Expression of protein phosphatases during postnatal development of rabbit heart

Protein phosphatases play a major role in the regulation of L-type calcium current (I_Ca) in heart cells. We previously showed developmental differences in the effects of inhibitors of protein phosphatases (PP's) on the modulation of I_Ca, with greater stimulatory effects on I_Ca observed in newborn than in adult ventricular cells. We hypothesized that this developmental difference might be due to greater expression and levels of PP 1 and PP 2A in newborn than in adult ventricular cells. We thus determined the mRNA expression of and subunits of PP 1 and the subunit of PP 2A in adult and newborn rabbit ventricles and levels of PP 1 and PP 2A in total homogenates, particulate membranes, and in soluble fraction prepared from isolated ventricular myocytes from adult and newborn rabbits. RT-PCR analysis demonstrated the presence of mRNA of these subunits of PP's in both newborn and adult ventricles. Northern blot analysis using ³²P labeled cDNA probes specific for PP 1, PP 1 and PP 2A showed that the expression of steady state mRNA levels for PP 1, PP 1 and PP 2A were much higher in newborn compared to adult rabbit ventricles. mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) in rabbit ventricles were measured as controls. GAPDH did not show significant developmental changes while mRNA for SERCA was higher in adult compared to newborns. Western blot analysis showed that PP 1 and PP 2A protein levels were also much higher in newborn compared to adult rabbit ventricular cells. Immunoblot analysis in particulate membranes and soluble fraction showed that PP1 was mainly membrane bound while PP 2 was present only in soluble fraction. These findings suggest that the two major protein phosphatases (PP 1 and PP 2A) in heart are expressed at much higher levels in newborn and decline to lower levels in adult ventricular myocytes. The presence of high levels of PP's and particularly PP 1 in newborn cells may be responsible for the greater dependence of newborn cells on the inhibition of PP as a mechanism of action of -agonist isoproterenol on I_Ca.     

Cytochemistry and biochemistry of acid phosphatases

Summary Acid phosphatases of the rat ventral prostate were studied cytochemically using different substrates. The results were compared to findings on isoelectric focussing gels stained for acid phosphatase activity. This is a highly specific and reproducible method which allows the distinction between secretory androgen-dependent and lysosomal acid phosphatases. Activity of lysosomal acid phosphatase was increased after castration, while the activity of the secretory enzyme gradually decreased after androgen deprivation. None of the substrates tested was selectively hydrolyzed by either secretory or lysosomal acid phosphatase. Phenylphosphate, creatine phosphate and choline phosphate were found to be inappropriate substrates for histochemical purposes, however, reproducible results were obtained with -naphthylphosphate, -glycerophosphate and p-nitrophenylphosphate. The method of isoelectric focussing (pH range 4.0–8.0) of enzymes with subsequent histochemical staining demonstrated lysosomal enzymes at pH 7.9 and 8.2 respectively. Small amounts of identical enzymes were found in liver, kidney, blood or epididymis. Secretory acid phosphatases were focussed at pH 5.5, 5.6, 5.65 and 7.15. Similar enzymes have been identified in epididymis, kidney, liver and pancreas. These results indicate that 1) at present no specific substrate for prostatic secretory or lysosomal acid phosphatases is available and 2) that no prostate-specific prostatic acid phosphatase (PAP) exists in the rat ventral prostate.Supported by the Deutsche Forschungsgemeinschaft (Au 48/6)     

Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5-untranslated region and the coding region, but the 3-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)⁺ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5- and 3-untranslated regions that might be important for PHYA mRNA degradation.     

On the heterogeneity of the slow reassociating (“unique”) DNA

Summary The slow reassociating fraction of mouse DNA (unique DNA), when allowed to reassociate in 0.14 m sodium phosphate buffer at 50 °C showed a biphasic melting curve with a transition at 78–80 °C. On the basis of this feature, the slow reassociating DNA was separated preparatively into two fractions: unique DNA I and II. Their duplexes showed differences with respect to thermal stability, S1 nuclease resistance and rate of reassociation. About one third of the sequences in each fraction were fraction-specific. The conclusion was drawn that for unique DNA I these should be the low repetitive or single copy related sequences (multigene families) and for unique DNA II—the unrelated single copy sequences or recent families of low repetitive not yet diverged sequences.     

Oligo-riboprobes

Summary In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. More-over, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes (oligo-riboprobes). These probes can be labelled to very high (10⁹ cpm/g) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from preprotachykinin cDNA.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

We have investigated the methylation status of the -tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the -tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub2 and tub4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two -tubulin genes, tub3 and tub4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.     

Sensitive 1H-31P correlations with 5′ methylene protons of DNA via homonuclear double-quantum coherence

A novel pulse sequence is presented for the correlation of 5 and 5 protons in DNA with phosphorus. Double-quantum coherence between the methylene protons is used to generate ¹H5-³¹P and ¹H5-³¹P cross peaks in an HMQC-type experiment. The resolution for these cross peaks is significantly improved over that of conventional HSQC experiments, as cross peaks between ¹H4 and ³¹P are largely suppressed and a 3D version of the experiment can be performed with little penalty in sensitivity. In addition, sensitivity is favoured by slower relaxation of the double-quantum coherence and a more favourable multiplet fine structure in the acquisition dimension.     

The general occurrence of “plastid initials” and their development into plastids in cortical cells of woody plants in winter

Summary Electron microscopic studies revealed that plastid initials, presumed precursors of plastids, occur in cortical cells of the following plants studied in February and March: Betula ermanii Cham.; Prunus sargentii Rhed.; Pyrus communis L.; Ribes sinanense F. Maekawa; Salix matsudana Koidz. forma tortuosa Rhed.; and Sambucus sieboldiana var. miquelii Hara. Since plastid initials were found previously in Malus pumila Mill., Morus bombycis Koidz. and Populus euramericana cv. gelrica (Sagisaka 1991), plastid initials have been found in all woody plants examined to date. In P. euramericana cv. gelrica, at later stages of the development of the initials in March, the conglomerates of plastid initials became heterogeneous in terms of size, extent of thylakoid formation and ability to form starch granules. The formation of prolamellar structures was frequently observed in cells of Magnolia kobus var. borealis Sarg., which was sampled on April 19. These observations suggest the course of events in the development of the plastid initial and the continuity of the life of amyloplasts over a year in the life of woody plants.     

Regeneration and Agrobacterium-mediated transformation of Forsythia×intermedia "Spring Glory"

Internode explants ofin vitro plants ofForsythia x intermedia Spring Glory were transformed with thegus andnpt II genes after inoculation with theA. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An average of 1% of explants regenerated transgenic shoots.-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed -glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - GUS andgus -glucuronidase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog - NOS nopaline synthase - NPT II andnpt II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - SSC sodium chloride-sodium citrate - X-Gluc 5-bromo-4-cbloro-3-indolyl glucuronide     

Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities

Summary Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these — white, white tipped green, patchy white, white virescent, white striping 1, white striping 2, white striping 4, fine striping, chlorina and yellow virescent showed monogenic recessive inheritance and the remaining three — yellow striping, yellow green and light green seedling phenotypes showed digenic recessive inheritance. The genes for (i) white tipped green (wr) and yellow virescent (yv) and (ii) patchy white (pw) and white striping 1 (wst 1) showed independent assortment. Further, the genes for white (w), white tipped green (wr) and yellow virescent (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants — white tipped green, patchy white, white striping 1, white striping 2, fine striping, chlorina, yellow virescent, yellow striping, yellow green and light green phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in white virescent there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F₁'s (mutant x control green). In F₁'s of six of the mutants — white tip, patchy white, chlorina, yellow virescent, fine striping and yellow striping the quantity of chlorophyll was almost equal to the wild type. In the F₁'s of three of the mutants — white striping 1, white striping 2 and light green an intermediate value between the mutant and wild types was observed. In yellow virescent retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants — white tipped green, white virescent, fine striping, chlorina, yellow striping, yellow green and light green defective plastids were also observed. In patchy white secondary destruction of chlorophylls and the presence of defective plastids were found to be associated with reduced chlorophyll quantity at maturity.Paper chromatographic studies of leaf flavonoids revealed some variation between the inbreds, but there were three common spots, 7, 8 and 9, except for PDP in which the spot 8 was absent. Chlorophyll deficient mutants differed from their respective controls in the absence of one or more of the spots present in the controls and in the presence of new spots in some of the mutants.Most of the chlorophyll mutants showed higher survival rate in the Kharif season than in Rabi season which was attributed to the higher mean day temperature and longer day light period in the Kharif season than in Rabi season.     

Reduced [3H]flunitrazepam binding in cingulate cortex and hippocampus of postmortem schizophrenic brains: Is selective loss of glutamatergic neurons associated with major psychoses?

Findings. Specific [³H]flunitrazepam binding to neuronal-type sites was significantly lower in anterior cingulate cortex, hippocampus, somatomotor cortex, cerebellar cortex, and globus pallidus in small postmortem samples of schizophrenic brains than in non-schizophrenic controls. Four of these five brain regions were reported by others to exhibit atrophy and/or neuronal loss in schizophrenia.Interpretation: Selective loss of hippocampal pyramidal neurons in postmortem schizophrenic brains has been reported (11). Pyramidal neurons are known to be glutamatergic (14, 26) and to exhibit high densities of benzodiazepine binding sites (25,31). Glutamatergic neurons are known to be abundant in most layers of the cerebral cortex, and most of these are pyramidal neurons (26). All layers of the cerebral cortex display high densities of benzodiazepine binding sites (24,25,31). The number of large pyramidal cells is little affected in most layers of the anterior cingulate cortex, but the number of small neurons is significantly lower, particularly in layer II (10). Pyramidal neurons range in size from very large to very small, and many very small pyramidal cells are often counted, together with small stellate neurons, as granule cells (28). Further, non-pyramidal glutamatergic neurons are reportedly also found in cerebral cortex (26). Thus, it seems possible that the large reduction in [³H]flunitrazepam binding we find in anterior cingulate cortex reflects the selective loss of glutamatergic neurons. The hypothesis that selective loss of glutamatergic neurons form various brain regions is associated with major psychoses can be easily tested by immunohistochemical studies of these regions using glutamate- and GABA-specific antibodies.     

Variability of bacterial gene-directed enzyme production in human genetically deficient cells

Summary Human -galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GM₁-gangliosidosis type I) were treated with phage plac DNA, coding for Escherichia coli -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23). New -galactosidase activity detected in cell extracts of phage DNA-treated GM₁-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant -galactosidase activity in plac DNA-treated cells resembled that of mutant E. coli -galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions.More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q-replicase, f 1-coat protein, or UDPG-4-epimerase.     

The inheritance of host plant resistance and its effect on the relative infection efficiency of Magnaporthe grisea in rice cultivars

The inheritance of host plant resistance and its effect on the relative infection efficiency for leaf blast was studied in the crosses IR36/CO39 (partially resistant × highly susceptible) and IR36/IR64 (both partially resistant). On the natural scale, gene action appeared multiplicative. After log transformation, additive effects described most of the genetic variation in the cross IR36/CO39, while additive and dominance effects were about equal in magnitude in the cross IR36/IR64. Dominance was towards increased resistance. No transgressive segregation occurred in the cross IR36/CO39. The number of genes that reduce lesion number was estimated to be zero in CO39 and five or more in IR36. The cross IR36/IR64 showed transgressive segregation in both directions, and IR36 and IR64 each contain at least one gene that is not present in the other cultivar. The heritabilities (narrow sense) in the F₂ were low (range 0.06–0.16), while narrow sense heritabilities based on F₃ lines were much higher (range 0.41–0.68). Lesion numbers in F₃ lines were reasonably correlated with those in F₅ progenies derived from the same F₂ plant (r was±0.6 in both crosses). Partial resistance can be effectively improved by selecting the most resistant plants from the most resistant F₃ lines.     

The inhibitory effect of trilinolein on norepinephrine-induced β-myosin heavy chain promoter activity,reactive oxygen species generation,and extracellular signal-regulated kinase phosphorylation in neonatal rat cardiomyocytes

The myocardial protective effects of trilinolein, isolated from the traditional Chinese herbSanchi (Panax notoginseng), are thought to be related to its antioxidant activity. However, the intracellular mechanism underlying the protective effect of trilinolein in the heart remains unclear. In the present study, we investigated the effect of trilinolein on norepinephrine (NE)-induced protein synthesis in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with NE, then protein content, [³H]-leucine incorporation, and -myosin heavy chain (-MyHC) promoter activity were examined. The effect of trilinolein on NE-induced intracellular reactive oxygen species (ROS) generation was measured with a redoxsensitive fluorescent dye (2,7-dichlorofluorescin diacetate) and extracellular signal-regulated kinase (ERK) phosphorylation by Western blotting. Trilinolein inhibited NE-increased protein synthesis, -MyHC promoter activity, and intracellular ROS. Both trilinolein and the antioxidant, N-acetyl-cysteine, decreased NE- and H₂O₂-induced protein synthesis, -MyHC promoter activity, and ERK phosphorylation. These data indicate that trilinolein inhibits NE-induced protein synthesis via attenuation of ROS generation in cardiomyocytes.     

Analysis of the effect of rye chromosomes 4R, 6R and telomeric heterochromatin on 7R on homologous chromosome pairing in pentaploid hybrids (AABBR,AABBD)

Summary Meiotic pairing in Triticum turgidum cv. Ma (4x) with a mean chiasmata frequency of 27.16 per cell was compared with chiasmata frequencies in its hybrids with several triticale strains, Chinese Spring wheat and its addition lines for Imperial rye chromosomes 4R and 6R. In hybrids between Ma and x Triticosecale cv. Rosner the chiasmata frequency was marginally reduced by an average of 1.25%, by 8.8% in hybrids with x Triticosecale cv. DRIRA HH and by 6.7% with DRIRA EE (lacking 90% telomeric heterochromatin from chromosome arm 7RL). In pentaploid hybrids between Ma and T. aestivum cv. Chinese Spring the reduction was an average of 10.30%, while addition lines with rye chromosome 6R reduced chiasmata frequencies by an average of 7.4% and rye addition line for 4R showed the greatest depression in chiasmata frequency in hybrids by a 25.04% reduction. An interchange difference involving long chromosome segments was observed between Ma and Rosner.Contribution No. 819 Ottawa Research Station     

Study on the vesicular-arbuscular mycorrhiza of three cultivars of potato (Solanum tuberosum L.)

Summary The establishment and development of vesicular-arbuscular mycorrhizal (VAM) fungi were studied in three cultivars of potato, which differed in susceptibility to Late blight, in a field experiment on a lateritic sandy-loam during two growing seasons (1980 and 1981). The cultivars SSC 1174 (highly resistant) and Kufri Jyoti (resistant) showed an earlier establishment and more rapid development of VAM fungi than up-to-date (highly susceptible). The first mycorrhizal infection in both SSC 1174 and Kufri Jyoti was observed after 12 days in 1980 and 8 days in 1981, whereas in up-to-date it was observed after 19 and 12 days respectively. The mycorrhizal infection increased with the age of the plants in all the three cultivars.     

β-Adrenergic receptors of frog erythrocytes

Following persistent stimulation of -adrenergic receptors of frog erythrocytes with (–)-isoproterenol, the cyclic adenosine 3,5-monophosphate-dependent protein kinase (cAMP-dependent protein kinase) (EC 2.7.1.37) was activated for several hours. This activation outlasted the duration of the increase of cAMP content. Following a persistant stimulation of -adrenergic receptors with isoproterenol, the phosphorylation of selective membrane proteins was increased. This increase in phosphorylation lasted longer than 4 hr but less than 12 hr. Between 2 and 4 hr after receptor stimulation the loss of -adrenergic receptor form plasma membrane was maximal, and the phosphorylation of two membrane proteins characterized by molecular weights of 60,000 and 38,000 daltons was selectively enhanced. In addition we found that isolated erythrocytes are capable of synthesizing RNA and polypeptides and that incubation with (–)-isoproterenol induces a longterm delayed increase of the synthesis of erythrocyte proteins. This increase in the synthesis of proteins appears to require new RNA synthesis. Thus the possibility can be entertained that this delayed increase in protein synthesis participates in the new synthesis of receptor and is operative in the termination of -adrenergic receptor subsensitivity elicited by a persistent stimulation with (–)-isoproterenol.     

Geographische Herkunft und Anpassung beim Apfel

Zusammenfassung In den Jahren 1961 und 1962 wurden Untersuchungen mit Hilfe der Exosmose-Methode durchgeführt, um sich über die Frostresistenz von nahezu 100 Apfelsorten zu orientieren. Die Versuchsergebnisse (ausgedrückt in RLF-Werten, d. h. als Proportion des elektrolytischen Widerstandes zwischen den Kontrollen und den kältebehandelten Pflanzenteilen) zeigen gute Übereinstimmung mit den Resultaten anderer Härtebeurteilungsmethoden, z. B. mit den Gefrierversuchen in Gefrierkammern und mit den amerikanischen, deutschen und schweidischen Freilandbeobachtungen. Die berechneten Korrelationskoeffizienten gaben Werte von r=>+0,750 mit einer statistischen Sicherheit von P=<0,001. Es gibt jedoch Sorten, welche sich unter den verschiedenen Versuchsbedingungen nicht gleichartig verhielten. In diese Gruppe gehören die Sorten Goldparmäne, James Grieve, Lanes Prince Albert, Schöner aus Nordhausen, Slava Petersburga und Transparente de Croncels. Der Zusammenhang zwischen der geographischen Herkunft und der Frostresistenz ist augenfällig. Die durchschnittlichen RLF-Werte der frostempfindlichen oder sehr frostempfindlichen französischen Sorten liegen bei ungefähr 150, die der finnischen und baltischen frostharten Sorten dagegen unter 110. Die Rangordnung der verschiedenen Klimagebiete erfolgte entsprechend der geographischen Lage. Es gab Sorten, die der allgemeinen Tendenz nicht folgten und sich abweichend verhielten. Hierzu gehören: Transparente de Croncels, (Frankreich), Wealthy (USA), Cox Pomona und Ribston (Großbritannien). Über eine wesentlich schlechtere Resistenz, als auf Grund des Ursprungsgebietes zu erwarten war, verfügt Ontario (Kanada).     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.