Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.

Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.

Detection of CRISPR/Cas9-Induced Genomic Fragment Deletions in Barley and Generation of Homozygous Edited Lines via Embryogenic Pollen Culture.

The CRISPR/Cas9 system from Streptococcus pyogenes is an increasingly popular tool for genome editing due to its ease of application. Here we demonstrate genomic DNA fragment removal using RNA directed Cas9 nuclease in barley. The high mutation frequency confirms the exceptional efficiency of the system and its suitability for generating loss-of-function mutant lines that may be used in functional genetics approaches to study endomembrane trafficking pathways and posttranslational protein modifications. The generation of doubled haploids from genome edited plants allows the recovery of true breeding lines that are instantly homozygous for the edited alleles.

Differential Expression Profiling of Microspores During the Early Stages of Isolated Microspore Culture Using the Responsive Barley Cultivar Gobernadora.

In barley, it is possible to induce embryogenesis in the haploid and uninucleate microspore to obtain a diploid plant that is perfectly homozygous. To change developmental fates in this fashion, microspores need to engage in cellular de-differentiation, interrupting the pollen formation, and restore totipotency prior to engaging in embryogenesis. In this work, we used the barley cultivar Gobernadora to characterize the transcriptome of microspores prior to (day 0) and immediately after (days 2 and 5) the application of a stress pretreatment. A deep RNA-seq analysis revealed that microspores at these three time points exhibit a transcriptome of ∼14k genes, ∼90% of which were shared. An expression analysis identified a total of 3,382 differentially expressed genes (DEGs); of these, 2,155 and 2,281 DEGs were respectively identified when contrasting expression at days 0 and 2 and at days 2 and 5. These define 8 expression profiles in which DEGs share a common up- or down-regulation at these time points. Up-regulation of numerous glutathione S-transferase and heat shock protein genes as well as down-regulation of ribosomal subunit protein genes was observed between days 0 and 2. The transition from microspores to developing embryos (days 2 vs. 5) was marked by the induction of transcription factor genes known to play important roles in early embryogenesis, numerous genes involved in hormone biosynthesis and plant hormonal signal transduction in addition to genes involved in secondary metabolism. This work sheds light on transcriptional changes accompanying an important developmental shift and provides candidate biomarkers for embryogenesis in barley.

Dynamics of post-translationally modified histones during barley pollen embryogenesis in the presence or absence of the epi-drug trichostatin A.

KEY MESSAGE: Improving pollen embryogenesis. Despite the agro-economic importance of pollen embryogenesis, the mechanisms underlying this process are still poorly understood. We describe the dynamics of chromatin modifications (histones H3K4me2, H3K9ac, H3K9me2, and H3K27me3) and chromatin marks (RNA polymerase II CDC phospho-Ser5, and CENH3) during barley pollen embryogenesis. Immunolabeling results show that, in reaction to stress, immature pollen rapidly starts reorganizing several important chromatin modifications indicative of a change in cell fate. This new chromatin modification pattern was accomplished within 24 h from whereon it remained unaltered during subsequent mitotic activity. This indicates that cell fate transition, the central element of pollen embryogenesis, is completed early on during the induction process. Application of the histone deacetylase inhibitor trichostatin A stimulated pollen embryogenesis when used on pollen with a gametophytic style chromatin pattern. However, when this drug was administered to embryogenic pollen, the chromatin markers reversed toward a gametophytic profile, embryogenesis was halted and all pollen invariably died.

Transcriptome analysis reveals translational regulation in barley microspore-derived embryogenic callus under salt stress.

KEY MESSAGE: Transcriptome analysis of barley embryogenic callus from isolated microspore culture under salt stress uncovered a role of translation inhibition and selective activation of stress-specific proteins in cellular defense. Soil salinity is one of the major abiotic stresses which constrains the plant growth and reduces the productivity of field crops. In this study, it was observed that the salt stress in barley isolated microspore culture impacted not only on the quantity of embryogenic callus but also on the quality for later differentiation. The barley microspore-derived embryogenic callus, a transient intermediate form linked cells and plants, was employed for a global transcriptome analysis by RNA sequencing to provide new insights into the cellular adaptation or acclimation to stress. A total of 596 differentially expressed genes (DEGs) were identified, in which 123 DEGs were up-regulated and 473 DEGs were down-regulated in the embryogenic callus produced from microspore culture under salt stress as compared to the control conditions. KEGG pathway analysis identified 'translation' (27 DEGs; 12.56 %) as the largest group and followed by 'folding, sorting and degradation' (25 DEGs; 11.63 %) in 215 mapped metabolic pathways. The results of RNA-Seq data and quantitative real-time polymerase chain reaction validation showed that the genes related to translation regulation (such as eIF1A, RPLP0, RPLP2, VARS) were down-regulated to control general protein synthesis, and the genes related to endoplasmic reticulum stress response (such as small heat shock protein genes) were selectively up-regulated against protein denaturing during microspore embryogenesis under continuous salt stress. These transcriptional remodeling might affect the essential protein synthesis for the cell development to fulfill totipotency under salt stress.

Carotenoids of aleurone, germ, and endosperm fractions of barley, corn and wheat differentially inhibit oxidative stress.

The antioxidant potential of carotenoids from aleurone, germ, and endosperm fractions of barley, corn, and wheat has been evaluated. HPLC analysis confirmed the presence of lutein and zeaxanthin carotenoids (nd-15139 µg/kg) in extracts of cereal grain fractions. The antioxidant properties using 2,2-diphenyl-1-picrylhydrazyl, oxygen radical absorbance capacity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays revealed significantly higher (P<0.001) antioxidant activity in the germ than in the aleurone and endosperm fractions. Using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,2'azobis (2-amidinopropane)dihydrochloride (AAPH)-induced cell loss was effectively reduced by preincubating Caco-2, HT-29, and FHs 74 Int cells with carotenoid extracts. Moreover, carotenoid extracts reduced (P<0.001) AAPH-induced intracellular oxidation in the cell lines, suggesting antioxidant activity. Of the 84 antioxidant pathway genes included in microarray array analysis (HT-29 cells), the expressions of 28 genes were enhanced (P<0.05). Our findings suggest that carotenoids of germ, aleurone, and endosperm fractions improved antioxidant capacity and thus have the potential to mitigate oxidative stress.

Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism.

Two alternative cytokinins, thidiazuron and meta-topoline, were tested in isolated microspore culture on recalcitrant barley genotypes (six-row, spring), and green plant regeneration was improved substantially. Doubled-haploid (DH) plants are coveted in plant breeding and in genetic studies, since they are rapidly obtained and perfectly homozygous. In barley, DHs are produced mainly via androgenesis, and isolated microspore culture (IMC) constitutes the method offering the greatest potential efficiency. However, IMC can often be challenging in some genotypes because of low yield of microspores, low regeneration and high incidence of albinism. Six-row spring-type barleys, the predominant type grown in Eastern Canada, are considered recalcitrant in this regard. Our general objective was to optimize an IMC protocol for DH production in six-row spring barley. In particular, we explored the use of alternative hormones in the induction medium (thidiazuron and dicamba), and in the regeneration medium (meta-topoline). This optimization was performed on two typical six-row spring (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) barley cultivar. When 6-benzyl-aminopurine (BAP) was replaced by a combination of thidiazuron and dicamba in the induction medium, a 5.1-fold increase (P < 0.01) in the production of green plants resulted. This increase was mainly achieved by a reduction of albinism. Moreover, a 2.9-fold increase (P < 0.01) in embryo differentiation into green plants was obtained using meta-topoline instead of BAP in the regeneration medium. Together, these innovations allowed us to achieve a substantial improvement in the efficiency of IMC in this recalcitrant type of barley. These results were later successfully validated using sets of F1s from a six-row spring barley breeding program.

Improving the efficiency of isolated microspore culture in six-row spring barley: I-optimization of key physical factors.

An improved isolated microspore culture protocol alleviating the recalcitrance typically observed in six-row spring barley was developed by optimizing four key physical factors to increase embryogenesis and reduce albinism. Doubled haploid (DH) plants are completely homozygous individuals that can be generated in just a few months via androgenesis in vitro. DHs are useful tools in genetic research and in plant breeding. Isolated microspore culture (IMC) is the most efficient way to produce DHs, but a strong genotype dependency imposes limitations to its wide application. Six-row, spring barley genotypes are considered as particularly recalcitrant due to a low frequency of embryogenesis and a high rate of albinism. Seeking to develop an efficient IMC protocol for this type of barley, we explored four important factors: (1) the harvest stage of immature spikes, (2) the type of pretreatment applied, (3) the osmotic potential in the induction medium, and (4) the plating density of microspores. This work was first performed using four barley genotypes: two typical six-row spring cultivars (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) cultivar. First, by optimizing the harvest stage for each genotype we obtained a twofold to fourfold increase in the yield of embryogenic microspores. Second, two pretreatments (0.3 M mannitol for 2 days, or a combination of cold and heat over 15 days) both performed significantly better than the commonly used cold pretreatment (28 days at 4 °C). Third, an induction medium-containing mannitol (32 g/l) doubled green plant regeneration. Fourth, a plating density of 10(6) microspores/ml yielded the highest number of green regenerated plants. Our most important findings were then confirmed using sets of F1s from a six-row, spring-type breeding program.

Time-lapse imaging of the initiation of pollen embryogenesis in barley (Hordeum vulgare L.).

Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.

[Estimation of the efficiency of callus formation and regeneration in barley spring varieties zoned in Ukraine].

The data on in vitro culture establishment and on estimation of callus formation and plant regeneration for eight barley Hordeum vulgare varieties (Getman, Tabora, Adagio, Galactic, Europrestige, Korona, Nevada and Stalker) zoned in Ukraine is represented. Mature embryos of these genotypes were used to study the callus induction and plant regeneration. Using of this approach can rather simplify and intensify the work. Medium, that consists of MS salts supplemented with caseine hydrolysate (1 g/L), L-proline (690 mg/L), thiamine-HCl (1 mg/L), maltose (30 g/L), 2,4-D (2 mg/L), CuSO4 (12.5 mg/L), myoinositol (250 mg/l), gerlite (3.5 g/L) pH 5.6-5.8 was used for both callus formation and plant regeneration. Formation of callus from mature embryos was observed in all studied varieties and showed high frequency (from 65 +/- 3.4% to 100%). It was found that cultivars Korona (88 +/- 2.8%), Europrestige (89 +/- 6.5%), Tabora (93 +/- 3.4%), Getman (99 +/- 0.8%) and Nevada (100%) had the highest regeneration potential. Organogenesis and somatic embryogenesis were the two ways of plant regeneration from callus tissues in barley. The highest general regeneration potential of cultivar Getman (50 +/- 5%) was observed. This cultivar was selected for the further work for development of effective genetic transformation protocol.

Non-invasive mapping of lipids in plant tissue using magnetic resonance imaging.

Plant oil has become an important component in the search for a replacement for non-renewable energy sources, as well as being used for a wide range of industrial purposes, all in addition to its vital importance for human diet. Detailed knowledge of the lipid distribution in plants is fundamental for the understanding of local regulatory networks covering storage metabolism, and for the development of new approaches for plant breeding and transgenic research. We here review a measurement protocol or "tool" based on magnetic resonance imaging (MRI), which allows the non-invasive detection and quantitative visualization of lipid in living plant tissue. The method provides quantitative lipid maps with a resolution close to the cellular level and can be used on a wide range of plants and is applicable at the level of individual tissues, organs, or entire plants during ontogeny. Lipid imaging is designed for both biotechnology and basic science and can be combined with histological, biochemical, and gene expression analysis. Here we present the method as practiced in our group, and discuss unique advantages and limitations of the lipid-imaging tool. Seeds of barley and rapeseed were used as a model for visualization of local oil accumulation at the organ- and tissue-specific scale.

Microspore embryogenesis: assignment of genes to embryo formation and green vs. albino plant production.

Plant microspores can be reprogrammed from their normal pollen development to an embryogenic route in a process termed microspore embryogenesis or androgenesis. Stress treatment has a critical role in this process, inducing the dedifferentiation of microspores and conditioning the following androgenic response. In this study, we have used three barley doubled haploid lines with similar genetic background but different androgenic response. The Barley1 GeneChip was used for transcriptome comparison of these lines after mannitol stress treatment, allowing the identification of 213 differentially expressed genes. Most of these genes belong to the functional categories "cell rescue, defense, and virulence"; "metabolism"; "transcription"; and "transport". These genes were grouped into clusters according to their expression profiles among lines. A principal component analysis allowed us to associate specific gene expression clusters to phenotypic variables. Genes associated with the ability of microspores to divide and form embryos were mainly involved in changes in the structure and function of membranes, efficient use of available energy sources, and cell fate. Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration.

Microspore embryogenesis in barley: anther pre-treatment stimulates plant defence gene expression.

Microspore embryogenesis (ME) is a process in which the gametophytic pollen programme of the microspore is reoriented towards a new embryo sporophytic programme. This process requires a stress treatment, usually performed in the anther or isolated microspores for several days. Despite the universal use of stress to induce ME, very few studies have addressed the physiological processes that occur in the anther during this step. To further understand the processes triggered by stress treatment, we followed the response of anthers by measuring the expression of stress-related genes in two barley (Hordeum vulgare L.) cultivars differing in their ME response. Genes encoding enzymes involved in oxidative stress (glutathione-S-transferase, GST; oxalate oxidase, OxO), in the synthesis of jasmonic acid (13-lipoxygenase, Lox; allene oxide cyclase, AOC; allene oxide synthase, AOS) and in the phenylpropanoid pathway (phenylalanine ammonia lyase, PAL), as well as those encoding PR proteins (Barwin, chitinase 2b, Chit 2b; glucanase, Gluc; basic pathogenesis-related protein 1, PR1; pathogenesis-related protein 10, PR10) were up-regulated in whole anthers upon stress treatment, indicating that anther perceives stress and reacts by triggering general plant defence mechanisms. In particular, both OxO and Chit 2b genes are good markers of anther reactivity owing to their high level of induction during the stress treatment. The effect of copper sulphate appeared to limit the expression of defence-related genes, which may be correlated with its positive effect on the yield of microspore embryos.

Effects of hyperbaric oxygenation on cultured barley embryos.

Changes in relative water content (RWC), lipid peroxidation, proline and antioxidant system in relation to the tolerance to oxidative stress enzymes mediated high pressure were investigated in Hordeum vulgare L. cv. Tokak. For this purpose, mature embryos cultured on MS media were treated in a hyperbaric oxygenation chamber (approx. 59.06 feets, 2 kp/cm2) with pure oxygen for 60 minutes/day for a growth period of ten days in a plant growth chamber. Constitutive activities of SOD, APOX, GR and POX were higher in hyperbaric oxygenated (HBO) explants, being 96.07%, 28.57%, 77.77% and 54.14% for the 5th days; 95.78%, 40%, 37.5%, and 94.98% for the 10th days of culture, respectively, than in the control plants. Increase in SOD activity was also shown on polyacrilamide gel electrophoresis on the 10th day of application. Proline accumulation was increased in HBO-treated explants both on the 5th days (85.71%) and 10th days (37.14%) of treatment. MDA content was found to be higher in HBO treated explants both on the 5th (53.84%) and 10th (59.83%) days of culture.

Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development.

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.

Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature.

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.

Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis.

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.

Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores.

A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 degrees C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 degrees C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55-60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 degrees C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 degrees C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85-97% of these ELS regenerated green shoots. The frequency of doubled haploids (74-83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 degrees C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.

Characterization of cDNAs expressed in the early stages of microspore embryogenesis in barley (Hordeum vulgare) L.

To gain insight into the molecular events occurring in the very early stages of barley microspore embryogenesis, cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture were isolated and characterized. A cDNA library established from barley microspores cultured for three days was differentially screened against probes generated from freshly isolated microspores. Three cDNAs representing genes not previously identified in barley were isolated. ECA1 (early culture abundant 1) lacked significant homology to known genes or proteins, and the transcript was only expressed during the early stages of culture. Expression was also reduced in low-density control cultures, therefore this gene may play a role in the early stages of barley microspore embryogenesis. ECGST (early culture glutathione S-transferase) had homology to parA-like genes, which are members of a newly discovered group of glutathione S-transferases (GSTs). The protein corresponding to ECGST may be important in protecting cells from oxidative stress during the culture process. ECLTP (early culture lipid transfer protein) had homology to lipid transfer proteins (LTPs), and had an expression pattern similar to that of an LTP known to be a marker of the early stages of embryogenesis in the carrot somatic embryogenesis system. The identification and characterization of the clones isolated in this study provides new information on the events involved in barley microspore embryogenesis.