Microspore cultures as donor tissue for the initiation of embryogenic cell suspensions in barley

We have initiated embryogenic cell suspension cultures of barley (Hordeum vulgare L.) Igri from isolated microspore cultures. Data were obtained on the time required for establishment, frequency of establishment, i.e. number of calluses out of the total number of initiations giving rise to suspensions, and embryogenic capacity of the suspension cultures. For comparison, establishment of embryogenic cell suspensions from callus derived from immature zygotic embryos of Igri, Dissa and Golden Promise was also carried out. The results revealed that embryogenic suspension cultures were established in half the time and with a seven-fold higher frequency from microspore cultures than from zygotic embryo-derived calluses. The suspension cultures were still capable of embryo formation after two years. However, only albino plantlets were regenerated. For comparison, long term callus cultures derived from microspores, anthers and zygotic embryos were established. From the anther and zygotic embryo-derived callus cultures green plants were continuously regenerated, whereas the microspore-derived callus cultures lost this ability after the second subculture.     

Quantitative resistance to barley leaf stripe (Pyrenophora graminea) is dominated by one major locus

A major gene underlying quantitative resistance of barley against Pyrenophora graminea, a seedborne pathogen causing leaf stripe, was mapped with molecular markers in a barley doubled haploid (DH) population derived from the cross Proctor x Nudinka. This quantitative trait locus (QTL) accounts for r ²= 58.5% and was mapped on barley chromosome 1, tightly linked to the naked gene. A second resistance QTL accounting for 29.3% of the variation in the trait was identified on the P arm of barley chromosome 2. Another two minor QTLs were detected by further analysis. None of the QTLs was found in the barley chromosome 2 Vada region studied by Giese et al. (1993).     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Triple hybridization with cultivated barley (Hordeum vulgare L.)

Summary A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. Gull, Golden Promise and Vada. Generally, cv Gull had high crossability in crosses with related species in the primary hybrid. It is suggested that Gull has a genetic factor for crossability not present in cv Vada and cv Golden Promise. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv Vada but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x Gull (2n=7–30) and (H. jubatum x H. lechleri) x Gull (2n=20–22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19–22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.     

Production of dodecaploid plants of Japanese persimmon (Diospyros kaki L.) by colchicine treatment of protoplasts

Summary Dodecaploid plants of Japanese persimmon (Diospyros kaki L.) were obtained by colchicine treatment of protoplasts. Callus protoplasts of Jiro (2n=90, x=15) were cultured in modified KM8p medium with 0.1% colchicine for 3–9 days. After colchicine treatment, they were cultured using agarose bead culture. Microcalli were recovered from the protoplasts after 3 months. Flow cytometric measurement showed that nine of 31 callus lines obtained from 6 days of colchicine treatment had twice the nuclear DNA content as non-treated controls. Plantlets were regenerated from the calli with twice the nuclear DNA content. Microscopic observation of root tip cells showed that their somatic chromosome number was 2n=180 (x=15). Compared with Jiro, dodecaploid plants had longer stomatal guard cells and lower stomatal densities, consistent with increased ploidy.     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Chromosomal instability in Lathyrus sativus L.

Summary In Lathyrus sativus (2n=14), variety LSD-1 shows an instability of somatic chromosome number which can be observed in root tip and shoot tip mitoses. In this variety, approximately 54% of the seedlings showed intra-individual variation in chromosome number ranging from 2n=14–3. This variability in chromosome number was recorded in approximately 60% of the dividing cells. Two seedlings were triploid with 21 chromosomes. Variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result in spindle abnormalities, chromosome degradation and minute chromosomes. The variation in chromosome number is probably responsible for the pollen polymorphism noted in this particular strain. The possible mechanism of intra-individual variability and the occurrence of the phenomenon vis-a-vis its applications are discussed.     

Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue

Summary An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a direct fluorescence procedure using FTC-toxin, but not by an indirect procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the direct and indirect procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the indirect procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a direct procedure using FTC-antiprofilaggrin but only weakly by an indirect double antibody procedure. Insensitivity to indirect procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.     

Analysis of meiosis in triticale (XTriticosecale Wittmack) X rye (Secale cereale L.) F1 hybrids at three ploidy levels

Summary Triticales (XTriticosecale Wittmack) at three ploidy levels (8x, 6x, 4x, x=7) were crossed with diploid rye (Secale cereale L.) to produce a solitary hypopentaploid hybrid (2n=32), and a number of tetraploid (2n=4x=28) and triploid (2n=3x=21) hybrids. The hybrids exhibited a morphology which was intermediate between the parents. The number of bivalents ranged from 1–7 (4.65 per cell) in hypopentaploid, from 2–12 (7.13 per cell) in tetraploid and from 4–9 (6.84 per cell) in triploid hybrids. In 4x and 3x hybrids, trivalents and quadrivalents were also observed at low frequencies (range 0–1; mean 0.01–0.03 per cell). Chiasmata frequency was highest in triploid hybrids (12.44 per cell), lowest in hypopentaploid (5.37 per cell) and intermediate in tetraploids (10.54 per cell). More than 7¹¹ were found in 39.7% pollen mother cells (PMC's) in the 4x hybrids and in 5.0% PMCs in 3x hybrids. It is concluded that an increase in the relative proportion of wheat chromosomes in the hybrids had a slight suppression effect on homologous as well as homoeologous pairing of rye chromosomes. Contrary to this, the relative increase in rye complement promoted homoeologous pairing between wheat chromosomes. In triploid hybrids, the chiasmata frequency as well as the c value were the highest, suggesting that in tetraploid hybrids rye chromosomes had a reduced pairing (low frequency of ring bivalents).     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Ecological theories and Dutch nature conservation

This paper aims to achieve insight into various ecological theories in the Netherlands which have different, and sometimes opposing, views on the conservation of nature. Interviews, publications and archival research brought to light four separate theories: vitalistic/holistic, dynamic, cybernetic and chaos. Diversity is reached through stability according to vitalistic/holistic and cybernetic theories, but through change and instablility according to the dynamic and chaos theories. These two groups are working apart, and continue to have their own ideas. Prediction of the future is only possible with the vitalistic/holistic and cybernetic theories. Ecologists who adhere to these theories feel responsible and able in different ways to change ecological nature towards desirable end goals. The other two theories, dynamic and chaos, appear to be less activist.     

Morphological and cytological characteristics of some wheat x barley hybrids

As initial step in the transfer of dwarf bunt resistance from barley into wheat, the two cereal crops were hybridized. Using the wheat cultivars Fukuhokomugi and Chinese Spring (AABBDD genomes) as female parents and barley cultivar Luther (II genome) as male, we synthesized 9 euploid hybrids (2n = 4x = 28; ABDI genomes). The hybrids were vigorous, but highly sterile. Meiotic analyses of seven hybrids showed considerable variation in chromosome pairing. Of the hybrids involving Fukuhokomugi 3 had high pairing with a mean of 5.08–6.72 chiasmata per cell, while others had 2.16–3.52 chiasmata per cell. As many as 12 bivalents in some pollen mother cells would suggest at least some pairing between wheat and barley chromosomes. This level of homoeologous pairing, coupled with some, albeit low, female fertility of the F₁ hybrids, could offer an opportunity for intergeneric gene transfers from barley into wheat and vice versa.     

Robertsonian change in allies ofZebrina (Commelinaceae)

Meiotic pairing inTradescantia soconuscana 2n=26 (6M+16A+4T) suggests that it has a tetraploid constitution which is not apparent in its chromosome number. Its nombre fondamental of 32 indicates that it could have evolved from an ancestor with x=8 by a combination of Robertsonian fusion, hybridization and polyploidy. The 2n=16 (8A+8T) karyotype of a closely related diploid supports this. The allied genusZebrina may have followed a similar method of chromosome evolution.     

Complex interspecific hybridization in barley (Hordeum vulgare L.) and the possible occurrence of apomixis

Summary Several complex hybrids were produced from the combination [(Hordeum lechleri, 6x xH. procerum, 6 x) × H. vulgare, 2 x]. Crosses with six diploid barley lines resulted in triple hybrids, most of which had a full complement of barley chromosomes (no. 1–7), but were mixoploid with respect to alien chromosomes (19–22). In one combination, chromosome no. 7 was duplicated. Meiosis in triple hybrids showed low, but variable pairing (1.3–5.5 chiasmata per cell). The syndesis probably did not include the barley chromosomes. Direct back-crosses to di- and tetraploid barley lines were unsuccessful. Chromosome doubling of the triple hybrid based on cv Pallas resulted in a plant with 2n = 53–56, which had an increased fertility. Backcrosses to one di- and one tetraploid barley line resulted in offspring. The cross made with the tetraploid line (Haisa II), produced a 28-chromosomic plant in which the male parental genome was absent. We suspect that this plant may have arisen through parthenogenetic development of a reduced female gamete. The other cross with a diploid line (9208/9) resulted in plant with 2n = 51–53. The most likely explanation for this second plant is that an unreduced gamete from the amphiploid was fertilized by a normal gamete from the backcross parent, and during early embryo development, some chromosomes were eliminated.     

Ploidy dependence of chromosomal variation in callus cultures ofHyoscyamus muticus L.

Summary Remarkable variation for chromosome number was observed in both diploid (2x) and autotetraploid (4x) callus cultures continuously examined for 12 months at monthly subsculture intervals. Initially, the subcultures exhibited predominantly the genomic level of the starting material which subsequently and gradually developed into heterogenous populations of euploid and aneuploid cells imparting the subcultures a aneusomatic status. The comparison of chromosomal instability recorded in 2x vs. 4x callus cultures revealed that with time both types of calli stabilized at a chromosome number around the 4x level. However, the chromosomal examination of the adventitious roots emerging from the disorganized calli revealed the euploid (both 2x and 4x) chromosome levels suggesting the occurrence of amorphogenetic sieve.This paper is dedicated to Professor A. K.Sharma of Calcutta University on his 65th birthday by his former student U. C. L. and grandstudent S. S. (CIMAP Publication no. 739).     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.