Zinc sulphate improved microspore embryogenesis in barley

The effect of ZnSO₄ concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control) to 600 μM in the stress pre-treatment medium alone or in combination with 30 (control) to 600 μM in the embryo induction medium were assayed in anther culture. Incorporation of Zn²⁺ in the pre-treatment medium itself did not affect microspore embryogenesis. The optimum concentration in the stress pre-treatment and induction media was 180 μM for cultivars (cvs.) Igri and Reinette, and 90 μM for cv. Hop. A significant increase of 30 and 300% in cv. Igri and Reinette, respectively, were produced with 180 μM ZnSO₄ in both the number of embryos and green plants. In order to confirm the effect of Zn²⁺ on microspore embryogenesis this micronutrient was incorporated in the induction medium of isolated microspore cultures of cv. Igri. Concentrations of 90–300 μM ZnSO₄ resulted in an increase of 40–53% in the number of embryos and green plants. All these results indicate that the beneficial effect of Zn²⁺ is exerted mainly during the culture phase, increasing the number of embryos, leading to an increased number of green plants, but it had no effect on percentage of regeneration or green plants.     

Androgenic response to preculture stress in microspore cultures of barley

Summary. Various stresses such as starvation and cold or heat shocks have been identified as triggers in the induction of the microspore embryogenesis. This study attempts to quantify the effects of different pretreatment conditions for successful microspore culture of malting barley (cv. Scarlett). While the sporophytic microspore development could be induced from treated and nontreated microspores, abiotic stress was essential for embryo formation and plant regeneration. The type of stress treatment applied affected the numbers and the ratios of albino and green plants regenerated, as well as their fertility. The highest number of green plants was obtained after the treatment of anthers in 0.3 M mannitol at 32 °C for 24 h before microspore culture. Correspondence and reprints: Department of Plant Biotechnology and Cytogenetics, Institute of Plant Breeding and Acclimatization, Radzików, 05-870 Blonie, Poland.     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

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 reorientated 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.     

Effect of growth regulators on microspore embryogenesis in coconut anthers

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y₃ medium containing 66 μM 2,4-D), followed by maturation medium (Y₃ medium without growth regulators) and germination medium (Y₃ medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA₃), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).     

Effects of Mannitol Pretreatment on Androgenesis of Barley (Hordeum vulgare L.)

The effects of mannitol pretreatment on androgenesis of barley were systematically studied in comparison with that of cold pretreatment and control. The results showed that mannitol pretreatment could significantly increase the frequency of pollen survival reaching 19.0% on the eighth day, while in cold pretreatment and control they were 8.4% and 6.6 %, respectively. Mannitol pretreatment could also improve the quality of pollen and inhibit starch production from microspore, which were quite advantageous to microspore division and development. The developing period was shortened 2--3 days as compared with cold pretreatment and control. The major developmental pathways of androgenesis after mannitol pretreatment were the equal division (B pathway). In addition, the majority of microspore nuclei were diploids. On the contrary, the major microspores pretreated with low temperature had fewer chromosomes than with mannitol pretreatment, the microspore nuclei were haploids.     

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.     

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.     

Genetical analysis of microspore derived plants of barley (Hordeum vulgare)

Summary From an F₁ hybrid between the two barley (Hordeum vulgare L.) cultivars Golden Promise and Mazurka a series of doubled haploid (DH) lines were generated both from microspores by anther culture and from immature zygotic embryos after hybridization withH. bulbosum. The DH lines from both sources were used to monitor the segregation of the five major genes, rachilla hair length, DDT susceptibility, height, C hordein polymorphism and mildew resistance. Whereas the microspore-derived samples showed significant departures from the expected 11 ratio for three of the five genes, theH. bulbosum lines showed deviation for only one gene. Analysis of linkage data also showed differences between the two series of DH lines. Cytogenetic analysis revealed a mean chiasma frequency in theH. bulbosum lines which was very similar to the F₁ hybrid. In contrast, four of the ten microspore derived lines examined showed a reduced chiasma frequency. One showed evidence of translocation heterozygosity.     

Wheat microspore embryogenesis during in vitro anther culture

Summary Cytological analysis of microspore embryogenesis during in vitro culture reveals a high mortality in the first week and a latency phase of about one week before the first embryogénic mitosis. Genotypic differences observed during our wheat anther culture do not seem to originate at the induction level but are linked to the different abortion rates.     

Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

Structural changes during early embryogenesis in wheat pollen

Summary We have made a detailed cytological examination of the development of wheat embryoids, monitoring their initial divisions from two to ten cells by both light and electron microscopy. According to our observations the first embryogenic division is symmetrical. After the androgenesis induction treatment, there is a decrease in ribosome population with cells that have inactive nucleoli made up almost exclusively of a dense fibrillar component. This population is restored after initial embryogenic divisions. During the initial divisions the embryogenic pollen grains do not appear to change in size and the pollen wall remains intact. The exine undergoes no modification but the intine thickens, and we have observed that the thickness of the intine can be used as a cytological marker of androgenesis. The walls separating the cells obtained after embryogenic division contained numerous plasmodesmata. The beginnings of embryo polarization and cell differentiation could be made out in the very early pollen embryoids.     

Acetyl-CoA carboxylase during development of plastids in wild-type and mutant barley seedlings.

A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150,000 X g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mutants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Dark-grown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carboxylase activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.     

Several QTLs involved in osmotic-adjustment trait variation in barley (Hordeum vulgare L.)

 Osmotic adjustment (OA) was previously demonstrated to be an important adaptive mechanism of drought tolerance in cereals. In order to determine which genomic regions are involved in OA variation, 187 barley (Hordeum vulgare L.) recombinant inbred lines (RILs) derived from a cross between Tadmor (drought tolerant) and Er/Apm (susceptible) were studied in a growth chamber for their OA capacity (through correlated traits and by calculation), at an early growth stage and under two water treatments (soil moisture of 14% and 100% of field capacity). The continuous distribution of the traits and their broad-sense line heritabilities, ranging from 0.04 to 0.44, indicated that OA and related traits should have a polygenic nature. A subset of 167 RILs were also genotyped using 78 RFLP, 32 RAPD and three morphological markers and a linkage map was constructed. Despite strong environmental effects acting on the traits, interval mapping and single-marker ANOVA allowed the detection of three QTLs for relative water content (RWC), four QTLs for osmotic potential (ψ_π), two QTLs of osmotic potential at full turgor (ψ_π100) and one QTL for osmotic adjustment at a soil moisture of 14% field capacity. For the irrigated treatment, only two QTLs were detected: one for RWC and one for ψ_π100. Two chromosomal regions were involved in several OA-related trait variations and could be considered as regions controlling OA; these were present on chromosome 1 (7H) and chromosome 6 (6H), whereas other regions were specific for one trait. No major QTL was found. However, the genomic region involved in OA-related traits on chromosome 1 (7H) in barley seemed to be conserved for OA variation among cereals. Epistatic effects, with or without additive effects, acted on the traits. Received: 15 July 1997 / Accepted: 29 October 1997     

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

 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. Received: 28 September 1998 / Revision received: 27 November 1998 / Accepted: 5 January 1999     

Haploid formation in maize, barley, flax, and potato

Summary. The article is reviewing some significant features and issues in the process of haploid formation in two important monocotyledonous crop plants – maize and barley – and in two dicotyledonous plants – flax and potato. Exotic maize lines with higher androgenic response turned up as a good source for this heritable trait and this valuable trait can be incorporated into elite maize lines via crossing. Lots of attempts were devoted to identifying some cytological and/or morphological markers for androgenic response in maize microspore cultures. The “starlike” organization of the cytoplasm inside the induced maize microspores together with the enlarged size of induced microspores can be considered as morphological markers for androgenic response. In barley, microspores with rich cytoplasm that was of granular appearance with the nucleus located near the cell wall and with no visible vacuole had the largest survival rate and many of these cells continued in development and produced embryos. In flax, a dramatic increase of induction rate in anther cultures (up to 25%) was achieved when flax anthers were pretreated for 3 days at 4 °C and afterwards kept for 1 day at 35 °C. Also gynogenesis in flax has been reported already and complete plants were obtained. In potato microspore cultures, formation of two dissimilar cells indicated a strong polarization in the system and as a result of this polarization a prominent suspensor developed that persisted until the torpedo stage of the androgenic embryo. This was the first time the formation of a well developed suspensor was described in connection with androgenesis. Correspondence and reprints: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07 Nitra, Slovak Republic.     

Unequal distribution of plastids during generative cell formation in Impatiens

Summary This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts.     

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.     

Barley anther culture: effects of annual cycle and spike position on microspore embryogenesis and albinism

The effect of donor plants annual cycle and anther/spike position on the production of microspore-derived plants and albinism were studied. We used the winter cv. Igri and the spring cv. Cork, known to respond similarly in anther culture but to produce 78% and 2% of green plants, respectively. In both cvs. the number of microspore-derived plants was significantly higher when the anthers were collected from January to July than from August to December. However, during this period the proportion of albino plants was not altered. Conversely, the anther response decreased from 76.6 to 31.5% in Igri and from 58.8 to 32.0% in Cork when the donor spike originates from the main shoot or the fourth tiller. Significantly, anthers collected from spike of the second tiller enabled us to drastically increase the proportion of regenerated green plantlets, by 16% in Igri and 1800% in Cork.