Barley Transformation Research Articles

Somatic Embryogenesis and Plant Regeneration in Barley (Hordeum vulgare L.)

Commercial cultivars and elite germplasms of barely (Hordeum vulgare L) are still recalcitrant to genetic transformation because of the lack of an efficient regeneration system. In this study, we established an efficient plant regeneration procedure from embryogenic calli derived from mature embryos. Callus induction from germinated mature embryos was best as over 95% in CIM medium (CI medium containing <TEX>$2.5mg/{\ell}$</TEX> dicamba) under dark incubation. Development of embryogenic callus was highest as over 50% in CI3D medium (EC medium supplemented with <TEX>$3mg/{\ell}$</TEX> 2,4-D). The highest regeneration of plants from embryogenic callus (40%) was obtained with CIS medium (<TEX>$SI+1mg/{\ell}IAA\;and\;2mg/{\ell}\;BA$</TEX>). These plant regeneration conditions could be useful in improving barley transformation efficiency.

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules

We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.

Application of antisense transformation of a barley chitinase in studies of arbuscule formation by a mycorrhizal fungus

Barley (Hordeum vulgare L.) plants of two commercial cultivars were transformed with sense and antisense constructs of a chitinase class II gene in order to develop a transformation system for this gene in barley. Transformation of embryos with the two antisense constructs resulted in ten regenerated plants, while no plants could be obtained using the sense construct. The presence of the inserted construct could be confirmed for six of the plants by PCR analysis. This system was used to study the role of class II chitinase in the regulation of mycorrhizal symbiosis. The colonization of two of the antisense transformants by the arbuscular mycorrhizal fungus Glomus intraradices was investigated microscopically and by use of signature fatty acids. The arbuscular incidence increased in transformed barley, and one transformant supported higher extraradical mycelium biomass. It is concluded that antisense transformation of barley could be a useful tool in investigations on the symbiosis between barley and mycorrhizal fungi.

Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.

Effects of Three Promoters in Barley Transformation by Particle Bombardment of Mature and Immature Embryos

ABSTRACTThree different promoters, cauliflower mosaic virus 35S (CaMV 35S), polyubiquitin of maize (Ubi1) and actin1 of rice (Act-1) were compared in a transient assay using particle bombardment of barley mature and immature embryos. The effectiveness of different promoters was compared using a reporter gene expressing β-glucuronidase (GUS). Different levels of GUS gene activity in barley embryos were detected by histochemical analyses. The CaMV 35S promoter showed the lowest level of transient expression in barley cells. The highest activity was observed for the rice (Act-1) promoter and assumed to be a strong promoter in barley. All studied promoters induced about 1% higher number of blue spots in immature barley embryos vs. mature. This study shows that an efficient transformation system could be developed in barley through particle bombardment using constructs based on rice (Act-1) promoter.

Development of the Simple Gene Gun Apparatuses Systems

ABSTRACTThree models of a simple and inexpensive home-made gene gun apparatus was designed for genetic transformation of barley, wheat, maize and rice mature embryos. The compact and efficient design for gene transfer in plants was improved. The biological, physical parameters influencing transformation efficiency and bombardment medium suitable for gene guns apparatus were optimized. High levels of transient expression of the β-glucu-ronidase gene in the mature embryos of cereals were achieved.

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

Somatic Embryogenesis and Morphogenetic Potential of Spring Barley (Hordeum vulgare L.) in the System of Technological Genetic Transformation

We studied somatic embryogenesis and morphogenetic potential in young embryos of 17 spring barley cultivars. A considerable effect of the genotype as well as of certain biologically active compounds in the growth medium on the frequency of embryogenic callus formation and regenerative capacity was observed. The Murashige and Skoog medium complemented with myoinositol and casein hydrolysate is suitable for embryogenic callus induction. The regeneration medium should be complemented by hormones, 2.5 mg/l auxin 2,4-D and 0.1–0.5 mg/l cytokinin 6-BAP, to increase the frequency of somatic embryo maturation. The presence of cefotaxime and high level of copper are desirable to increase the efficiency of callogenesis, regeneration, and callus quality. Three cultivars with economically valuable characters and high morphogenetic potential were recommended for development of efficient technology for barley transformation.

An Evaluation of Feed-Back Insensitive Aspartate Kinase as a Selectable Marker for Barley (Hordeum Vulgave L.) Transformation

In the present study we have evaluated the use of feedback insensitive aspartate kinase as a selectable marker for barley transformation. Immature embryos were bombarded with plasmids containing an Escherichia coli mutant lysC gene encoding a lysine—threonine feedback insensitive form of aspartate kinase (AK). The heterologous gene was fused to the sequence coding for the transit peptide of the small subunit of ribulose 1,5-bisphosphate carboxylase of pea in order to target AK into the chloroplast. Either the cauliflower mosaic virus (CaMV) 35S or the maize ubiquitin promoter directed expression of lysC. Transgenic, regenerable cell cultures were obtained after bombardment with both constructs and selection on MS medium with millimolar concentrations of lysine plus threonine. Transgenic regenerants were, however, only obtained from cultures where the ubiquitin promoter directed expression of the lysC gene. Additional parameters of importance were omission of glutamine from the selection medium, a small embryo size and the application of selection immediately following isolation of embryos. From a total of 720 embryos bombarded with the ubiquitin—lysC construct, 13 transgenic cell lines were selected. Two of these regenerated green plants, while 8 lines produced only albino plants. All regenerated plants were transgenic as evidenced by PCR amplification of the heterologous gene. In two individual lines of green transgenic plants, enzyme assays revealed a 3.5 and 4.0-fold increase in AK activity in the leaves, respectively while in two independent transgenic albino plants, the increase in AK activity was 6.5 and 11.5-fold, respectively. The potential of this selection system for barley transformation is discussed.

Optimisation of Steeping Program in Malt Industry

Abstract Malt is the major cereal grain used in beer processing. It results from the transformation of Barley along the three main steps of the malting process: steeping, germination and kilning. The malt quality is strongly correlated to the set-up of the steeping phase. The later consists of 2 or 3 sequential cycles of wet steeps and air rests. In this work the steeping program durations which optimise the malt quality, expressed as friability, are determined. In order to perform this optimisation, a mathematical model is constructed using an iterative experimental design. Both model and the resulting optimal steeping conditions are experimentally validated.

Novel plasmid vectors for homologous transformation of barley (Hordeum vulgare L.) with JIP23 cDNA in sense and antisense orientation

The most abundant jasmonate-induced protein (JIP) in barley leaves is a 23 kDa protein (JIP23). Its function, however, is unknown. In order to analyze its function by homologous transformation, new plasmid vectors have been constructed. They carry the cDNA coding for JIP23 in sense or antisense orientation under the control of the Ubi-1-promoter as well as the pat resistance gene under the control of the 35S promoter. Barley mesophyll protoplasts were transiently transformed with the sense constructs. PAT activity and immunological detection of JIP23 could be achieved in transformed protoplasts but not in untransformed protoplasts indicating that the construct was active. Thus, these new vectors are suitable for stable transformation of barley. Carrying a multiple cloning site (MCS), these vectors can be used now in a wide range of transformation of barley.

Chopra, V.L., and Prakash, S. (eds) Evolution and adaptation of cereal crops

Chopra, V.L., and Prakash, S. (eds) Evolution and adaptation of cereal crops

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l−1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.

Genetic transformation of the commercial barley ( Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus

Commercial barley cultivars are difficult to transform because of the lack of an efficient regeneration system. By modifying certain components in the standard culture medium, we have developed a reproducible and more efficient regeneration system. Herbicide-resistant transgenic plants from barley (Hordeum vulgare L. cv. Conlon) were obtained using this medium. Embryo-derived callus was bombarded with pAHC25, which contains the screenable marker gus (β-glucuronidase) and the selectable marker bar (bialaphos resistance gene), both driven by the maize ubiquitin promoter (Ubi1) and followed by the nos terminator. Following bombardment, callus was transferred to callus-induction medium supplemented with 5 mg/l bialaphos for selection. Resistant calli were subsequently transferred to maintenance medium containing 5 mg/l bialaphos for further selection and finally transferred to regeneration medium with 5 mg/l bialaphos. Green shoots that developed on the regeneration medium were transferred to rooting medium containing 3 mg/l bialaphos. Eighty-five transgenic plants were obtained from 13 independent transformation events. Progeny tests showed Mendelian inheritance for the transgenes. This is the first report of the production of large numbers of transgenic plants from a commercial cultivar adapted to Midwestern US barley production.

Agrobacterium-mediated barley ( Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA∝barley genomic DNA junctions

Summary Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with the hpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a non-destructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants.

Matrix attachment regions (MARs) enhance transformation frequencies and reduce variance of transgene expression in barley.

Nuclear matrix attachment regions (MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. They are suggested to play a role in the cis unfolding and folding of the chromatin fibre associated with the regulation of gene transcription. Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. The present study is the first to investigate the influence of MAR sequences on transformation frequencies and transgene expression in barley, which is highly relevant to the future improvement of this crop by biotechnology. Two plant MAR sequences were tested both for their ability to bind to the nuclear matrix of barley leaf nuclei and to regulate the expression of a reporter gene in transgenic barley. Competitive in vitro MAR binding assays with the 520 bp P1-MAR from soybean and the 516 bp TBS-MAR from petunia revealed that only the P1-MAR had specific binding affinity for barley nuclear matrices. The barley transformation frequency with the uidA reporter gene was increased 2-fold when the gene was flanked with either the P1-MAR or TBS-MAR, while the gene copy number was strongly reduced. The presence of P1-MAR sequences increased the mean activity and reduced the variance in expression of a co-integrated reporter gene in barley consistent with the proposed model of MAR activity.

Transgenic Approaches to Combat Fusarium Head Blight in Wheat and Barley

Fusarium head blight (FHB) and contamination with deoxynivalenol (DON) produced by the primary pathogen Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.)] have caused devastating losses to wheat (Triticum aestivum L.), durum (Triticum turgidum L. ssp. durum Desf.Husr.), and barley (Hordeum vulgare L.) growers across the USA since the early 1990s. Evaluation of barley, wheat, and related germplasm yielded only a few accessions with partial resistance. This resistance appears, in most cases, to be under polygenic control, making the development of resistant cultivars with suitable agronomic and quality traits a challenge. The insertion of individual antifungal and antitoxin genes via genetic transformation has the potential to aid in development of resistant wheat and barley cultivars. Although wheat and barley transformation has been achieved in several laboratories, the development of a high throughput wheat and barley transformation systems has been slowed by genotype effects on plant regeneration, low transformation efficiencies, somaclonal variation, and problems with transgene inheritance and stability of expression. Among the antifungal genes targeted to combat FHB are coding sequences for proteins that degrade fungal cell walls, disorganize fungal membranes, bolster the host defense response systems, and interfere with fungal protein synthesis, pathogenesis, and/or accumulation of DON. Promoter sequences have been selected that confer high levels of expression to the antifungal constructs, particularly in the spike tissues which are susceptible to FHB. As more antifungal genes are inserted into wheat and barley, field and greenhouse evaluation will show whether transgenes achieve their potential in the fight against FHB.

Trichothecene toxin effects on barley callus and seedling growth

Fusarium head blight (FHB) has caused devastating losses to barley growers in ND, MN and SD since 1993. A major component of these losses has been the formation of protein synthesis inhibitor trichothecene toxins such as deoxynivalenol (DON) by the Fusarium species that cause FHB. Genetic transformation of barley with genes that chemically inactivate or transport the toxins out of cells has potential for reducing DON levels. The ability to directly select for callus cells transformed with these genes would simplify production of transgenic plants and avoid the introduction of a second selectable gene. Differences in seedling germination and growth on medium containing toxin would help identify transgenic plants expressing the antitoxin genes. The objective of this study was to determine the effects of DON and the related toxin diacetoxyscirpenol (DAS) on barley callus growth and seedling germination, root and shoot growth. The effects of the toxins on callus growth were small. Although statistically significant differences were seen at the higher toxin levels after eight weeks, the differences were not large enough for selection between toxin resistant and toxin susceptible calli. Neither toxin had an effect on seed germination. DON at 5 and 10 mg/L significantly reduced root growth by day 7, and 10 mg/L significantly reduced shoot growth by day 4. The effects of DAS were greater, with all toxin levels significantly reducing root growth by day 2 and shoot growth by day 4. The results indicate that DAS may be usable for screening progeny of transgenic plants for expression and segregation of anti-toxin genes.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

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.