Albinism in barley androgenesis

Androgenesis is highly useful for plant breeding, significantly reducing breeding cycle times, as well as in a wide range of biological research. However, for widespread use this process must be efficient. Despite several decades of research on the phenomenon of androgenesis, many processes involved are obscure and there is much to be understood about androgenesis. One of the problems inherent in androgenesis, and reducing its efficiency, is albinism. This article reviews albinism in barley anthers and microspores in vitro cultures. Of special interest is the fate of plastids throughout androgenesis, which is important at several levels, including the genes responsible for driving the green-to-albino ratios. We also summarize the external factors that reduce the incidence of albino plants that are regenerated via androgenesis.     

Effects of free and chelated iron on in vitro androgenesis in barley and wheat

A suitable form of iron supplement in the induction medium was found to be important for further development of induced pollen embryos in barley and wheat cultivars (genotypes), especially those providing few green plants viain vitro androgenesis. Genotypes able to regenerate many green plants were less susceptible to the lack of iron in induction medium. Although Fe-EDTA was found to be a suitable form of iron in the induction medium, androgenesis was also induced on media containing non-chelated iron (Fe²⁺ and Fe³⁺ ions). EDTA alone without iron inhibited the androgenic response even in the wheat cv. Florida, a model cultivar for androgenesis in wheat. In all barley cultivars under study including cv. Igri, a model cultivar for androgenesis in barley, EDTA alone caused an almost total suppression of androgenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of combinations of alien translocations on in vitro androgenesis in near-isogenic lines of spring bread wheat

The features of in vitro androgenesis were studied in cultured anthers of spring bread wheats L503 and Dobrynya, having 7DS-7DL-7Ae#1L translocation with genes Lr19/Sr25 (Lr19 translocation) from Agropyron elongatum (Host.) P.B. and their near-isogenic lines carrying combinations of Lr19 translocation with translocations: 1BL-1R#1S with genes Pm8/Sr31/Lr26/Yr9 (Lr26 translocation) from Secale cereale L., 4BS-4BL-2R#1L with genes Lr25/Pm7 (Lr25 translocation) from Secale cereale, 3DS-3DL-3Ae#lL with genes Lr24/Sr24 (Lr24 translocation) from Agropyron elongatum and 6BS-6BL-6U#1L with gene Lr9 (Lr9 translocation) from Aegilops umbellulata Zhuk. In comparison with those varieties having received the Lr19 translocation, the following was established: (1) the combination of translocations Lr19 + 26 increased embryo frequency and green plant regeneration; (2) the combination of translocations Lr19 + 9 decreased embryo frequency but increased green plant regeneration; (3) the combination of translocations Lr19 + 24 decreased embryo frequency but increased green and albino plant regeneration; (4) the combination of translocations Lr19 + 25 increased embryo frequency and green plant regeneration but decreased albino plant regeneration. Thus, on near-isogenic lines of spring bread wheat, the influences of genotypes of four alien translocation combinations on in vitro androgenesis were determined.     

Identification of candidate genes for in vitro androgenesis induction in maize

Extensive studies have been conducted to understand the genetic control of in vitro androgenesis, but little is know about the genes and the mechanisms involved in the switch that allows an immature pollen grain to develop as an embryo. We have developed two maize isogenic lines with high androgenetic aptitude, named AH5-44 and AH5-49, through backcross and selection from a high-responsive DH229 line on the non-responding A188 line genetic background. The genomic structure of these two lines was precisely described with microsatellite markers. Five regions retained from the parent DH229 highly responsive to androgenesis were localised in both AH5-44 and AH5-49. Sequences expressed on microspores extracted from the four lines were amplified using a cDNA-AFLP protocol. For each line, eight culture conditions were compared: microspores extracted after tassel recovery, after 7 or 14 days in cold room and after 1–4 days of in vitro culture. This genetic and developmental screening allowed us to identify four sequences, including a new HSP70-like candidate gene. Possible implication of the identified sequences in androgenesis response is discussed.Correspondence and request for material (near isogenic line seeds and pre-amplified cDNA-AFLP samples are available for other groups to identify new sequences potentially involved in androgenesis using a larger set of primer combinations on cDNA-AFLP samples) should be addressed to P.B. (e-mail: barret@clermont.inra.fr) Sequence data from this article have been deposited in the EMBL/GenBank data libraries under accession nos. AY222837 (HSP70-like), CD766057 (E9 M1-130), CD766058 (E13 M1-550), and CD766059 (E9 M5-320)     

In vitro induced androgenesis inMelandrium album

Haploid and dihaploid female and rare dihaploid male plants were produced inMelandrium album throughin vitro induced androgenesis. In the seed progeny obtained from cross-hybridization between dihaploid androgenic males (supermales) and standard females only male plants were observed. The microspores containing Y chromosome gave rise to supermales.     

Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos

Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure into an embryo-like structure during barley androgenesis.     

Effect of amino acids,growth regulators and genotype on androgenesis in barley

The effects of an amino acid mixture and of plant growth regulators added to the FHG barley anther culture medium were examined using three barley cultivars (Cadette, Léger, and Igri) grown in two environments (growth cabinet and glasshouse). ‘Léger’ and ‘Igri’ were known as responsive, and ‘Cadette’ as recalcitrant to androgenesis. Our first experiment showed that the amino acid-supplemented medium was best for embryogenesis and regeneration of ‘Cadette’ and ‘Igri’ in both environments, and if ‘Léger’ in the growth cabinet. The addition of ABA and TDZ did not improve embryogenesis and plant regeneration, and PAA decreased them in the growth cabinet. The addition of the amino acid mixture in the FHG medium also reduced the percentage of albino plants in the growth cabinet, but growth regulators did not improve the percentage of albino plants, and in some cases increased it. In the growth cabinet, disregarding media, ‘Léger’ produced more embryos than ‘Cadette’ and ‘Igri’, and Léger' and ‘Igri’ produced more green plants than ‘Cadette‘. Percentages of albino plants were higher or ‘Cadette’ than for ‘Igri’ or ‘Léger’. In a second experiment, we compared seven hybrids with their parents for androgenic responsiveness. Hybrids had a higher ability to generate green plants than expected based upon the weighted average reflecting the contribution of each parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

The nitrogen fertilization of spring barley

Summary From 1971 to 1979 field trials with increasing rates of fertilizer nitrogen on spring barley with sugar beet as the preceding crop were conducted on a farm on sandy loam in the south western part of The Netherlands. Prior to sowing and fertilizing soil samples were taken and analysed for mineral nitrogen (N_min). The average yield increase through application of fertilizer nitrogen was only 750 kg of grain per ha per year, the maximum yield being about 5 tonnes per ha. In the case of a fixed rate of fertilizer nitrogen per annum it can be derived from the response curves that 60 kg of N would have given the smallest average yield deficit (170 kg grain per ha) in comparison with maximum yields. With an N-advisory system based on soil analysis the average yield deficit would be at a minimum (163 kg of grain per ha) with a value for mineral soil nitrogen+fertilizer nitrogen totalling 120 kg N per ha.No relationship was found between optimum rate of fertilizer nitrogen and the amount of mineral soil nitrogen at the end of the winter. This was ascribed to the relatively small variation in mineral soil nitrogen and the weak response of the crop to fertilizer nitrogen.Promising results from nitrogen fertilizing systems based on soil analysis can be expected from more responsive crops like winter wheat, sugar beets and potatoes.With the average yield deficit compared with maximum yield as a characteristic, the usefulness of any N-advisory system can be compared, taking a fixed rate of nitrogen system as a standard.Seconded by the Agricultural Bureau of the Netherlands Fertilizer Industry (LBNM).     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fusariosis of spring barley cultivated in Lublin region

Diseases of spring barley in 1986–1988 seasons have been examined on barley plantations in Lublin region. Observations in eight weeks after sowing each year spring showed the occurrence of root rot and sheath rot in seedlings. As a result of mycological examination of infected seedlings 34 species of fungi were isolated:Fusarium spp. amounted up to 23% of all isolates. Each year,Fusarium culmorum andF.avenaceum were isolated, butF graminearum only in 1987. On all inspected fields there occurred plants with eye — spots or necrotic stripes on lower internodes. As a result of fungi isolation the colonies belonging to 30 species were identified from stems and roots of examined plants. There was about 35% of fusaria between isolates each year.Fusarium culmorum was most frequently isolated. This fungus both from stems with two mentioned kinds of symptoms and from roots was isolated.Fusarium avenaceum each year andFusarium graminearum in 1986 and 1988 were isolated. Mentioned there species were also isolated from kernels.     

Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

 Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B₁- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B₁- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T₀ leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T₁ progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T₁ progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T₅ progeny in one line, T₄ progeny in one line, T₃ progeny in three lines and T₂ or T₁ progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B₁- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T₁ progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T₂ or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T₂ or later generations. We conclude that the B₁- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering. Received: 28 May 1998 / Accepted: 19 December 1998     

Regulation ofin vitro androgenesis in tobacco through iron-free media

Globular embryoids were continually produced in anther cultures of tobacco (Nicotiana tabacum L. cv. Samsun) from the pool of resting microspores if the iron-free medium was used. The supplement of iron stopped the development of fresh early embryoids still inducing continual conversion of the resting globular embryoids into torpedo-shaped embryoids, and into haploid plants. Globular embryoids in the anthers responded to the iron supply even after eight months’ cultivation on iron-free media. Isolated embryoids showed the same response. Haploid plants were regenerated from the anthers on the minimal medium consisting of agar, sucrose, iron and distilled water. Iron requirements of preglobular, globular and postglobular embryoids are discussed.     

Cross prediction studies on spring barley

Summary Additive genetic, dominance genetic and environmental correlations between pairs of agronomically important characters in five spring barley crosses were calculated from estimates of the components of variance and covariance, obtained by Triple Test Cross analysis. Phenotypic correlations were calculated from the Triple Test Cross family means and compared to the additive genetic correlations. Phenotypic correlations were generally lower than the additive genetic correlations and, occasionally, of different sign. The highest phenotypic correlations between single plant yield and its components were found with number of tillers whereas these were the lowest additive genetic correlations, thousand grain weight giving the highest. High dominance genetic correlations were found between single plant yield and both grain number and thousand grain weight thus indirect early generation selection for single plant yield using these two characters would be ineffective. Additive and dominance genetic correlations confirm association of the erectoides dwarfing gene with low thousand grain weight and plant yield.     

Molecular breeding for grain yield in barley: an evaluation of QTL effects in a spring barley cross

 We report results from a breeding strategy designed to accumulate favorable QTL alleles for grain yield identified in the SteptoeבMorex’ (SM) barley germplasm. Two map lines (SM73 and SM145) from the original mapping population were selected based on their marker genotype and QTL structure. When crossed, these lines would be expected to produce progeny with most favorable QTL alleles. One hundred doubled haploid (DH) lines from the F₁ hybrid of this cross were genotyped with ten RFLP markers and one morphological marker defining grain yield to monitor QTL segregation. A subset of 24 lines representing various combinations of putatively favorable and unfavorable QTL alleles, together with Steptoe, ‘Morex’, SM73, and SM145, were phenotyped for grain yield in five environments. Multiple regression procedures were used to explore phenotype and genotype relationships. Most target QTLs showed significant effects. However, significance and magnitude of QTL effects and favorable QTL allele phase varied across environments. All target QTLs showed significant QTL-by-environment interaction (QTL×E), and the QTL on chromosome 2 expressed alternative favorable QTL alleles in different environments. Digenic epistatic effects were also detected between some QTL loci. For traits such as grain yield, marker-assisted selection efforts may be better targeted at determining optimum combinations of QTL alleles rather than pyramiding alleles detected in a reference mapping population. Received: 2 June 1998 / Accepted: 17 September 1998