA uptake into protoplasts isolated directly from scutella and the regeneration of transgenic plants is reported. Scutellum protoplasts (cv. Clipper, an Australian malting cultivar) were co-transformed with plasmids Act 1-DGUS, containing the marker uidA gene, and pCaIneo, which contains the selectable marker neomycin phosphotransferase gene. Protoplast-derived calluses were selected on medium containing the antibiotic G418 (25 and 15 mg.l^–1) and macroscopic antibiotic resistant colonies were recovered. Fertile plants were regenerated from a callus line and molecular analysis confirmed transgene integration. Received: 11 October 1999 / Revision received: 11 February 2000 / Accepted: 11 February 2000     

Further evidence supporting Morocco as a centre of origin of barley

 Thirty-five populations of H. spontaneum from nine countries, encompassing almost all the known range of distribution of the species, Afghanistan, Crete (Greece), Cyprus, Iran, Iraq, Israel, Libya, Morocco and Turkey, were studied utilizing RFLP markers (21 probes with three restriction enzymes) distributed across all seven barley chromosomes in an attempt to unveil the genetic dissimilarities existing among them. UPGMA clustering, based on the Nei and Li (1979) similarity coefficient, produced a dendrogram where three clusters could be defined: two with a clear geographical distinction (Morocco and Cyprus) and another one grouping all the Asian/Middle Eastern populations, except for an accession from Iran that clustered separately. These results confirm our previous work and suggest that barley domestication could also have taken place outside the Fertile Crescent, particularly in Morocco. Received: 10 October 1998 / Accepted: 27 October 1998     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.     

Stability of transgene expression, field performance and recombination breeding of transformed barley lines

Stable inheritance of the transgene, consistent expression and competitive agronomic properties of transgenic crops are important parameters for successful use of the latter. These properties have been analyzed with 18 homozygous transgenic barley lines of the cultivar Golden Promise. The lines originated from three independent primary transformants obtained by the biolistic method with three plasmids containing respectively, the bar gene, the uidA gene and the gene for a protein-engineered heat-stable (1,3–1,4)-β-glucanase. Three production levels of recombinant β-glucanase were identified in homozygous transgenic T₃ plants, and these remained constant over a 3-year period. In micro-malting experiments, the heat-stable enzyme reached levels of up to 1.4 μg·mg⁻¹ protein and survived kiln drying at levels of 70–100%. In the field trials of 1997 and 1998 the transgenic lines had a reduced 1000-grain weight as well as variable yield depressions compared to the Golden Promise progenitor. In 1999 large-scale propagations of the lines with the highest recombinant enzyme synthesis during germination and of Golden Promise were studied at three different locations. In an irrigated field transgenic lines yielded approximately 6 t·ha⁻¹ and Golden Promise 7.7 t·ha⁻¹. Cross-breeding was carried out to transfer the transgene into a more suitable genetic background. Crosses of the semi-dwarf ari-e mutant Golden Promise gave rise to the four morphological phenotypes nutans, high erect, erect, and ari-e. Two improvements were achieved: (1) F₃ lines homozygous for the expression of heat-stable (1,3−1,4)-β-glucanase were found among lines that were homozygous for each of the four morphological phenotypes; (2) improved 1000-grain weights and yields with respect to those of the original transformants were observed in some F₄ lines homozygous for the morphological phenotypes and for the transgene. In the case of a homozygous nutans line, the transgenic plants had a higher 1000-grain weight than those lacking the transgene. Like mutants providing useful output traits, transgenic plants will often have to be improved by relocating the gene into more suitable genotypes. Received: 6 March 2000 / Accepted: 14 April 2000     

A newly identified barley gene, Dhn12, encoding a YSK2 DHN, is located on chromosome 6H and has embryo-specific expression

Dehydrins are water-soluble lipid-associating proteins that accumulate during low-temperature or water-deficit conditions, and are thought to play a role in freezing- and drought-tolerance in plants. Dhn genes exist as multi-gene families in plants. Previously, we screened lambda genomic libraries of two barley cultivars in an effort to isolate all of the barley Dhn genes. We identified 11 unique Dhn genes and estimated a total of 13 Dhn genes in the barley genome. To extend the collection, we used an alternative source of clones, a 1.5×Morex barley BAC library. In this library, we found nine Dhn genes that we described previously and one new Dhn gene, Dhn12. The Dhn12 gene encodes an acidic YSK₂ dehydrin. The Dhn12 gene is located on chromosome 6H, and shows a different expression pattern from all other Dhn genes identified previously. RT-PCR results show that Dhn12 expression is embryo-specific. Dhn12 is not expressed in seedling shoots under any of the conditions tested, including non-stressed as well as dehydrated, or cold-, ABA- or NaCl-treated seedlings. Received: 6 June 1999 / Accepted: 3 November 1999     

Map locations of barley Dhn genes determined by gene-specific PCR

We previously identified 11 unique barley Dhn genes and found, using wheat-barley addition lines, that these genes are dispersed on four chromosomes 3H, 4H, 5H, 6H. In the present work, more precise positions of barley Dhn genes were determined using gene-specific PCR and 100 doubled haploid lines developed from a cross of Dicktoo and Morex barley. Dhn10 is located on 3H between saflp106 and ABG4. Dhn6 is at the previously determined position on 4H between SOLPRO and BCD265a. Dhn1 and Dhn2 are at the previously determined position on 5H between mR and saflp172. The Dhn locus previously called Dhn4a on barley 5H or Dhn2.2 on T. monococcum 5A is in fact Dhn9 and maps to a revised position between BCD265b and saflp218. Dhn3, Dhn4, Dhn7 and Dhn5 each map to the same position on chromosome 6H, suggesting that the previously reported separation of Dhn3, Dhn4 and Dhn5 may reflect limitations in the accuracy of Southern blot data. In addition to clarifying the map positions of these important stress-related genes, these results illustrate the advantage of gene-specific probes for the mapping of individual genes in a multi-gene family. Received: 11 August 1999 / Accepted: 16 December 1999     

Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues

 Highly regenerative tissues of tall fescue and red fescue produced from mature seed-derived embryogenic callus were induced and proliferated on medium containing 2,4-dichlorophenoxyacetic acid (4.5 or 9.0 μM), 6-benzylaminopurine (0, 0.044, 0.44 or 2.2 μM) and cupric sulfate (0.1 or 5.0 μM) under dim-light conditions (10 to 30 μE m^–2 s^–1, 16 h light). Tall fescue tissues were transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA;gus), and red fescue with three plasmids containing hpt, uidA and a synthetic green fluorescent protein gene [sgfp(S65T)]. DNA from T₀ plants of eight independently transformed lines from tall fescue and 11 from red fescue were analyzed by PCR and DNA blot hybridization. The co-expression frequency of all three transgenes [hpt/bar/uidA or hpt/uidA/sgfp(S65T)] in transgenic tall fescue and red fescue plants was 25–27%; for two transgenes [hpt/bar or hpt/uidA for tall fescue and hpt/uidA or hpt/sgfp(S65T) for red fescue], the co-expression frequency was 50–75%. Received: 28 September 1999 / Revision received: 13 March 2000 / Accepted: 16 March 2000     

Development of YLM, a codominant PCR marker closely linked to the Yd2 gene for resistance to barley yellow dwarf disease

 The Yd2 gene in barley provides protection against barley yellow dwarf luteovirus (BYDV), the most economically devastating virus of cereals worldwide. Because resistance assays to identify Yd2-containing individuals from breeding populations are often difficult, we have developed a closely linked, codominant PCR-based marker for Yd2 using AFLP marker technology. The marker, designated YLM, can be amplified from barley genomic DNA prepared using a rapid and simple extraction procedure and, in a survey of more than 100 barley genotypes, was found to be polymorphic between most Yd2 and non-Yd2 lines. The YLM therefore shows excellent potential as a tool for selecting Yd2-carrying segregants in barley breeding programmes. Received: 15 August 1997 / Accepted: 1 December 1997     

The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants

Segregating T₁, T₂ and T₃ transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected β-glucuronidase (gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing the likelihood of an additive effect on transgene expression level is discussed. Received: 21 March 2001 / Accepted: 29 June 2001     

Identification of QTLs for partial resistance to leaf rust (Puccinia hordei) in barley

 The partial resistance to leaf rust in barley is a quantitative resistance that is not based on hypersensitivity. To map the quantitative trait loci (QTLs) for partial resistance to leaf rust, we obtained 103 recombinant inbred lines (RILs) by single-seed descent from a cross between the susceptible parent L94 and the partially resistant parent Vada. These RILs were evaluated at the seedling and adult plant stages in the greenhouse for the latent period (LP) of the rust fungus, and in the field for the level of infection, measured as area under the disease progress curve (AUDPC). A dense genetic map based on 561 AFLP markers had been generated previously for this set of RILs. QTLs for partial resistance to leaf rust were mapped using the “Multiple Interval Mapping” method with the putative QTL markers as cofactors. Six QTLs for partial resistance were identified in this population. Three QTLs, Rphq1, Rphq2 and Rphq3, were effective at the seedling stage and contributed approximately 55% to the phenotypic variance. Five QTLs, Rph2, Rphq3, Rphq4, Rphq5, and/or Rphq6 contributed approximtely. 60% of the phenotypic variance and were effective at the adult plant stage. Therefore, only the QTLs Rphq2 and Rhpq3 were not plant-stage dependent. The identified QTLs showed mainly additive effects and only one significant interaction was detected, i.e. between Rphq1 and Rphq2. The map positions of these QTLs did not coincide with those of the race-specific resistance genes, suggesting that genes for partial resistance and genes for hypersensitive resistance represent entirely different gene families. Also, three QTLs for days to heading, of which two were also involved in plant height, were identified in the present recombinant inbred population. These QTLs had been mapped previously on the same positions in different populations. The perspectives of these results for breeding for durable resistance to leaf rust are discussed. Received: 15 July 1997 / Accepted: 30 December 1997