A procedure for mini-preparations of genomic DNA from needles of silver fir (Abies alba mill.)

We have adapted a procedure for the isolation of genomic DNA from needles of silver fir (Abies alba) to meet the requirements for large-scale analysis of the population genetics of forest trees. Our modifications permit the entire procedure to be carried out in Eppendorf tubes, which greatly minimizes time, plant material, and the amounts of chemicals. DNA is recovered with a mean efficiency of 80 μg/g needles, is suitable for restriction by the common endonucleases, and serves as a substrate for PCR.     

Monoterpene hydrocarbon contents of the resin from seeds of silver fir (Abies alba Mill.)

Summary Using gas-chromatographic methods, the variability of the contents of monoterpene hydrocarbons (-pinene, -pinene, ₃carene and limonene) in the resin of silver fir seeds (Abies alba Mill.) was studied. Resin cavities were characterized according to their position on the seed surface. It was estabilished that the terpene content of the resin of cavities localized on the abaxialadaxial surfaces of the seed differs significantly, creating a gradient of resin composition around the circumference of the seed. The differences between various resin cavities of single seeds were greater than the differences between different seeds of a single cone and between seeds of various cones on a single tree. An accurate definition of the resin cavity location on the seeds appears to be a fundamental condition for the collection of a sample representing the resin composition of individual trees. Resin biosynthesis in the course of organogenesis and the control of terpene contents in the resin of various locations on the seed and the cone are discussed.     

Stomatal patchiness in conifers: experiments with Picea abies (L.) Karst. and Abies alba Mill.

Summary Strong evidence for the occurrence of pronounced stomatal patchiness in needles of Picea abies (L.) Karst. and Abies alba Mill. was found using various indirect methods. Anatomical investigations revealed a septate leaf anatomy for both species, a phenomenon expected if a patchy distribution of stomatal aperture is present. Calculation of some photosynthetic characteristics (e.g. carboxylation efficiency) from gas exchange measurements is shown to be markedly affected by the patchy distribution of stomatal apertures on the needles. The importance of stomatal patchiness in connection with air pollution related forest decline symptoms as well as an hypothesis suggesting a possible role of the phenomenon as a protective mechanism against photoinhibition are discussed.     

Somatic embryogenesis from seed explants of Abies alba

Megagametophytes of Abies alba containing the immature embryos were dissected from the seed coats and divided by longitudinal and transverse sections. They were placed with the cut surface down on modified Schenk & Hildebrandt medium containing 50 mgl^-1 myo-inositol and 2% sucrose, supplemented with 1 mgl^-1 N⁶-benzyladenine (BAP). An embryogenic type of callus proliferated after one month of culture. Closer examination revealed the presence of structures resembling early stages of embryogenesis as well as of single elongated, vacuolated cells and clusters of cells with dense cytoplasm. Under appropriate conditions, some of the somatic embryos elongated and formed cotyledons.     

Interaction of plant growth regulators and organic C and N components in the formation and maturation of Abies alba somatic embryos

To promote SE maturation, the influence of different media components on different developmental stages was quantitatively evaluated. Advanced maturation was achieved with a sequence of culture media (prematuration medium and maturation medium) that contained various carbohydrates, organic nitrogen compounds and plant growth regulators. Application of lactose, BA, L-glutamine and casein hydrolysate in the prematuration medium enhanced the total number of SEs and promoted advanced differentiation. The highest number of late torpedo stage SEs was observed on maturation medium supplemented with 200 mM lactose and 29 mM sucrose. Lactose and sorbitol favoured SE maturation up to the early cotyledonary stage. With application of PEG and high ABA concentrations (20–40 M), only early torpedo stages were formed. The number of late torpedo stage SEs was significantly higher on hormone free media or with lower ABA concentrations (0–5 M). Formation of early and late cotyledonary SEs was significantly enhanced by adding BA in the maturation medium: neither Zeatin nor 2iP were effective. In addition, low sucrose concentrations in the proliferation medium (29 mM compared to 58 mM) also favoured the formation of cotyledonary SE in the maturation medium.     

Detection and identification of gibberellins in needles of silver fir (Abies alba Mill.) by combined gas chromatography-mass spectrometry

Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-year old (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected by the Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA₉ was present in small amounts, while a major component was a cellulase-hydrolysable GA₉ conjugate which was assumed to be GA₉ glucosyl ester. It is proposed that GA₉ glucosyl ester plays a key role in the regulation of endogenous GA levels in silver fir needles.     

Phytohormones in needles of healthy and declining silver fir (Abies alba Mill.): I. Indole-3-acetic acid

 Levels of indole-3-acetic acid (IAA) were determined in needles from silver fir (Abies alba Mill.) trees in the northern Black Forest. IAA was quantified by gas chromatography (GC) as 1-heptafluorobutyryl-IAA-methylester (HFB-IAA-ME) using electron capture detection. Prior to GC analysis, extensive purification of needle extracts was performed employing two HPLC steps. Peak identity of HFB-IAA-ME was confirmed by combined gas chromatography-mass spectrometry in selected samples. Levels of IAA in needles belonging to different needle age-classes exhibited a cyclic seasonal pattern with highest concentrations in winter and lowest levels in spring when bud-break occurred. Such a cyclic seasonal pattern of IAA levels was also observed in needles from declining fir trees or fir trees suffering from a strong sulfur impact (S-impact) in the field due to a local SO₂ source. Levels of IAA increased with increasing needle age. This age dependency of IAA concentrations was most pronounced in late autumn when IAA levels were high and nearly disappeared in spring when IAA levels reached their minimum. In needles from declining fir trees or fir trees suffering from a strong S-impact in the field, IAA levels hardly increased with increasing needle age. It is suggested that in healthy trees high levels of IAA protect older needles from abscission and that the considerable losses of older needles of declining fir trees or of fir trees under S-impact are a consequence of the low levels of IAA found in older needles of such trees. Received: 4 May 1995 / Accepted: 29 August 1995     

Identification by combined gas chromatography-mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings

Seven day old seedlings of Pisum sativum L., cv. Kleine Rheinländerin, were wilted for 3 days. After partially removing the roots, they were rewatered and at the same time radioactive abscisic acid([1-¹⁴C]ABA, spec. activity 1.7·10⁸d s^-1mmol^-1) was applied for 1 h via the xylem of the roots. After 24 h, 4 days, and 12 days the seedlings were extracted and the metabolites of ABA were analyzed by means of thin-layer and gas chromatography in combination with mass spectrometry, autoradiography, and scintillation counting. Phaseic acid (PA) and dihydrophaseic acid (DPA) were identified as metabolites of ABA. The presence of another ABA-metabolite was also demonstrated. From its mass spectrum it has been postulated that this metabolite is 4-desoxy-ABA. In addition to these substances, several other metabolites, which are more polar than ABA and its known degradation products, were present in the seedlings. The quantity and number of these unknown metabolites increased with time.Abbreviations ABA abscisic acid - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen bis(5-phenyloxazole)     

Identification and characterization of nuclear microsatellite loci in Abies alba Mill.

Eleven polymorphic nuclear microsatellite markers for Abies alba Mill. were developed from an enriched genomic library. An average of 5.2 alleles per locus and a mean expected heterozygosity of 0.532 were found in a sample of 24 Abies alba individuals from different populations within Europe. These loci can be used in studies of genetic diversity for parentage analysis and for estimation of gene flow in silver fir populations. Moreover, successful amplifications were obtained for eight other Mediterranean Abies species, suggesting that these loci may be useful for similar applications in other fir species.     

Radioimmunoassay for the determination of free and conjugated abscisic acid

The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum albumin-(±)-ABA conjugate have a high affinity for ABA (K_a=1.3x10⁹l mol^-1). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25x10^-12 mol ABA, the measuring range extends to 20x10^-12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi.Abbreviations ABA abscisic acid - BHT 2.6-di-t-butyl-4-methyl phenol - TLC thin-layer chromatography - HSA human serum albumin Part 7 in the Series: Use of Immunoassay in Plant Science     

Sensitivity of Commelina stomata to abscisic acid

Stomata of Commelina leaves pre-opened by incubation in moist air were found to close within 30 min when supplied with abscisic acid (ABA) via the transpiration stream. Radioactive ABA had similar effects, but allowed the distribution of the compound within the leaf to be measured and correlated with stomatal movements to give estimates of the sensitivity of Commelina stomata. On a whole-leaf basis, less than 163 fmol ABA per mm² leaf area were present at the time of complete stomatal closure. This was close to other published estimates. By taking epidermal ¹⁴C measurements, however, it was possible to increase the accuracy of the estimate on the assumption that only ABA present in the epidermis was physiologically active. Thus, less than 235 amol ABA for stomatal complex were present at complete closure, and statistically significant narrowing of the stomatal aperture had occurred when between 12.6 and 45.4 amol per complex were present. The distribution of ABA within the epidermal tissue after transpiration-stream application was studied using microautoradiography, and the compound appeared to have accumulated within the stomatal complex.Abbreviations ABA abscisic acid - TLC thin-layer chromatography     

The contact zone between two migration routes of silver fir,Abies alba (Pinaceae), revealed by allozyme studies

Enzyme data from 35 populations or regional samples ofAbies alba were obtained from different regions of Austria and Bavaria. The genetic analysis revealed a clear differentiation between the western and the north/eastern populations. A rather narrow transition zone (the river valleys of Salzach and Traun) with genetically intermediate populations connects these two regions. This west-east differentiation can be interpreted as the result of distinct migration routes from a glacial refuge in Central Italy. The transition zone is assumed to be the contact area of two routes north of the Alps. Each migration route of silver fir parallels the distribution range ofCardamine trifolia (east) andAposeris foetida (west) respectively. It is supposed that these two species were already associated with silver fir during colonization.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Endogenous levels of abscisic acid and gibberellins in leaf protoplasts competent for plant regeneration in Betula platyphylla and Populus alba

Differences in endogenous levels of abscisic acid and gibberellinsbetween Betula platyphylla and Populusalba leaf protoplasts were determined using micro-scale extractionand purification steps, including thin layer chromatography ormicro-high-performance-liquid-chromatography and quantification by enzymelinkedimmunosorbent assay or micro-bioassay. The content of abscisic acid was tentimes higher in B. platyphylla than in P.alba on the basis of both cell number and dry weight; in contrast,levels of gibberellins were lower in the former. Leaf protoplasts of bothspecies are competent for plant regeneration through the exogenous supply ofauxins and cytokinins. The function of abscisic acid in these protoplastcultures is discussed in relation to the need for a strong cytokinin,N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU) for colony proliferation inB. platyphylla, in contrast to a weak cytokininrequirementin P. alba.     

Quantitative relationship between indole-3-acetic acid and abscisic acid during leaf growth in Coleus blumei

Quantitative determinations by gas chromatography-mass spectrometry ofindole-3-acetic acid (IAA) and abscisic acid (ABA) in growing leaves ofColeusblumei plants show parallel declines in leaf concentrations of bothhormones,except in leaf number 3 (about three-fourths of full size) where IAA level wasthe lowest of those measured. Expansion of the most recently unfurled leaf tofull size serves, in effect, to dilute both IAA and ABA about 1.7 to 1.Althoughabsolute levels of leaf IAA varied as much as an order of magnitude from onebatch of plants to another, ABA levels were proportional to the IAA level withan overall correlation coefficient of 0.91. Evidence, both correlative andcausal, for the determination of ABA status by IAA—and of IAA status byABA—in leaves and other developing organs is summarized.     

Population genetics analysis in a relic population of silver fir (Abies alba Mill.) in southern Italy: A comparison with microsatellites and reference data

We investigated the genetic diversity of the last natural population of Abies alba Mill. in Campania region (southern Italy) using nuclear and chloroplast microsatellite markers. We found high genetic diversity despite small population size and geographic isolation, especially when comparing our microsatellite data with information from the literature.     

The geographical distribution of genetic variation of silver fir (Abies alba,Pinaceae) in relation to its migration history

Silver fir presently occurs in many mountainous regions of eastern, western, southern and Central Europe. In order to elucidate the biogeographic history of fir populations in different European areas, the distribution of area-specific alleles at eight enzyme loci and the allele frequency distributions at seven polymorphic enzyme loci were investigated in seed or bud samples from 48 provenances comprising 98 single stands. Due to great genetic divergence among different populations located near the ancient glacial retreats, it was concluded that silver fir survived in five refugia during the last glaciation but migrated from only three refugia to its present natural range. The occurrence of unique alleles in particular fir areas indicated its migratory routes to the north and the existence of a few introgression zones where silver fir from different refugia has met during its expansion.     

Identification and measurement of indoleacetic and abscisic acids in the cambial region of Picea sitchensis (Bong.) Carr. by combined gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were identified by combined gas chromatography-mass spectrometry (GCMS) in fractions obtained by diffusion and extraction from bark peelings of Sitka spruce. A procedure is described for the quantitative analysis of IAA and ABA levels in the same extract using the GCMS technique of single-ion current monitoring. This procedure was used to measure the diffusible, free, and bound fractions of IAA and ABA in the cambial region of Sitka spruce throughout one year; the range in concentration for these fractions was 0.06–0.30, 0.46–3.85, and 0.04–0.20 g/g oven-dry weight, respectively, for IAA, and 0–0.08, 0.03–2.21, and 0.13–0.66 g/g oven-dry weight, respectively, for ABA. Movement in the cambial region was found to be polar for endogenous IAA and nonpolar for endogenous ABA. Recoveries of [¹⁴C]IAA internal standards showed that 73–99.5% of the IAA was lost during purification, and that there could be up to 5-fold differences in recovery between purifications, indicating that IAA loss shold be measured in quantitative analyses.Abbreviations ABA aoscisic acid - GCMS combined gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PVP polyvinylpyrrolidone - SICM single ion current monitoring - TMS trimethylsilyl     

Uptake and distribution of abscisic acid in Commelina leaf epidermis

Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a range of KCl concentrations. Control apertures were greatest at high concentrations of the salt, and the effects of ABA, in terms of closure, were most pronounced below 100 mol m^-3 KCl. Stomata opened on strips floated on buffer plus 50 mol m^-3 KCl and closed within 10 min when transferred to the same medium plus 0.1 mol m^-3 ABA. [2-¹⁴C]ABA was used to study uptake and distribution of the hormone by the epidermal strips. It was calculated that no more than 6 fmol ABA were present per stomatal complex at the time of closure, although uptake continued thereafter. Microautoradiography indicated that radioactivity from [2-¹⁴C]ABA accumulated in the stomatal complex at or near the guard cells within 20 min. TLC was used to examine the state of the label after 1 h incubation. Efflux of label from preincubated tissue appeared to occur in three phases (t_1/2=7.2 s, 4.0 min, 35.2 min). Efflux was correlated with stomatal re-opening. The results confirm that ABA can accumulate in the epidermis of C. communis.Abbreviation ABA Abscisic acid     

Endogenous inhibitors and seasonal changes in abscisic acid in Dioscorea floribunda Mart. & Gal.

In the tubers of medicinal yam (Dioscorea floribunda Mart. & Gal.) abscisic acid (ABA) content was high in all the parts during the dormant condition in the winter, but it decreased to a very low level in the actively growing plants. ABA content of the entire tuber was negatively correlated with temperature and photoperiod. Growth inhibitors including batatasin-I and phthalic acid were identified in the dormant tuber.CIMAP Communication No. 673.