A. M. Castillo

Production of large number of doubled haploid plants from barley anthers pretreated with high concentrations of mannitol.

Pretreatment with increasing concentrations of mannitol, from 0.3 to 0.7 M, was used to induce stress in cultured anthers of barley (Hordeum vulgare L.). Three cultivars with varying degrees of androgenetic ability were studied. A positive linear relationship was found between concentration of mannitol in the pretreatment medium and the number of regenerated green doubled haploid plants in all the cultivars. The pretreatment also resulted in an increasing proportion of embryos to dividing microspores, and in green to albino plantlets. The optimum length of the pretreatment seemed to be genotype dependent. When Ficoll was used as an alternative stress agent a differential genotype response was observed.

Comparison of anther and isolated microspore cultures in barley: effects of culture density and regeneration medium

A comparison of anther and microspore culture efficiency for green doubled haploid plant production was undertaken using 17 F1 crosses with potential agronomic performance. Green doubled haploid plants were produced from all F1 crosses by anther and microspore cultures, although there was a great variation among crosses. On average, anther culture resulted in a production of green plant twice that of isolated microspore culture (30 and 14 greenplants/100 anthers, respectively).The effect of microspore culture density on green plant regeneration was studied with the cultivars Igri, Reinette and Hop which have a high, medium and low androgenic response. The highest number of dividing microspores was obtained at a density of 2.4 ×105 viable microspores/ml for the three cultivars. However, the optimal density for the percentage of embryos/dividing microspores and greenplants/103 microspores depended on the cultivar. The highest number of green plants/103microspores was produced at 1.2 × 105 viable microspores/ml for cv. Igri and 2.4 × 105 for cultivars Reinette and Hop. Percentage of green plants/total plants was raised when the culture density was increased up to 6.0 × 105 viable microspores/ml, especially for cv. Reinette. Six regeneration media differing in maltose concentration, organic nitrogen and type of auxin were assayed with embryos from cultivar Reinette. Media without organic nitrogen containing 31 g l-1maltose and the auxins IAA or NAA produced more vigorous green plants.

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures

Abstract The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10–3–10–5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M2 doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M2 doubled-haploid lines carrying mutations per line analysed was 3.8% when 10–4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10–5 and 10–4 M sodium azide were applied to freshly isolated microspores from model cultivars.

Genetic markers for doubled haploid response in barley

In order to analyse the genetic control of anther culture response in barley, a doubled-haploid (DH) population from the cross between a medium responsive cultivar ‘Dobla’ and the model cultivar ‘Igri’ was produced. A linkage map was constructed with 91 markers. A sub-population of 41 lines was characterised for different components of the anther culture response, and was used for quantitative trait loci (QTL) analysis. The vrs1 locus region on chromosome 2H, which determines inflorescence row type, was coincident with the largest putative QTL for number of embryos (nEMB) and albino plants. A region of chromosome 6H was associated with QTLs for nEMB and green plants. QTLs for number and percentage of green plants were located on the long arm of chromosome 5H. Therefore, new QTLs for main components of barley anther culture response were identified on chromosomes 2H, 5H and 6H, indicating that anther culture response in barley could be controlled by relative few genes of large effect. This work is a useful step towards the identification of new regions on the barley genome that could be associated with fundamental biological process implicated in the anther culture response.

Chromosome Doubling in Monocots

The development of efficient chromosome doubling protocols is essential for the useful application of doubled haploid (DH) plants in breeding programs, since frequency of spontaneous doubling is most of the time too low. Chromosome doubling has been traditionally applied to plantlets, being the colchicine the most widely anti-microtubule agent used in vivo and in vitro. However during the last 15 years, protocols have been developed for the incorporation of different anti-microtubule agents during the early stages of androgenesis or gynogenesis. Factors affecting frequencies of spontaneous and induced chromosome doubling are summarized. For a successful chemical induction, a compromise between toxicity (which can result from high concentration and/or long time of application) and genome doubling efficiency should be adopted in order to obtain the highest number of green DH plants.

Desiccated doubled-haploid embryos obtained from microspore culture of barley cv. Igri

Abstract Barley microspore-derived doubled-haploid embryos have been produced in vitro. The development of embryo desiccation technology will allow long-term storage, germplasm preservation and low delivery cost. Treatment of the microspore-derived embryos was essential to induce desiccation tolerance and to arrest further development and plant regeneration. At the concentrations used, a treatment with trehalose was more efficient than with sucrose, and mannitol was harmful to the embryos. Up to 80% of the desiccated embryos produced complete green plants when transferred to regeneration medium, by the application of a 0.6 m trehalose or a 10–5 m abscisic acid treatment to the embryos in the culture induction medium. The morphology of these plants was similar to plants produced directly from non-desiccated embryos.

Expression profiles in barley microspore embryogenesis

In barley, microarray technology has allowed to study the gene expression profiling associated with the stress pretreatment phase of microspore embryogenesis, where the reprogramming of microspores takes place. Transcriptome analysis of anthers before and after 4 days of mannitol pretreatment, revealed changes in the expression of 2,673 genes. A thorough study of these differentially expressed genes indicated that microspores maintained their cell cycle machinery in a steady state during stress pretreatment and underwent a major reorganization of metabolic pathways accompanied by a multi-dimensional stress response. Up-regulation of transcription factors related to stress responses and changes in developmental programmes took place during the pretreatment. Preliminary studies have indicated that YABBY5, ZML2, CURLY LEAF and ICE1 transcription factors have a stress pretreatment specific induction and, therefore, could play a direct role in microspore reprogramming.

A cytoplasmic male sterility (CMS) system in durum wheat

Cytoplasmic male sterility (CMS) systems are based in the incompatible interaction between nucleus and cytoplasm and are commonly used for hybrid seed production in many crop species. The msH1 CMS system in common wheat results from the incompatibility between the nuclear genome of wheat and the cytoplasm of Hordeum chilense. Fertility restoration of the CMS phenotype is associated with the addition of the short arm of chromosome 6Hch from H. chilense. In this work, we attempt to transfer the msH1 system to durum wheat and to evaluate its potential as a new source of CMS for the production of hybrid durum wheat. For that purpose, an alloplasmic durum wheat line was developed by substituting wheat cytoplasm by that from H. chilense. This line was completely male sterile. Also, the double translocation T6HchS·6DL was transferred from common wheat into durum wheat, to test its potential as a restorer line. Finally, the system was tested by using the double T6HchS·6DL translocation in durum wheat as pollen donor for the alloplasmic male sterile line, which confirmed the fertility restoration ability of 6HchS in durum wheat.