Feeding behavior and responsivity to food odors in Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> (Cichlidae) after chronic polisensory deprivation

The paper reports the study of the food searching behavior in the Nile tilapia Oreochromis niloticus before and after the experimental anosmia combined with visual deprivation. The threshold concentration of the water extract of the common food organisms (Chironomidae larvae) was found for the tilapia with intact olfaction to be 10^–4 g/L, which allows this fish to search for food under the restriction of visual reception. The food searching behavior in response to food odors is completely lost as a result of irreversible anosmia and do not be restored after a 4.5-month period even for the high concentrations of the extract (10^–1–10^–2 g/L). These findings point to the absence of external taste buds on the head, body, and fins, or to their extremely low numbers. It is assumed that tactile sense also plays a significant role in the food search and prey location in the Nile tilapia.     

Chemosensory mediation of food searching in the Buenos Aires tetra <Emphasis Type="Italic">Hyphessobrycon anisitsi</Emphasis> (Characidae)

Food searching behavior of the Buenos Aires tetra Hyphessobrycon anisitsi has been investigated before and after experimentally induced anosmia combined with visual deprivation. The threshold concentration of a water extract of familiar food (chironomid larvae) for fish with intact olfaction was 10^–6 g/L. Induced anosmia led to complete loss of the search reaction to food odor. The chemosensory behavioral response to the water extract of food used at high concentrations (10^-1-10^-2 g/L) was not restored in fish after 1.5, 5, or 13.5 months of anosmia. The obtained results show that the external sense of taste is absent or extremely poorly developed in the Buenos Aires tetra.     

Effects of the long-term anosmia combined with vision deprivation on the taste sensitivity and feeding behavior of the rainbow trout <Emphasis Type="Italic">Parasalmo</Emphasis> (=<Emphasis Type="Italic">Oncorhynchus</Emphasis>) <Emphasis Type="Italic">mykiss</Emphasis>

We compared taste preferences, taste sensitivity, and behavior in testing food objects in the group of intact and two groups of sensory deprived rainbow trout Oncorhynchus mykiss yearlings. We demonstrated that long-term anosmia (for 9 months), as well as anosmia (for 9 months) combined with enucleation (object vision deprivation for 4 months), does not change the taste preference of fish for the agar pellets containing amino acids (L-alanine, L-proline, L-histidine, or glycine; 0.1 M). For all groups of fish, the threshold L-alanine concentration in pellets that caused a significant increase in consumption is 0.01 M. We showed that sensory deprived fish change their behavior of gustatory testing, namely, the rate of repeated snaps decreases as well as the pellet retention time in the mouth cavity. These results demonstrate that long-term anosmia combined with a partial vision deprivation does not significantly change the taste preferences and sensitivity in the fish that have no external taste buds. However, the observed reduction in the testing time of food objects and other changes in fish feeding behavior may suggest some functional alterations in the intraoral sensory systems (gustatory and/or tactile).     

Taste preferences and feeding behavior of the stone loach <Emphasis Type="Italic">Barbatula barbatula</Emphasis> (Balitoridae,Cypriniformes) after partial deprivation of circum-mouth external gustatory and tactile receptors

Comparative study of the feeding behavior and gustatory preferences mediated by extraoral and intraoral gustatory reception in the stone loach Barbatula barbatula in the norm and 1–6 months after extirpation of all three pairs of barbels was performed. It was found that partial loss of external gustatory receptors and the sensory deficit caused by it do not lead to any noticeable disturbances of the ability of fish to evaluate taste properties of food objects (artificial agar-agar pellets containing L-isomers of alanine, lysine, cysteine or an water extract of chironomid larvae) and make an adequate decision of their grasping or ignoring and swallowing or refusing. The extirpation of the barbels does not influence feeding behavior related to determination by fish of the taste properties of pellets and completely retains in its ritual an obligatory preliminary examination of the food object using external gustatory reception. It is suggested that external taste buds having different localization are equally capable of providing for fish a preliminary evaluation of the taste properties of the object. After extirpation of the barbels, the efficiency of grasping pellets decreases in fish, which indicates an important role of tactile reception in the determination of the site of location of the food object and in providing of the accuracy of the hunter’s dart in fish with a weak vision development.     

Role of olfaction and vision cues in feeding behavior and alarm reaction in the catfish pintado, Pseudoplatystoma corruscans

Experiments were performed to investigate senses that are essential for mediating fright reaction and food behavior in Pseudoplatystoma corruscans, pintado. The dilemma “to feed or to flee” was also analyzed in fishes with intact and sectioned olfactory tracts, stimulated by alarm substance extracts and food. Fishes were arranged into five groups: fish with intact lateral olfactory tracts (LOT), fish with intact medial olfactory tract (MOT), fish with tracts totally sectioned (TOTAL, both LOT and MOT), sham operated, and nonoperated fish. The five groups were submitted to either alarm substance extract and food stimulus or to distilled water (control) and food stimulus. Fish reacted to food independently of which tract (LOT, MOT or TOTAL) was sectioned; vision seems necessary and elemental to detect and deflagrate food response. Latency of the responses to each reaction was different between groups. None of the fish with sectioned tracts reacted to alarm substance extract, while sham- and nonoperated fish showed the typical alarm behavior response, leading to the conclusion that olfaction is essential for mediating alarm response. These results indicate that others sense systems (e.g., vision) are sufficient to trigger and elicit feeding behavior and that olfaction is not necessary to fully maintain food detection to qualitative and quantitative extent. However, olfactory tract integrity seems to be required for mediation of alarm reaction in P. corruscans.     

Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4

Taste buds and the peripheral nerves innervating them are two important components of the peripheral gustatory system. They require appropriate connections for the taste system to function. Neurotrophic factors play crucial roles in the innervation of peripheral sensory organs and tissues. Both brain-derived neurotrophic factor (BDNF) null-mutated and neurotrophin-4 (NT-4) null-mutated mice exhibit peripheral gustatory deficits. BDNF and NT-4 bind to a common high affinity tyrosine kinase receptor, TrkB (NTRK-2), and a common p75 neurotrophin receptor (NGFR). We are currently using a transgenic mouse model to study peripheral taste system development and innervation in the absence of both TrkB ligands. We show that taste cell progenitors express taste cell markers during early stages of taste bud development in both BDNF^−/−xNT-4^−/− and wild-type mice. At early embryonic stages, taste bud progenitors express Troma-1, Shh, and Sox2 in all mice. At later stages, lack of innervation becomes a prominent feature in BDNF^−/−xNT-4^−/− mice leading to a decreasing number of fungiform papillae and morphologically degenerating taste cells. A total loss of vallate taste cells also occurs in postnatal transgenic mice. Our data indicate an initial independence but a later permissive and essential role for innervation in taste bud development and maintenance. This work was supported by NIH-NIDCD R01-RDC007628.     

The effect of olfactory deprivation on the thresholds of sensitivity of skin chemoreceptors of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

Using method of registration of electrical potentials caused by chemical stimulation of the skin surface of the head of the carp Cyprinus carpio, the effect of prolonged olfactory deprivation (3, 9, and 12 months after operation) and partial blinding of anosmiated fish (9 months) on the sensitivity thresholds of external chemoreceptors was studied. It was found that, 3 months following anosmiation, the thresholds decreased by orders of 0.1–0.5, and, after 9 and 12 months, they decreased by orders of 0.1–0.8 depending on the kind of the chemical stimulus and the zone of registration. The highest increase in the sensitivity of skin chemoreceptors was recorded in anosmiated fish deprived also of object vision, by orders of 0.3–1.5. The results obtained indicate the existence of a close functional relationship between the olfactory and visual systems, on the one hand, and the skin chemosensory system, on the other hand. The compensatory processes taking place in these systems in the case of the loss of one of them have an adaptive pattern and are directed at provision of reliability of behavioral contacts and survival of fish in the environment.     

Density and distribution of external taste buds in cyprinids

Synopsis The aim of the present qualitative and quantitative study was to survey the density and distribution of external taste buds (TB) in 10 common European cyprinid species. TB pores were stained with silver nitrate and counted in 7 sample areas on the body and 3 on the fins. TB densities decrease from rostral to caudal and from ventral to dorsal. Highest densities were encountered in the gular region of the minnow (297 per mm²), white bream (291) and vimba (285), or at the forehead, in white bream (147). Of the fins, the pectoral showed highest densities (150 per mm² in vimba,138 in the minnow). The overall TB density rank from high to low is: minnow Phoxinus phoxinus, white bream Blicca bioerkna, crucian carp Carassius carassius, vimba VVimba vimba, bream Abramis brama, roach Rutilus rutilus, bleak Alburnus alburnus, sun bleak Leucaspius delineatus, sabre carp Pelecus cultratus and bitterling Rhodeus sericeus. This is also a fair rank of life styles from benthivory to open water planktivory and surface feeding. A significant positive correlation exists between densities of external taste buds and the relative sizes of the brain stem facial lobes. An apparent compensatory relationship between vision and taste, but not lateral line, is discussed.     

The effect of food odor background on gustatory preferences and gustatory behavior of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> and cod <Emphasis Type="Italic">Gadus morhua</Emphasis>

It was shown that stimulation by food odor (aquatic extract of food organisms, 10^?2 and 10^?3 g/l) does not cause shifts in gustatory preferences in carp Cyprinus carpio and cod Gadus morhua but modifies gustatory behavior. The level of consumption by carp of control granules and granules with attractive, by taste, L-proline (0.1 M) or deterrent L-lysine (0.1 M) (item by item presentation of granules) and by cod of control granules and granules with indifferent, to it, L-asparagine (0.1 M) (presentation of 10 granules simultaneously) is similar prior to and during olfactory stimulation. In the presence of food odor, the duration of taste testing for most types of granules, as well as the number of repeated graspings of granules with an attractive taste do not change in fish. At the same time, granules with indifferent or repulsive gustatory properties are rejected and repeatedly grasped by fish against the background of food odor more frequently than in water without odor. Olfactory stimulation leads to a considerable increase in the average number of graspings per one grasped granule with an indifferent or repulsive taste. Such behavior manifested by fish in the presence of food odor in response to granules with unattractive gustatory properties is apparently caused by the contradiction between the information coming via different chemosensory canals—olfactory and gustatory. The obtained results indicate that food stimulation caused by food odor in nature can lead to an increase in the actual consumption of only those accessible food items that have an attractive taste for fish.     

Hormone Profiling by LC-QToF-MS/MS in Dormant <Emphasis Type="Italic">Macadamia integrifolia</Emphasis>: Correlations with Abnormal Vertical Growth

A method for analyzing multiple plant hormone groups in small samples with a complex matrix was developed to initiate a study of the physiology of abnormal vertical growth (AVG) in Macadamia integrifolia (cv. HAES344). Cytokinins (CKs), gibberellins (GAs), abscisic acid (ABA), and auxins were detected in xylem sap and apical and lateral buds using high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS). The extraction method separated compounds with high sensitivity in positive (CKs) and negative (ABA, auxins, GAs) modes of QToF-MS/MS. CK profiles differed in xylem sap and apical and lateral buds irrespective of AVG symptoms. Trans-zeatin riboside (t-ZR) was dominant in sap of normal and AVG trees (∼4 and 6 pmol g⁻¹ FW, respectively). In apical buds isopentenyl adenine (iP) (∼30 pmol g⁻¹ FW) was the most abundant CK, and in lateral buds trans-zeatin (t-Z) (22–24 pmol g⁻¹ FW) and iP (24–30 pmol g⁻¹ FW) were the most abundant. t-Z levels of AVG trees were higher in apical buds (13.88 vs. 6.6 pmol g⁻¹ FW, p < 0.05) and lower in sap (0.16 vs. 0.51 pmol ml⁻¹, p < 0.005) compared to normal trees. ABA in lateral buds was 1.9 times higher (p < 0.001) in AVG. IAA was below quantification, whereas indole-3-butyric acid (IBA) was consistently present. GA₇ was the dominant GA in apical and lateral buds of all trees (100–150 pmol g⁻¹ FW). GA_{3, 4, & 9} were consistently present at low concentrations (<12 pmol g⁻¹ FW) in buds. GAs_{1, 3, & 9} were detected in xylem sap at low concentrations (<0.5 pmol g⁻¹ FW). Differences in sap amino acids (AA) were also assessed. In sap from AVG trees, asparagine and glutamine increased significantly (p < 0.05) in their contribution to total AA. Potential AVG hormone correlations are discussed.     

Plant secondary metabolites as mammalian feeding deterrents: separating the effects of the taste of salicin from its post-ingestive consequences in the common brushtail possum (Trichosurus vulpecula)

The effect of the phenolic glycoside, salicin, on food intake of the common brushtail possum (Trichosurus vulpecula) was studied in a series of feeding experiments. Increasing the concentration of salicin in a diet of fruits and cereals led to significant reductions of food intake in the short term (6 days). After prolonged (20 days) exposure to salicin, food intake (19 g kg^−0.75 day⁻¹) was still reduced relative to controls (31 g kg^−0.75 day⁻¹) but not reduced to the same extent as in the short-term experiments. Nonetheless, over these 20 days, common brushtail possums regulated their intake of salicin so as not to exceed a threshold limit of 1.9 ± 0.1 g kg^−0.75 day⁻¹. Manipulative experiments sought to determine whether this threshold intake was in response to pre-ingestive factors (taste) or the post-ingestive consequences of ingesting salicin. Dietary salicin (0.17–5.0% DM) had no significant effect on nitrogen balance or urea metabolism and injection of a specific serotonin receptor antagonist, ondansetron, did not lead to increases in salicin intake as has been found for some other plant secondary metabolites. Similarly, administration of 1.3 g salicin by gavage had no significant effect on the subsequent intake of salicin compared to controls that were gavaged with water. We concluded that pre-ingestive factors were responsible for common brushtail possums limiting their intake of salicin-rich diets rather than any measurable post-ingestive consequence of feeding. Accepted: 7 December 1999     

Fed-batch production of a bioflocculant from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l⁻¹ urea solution, the flocculating activity was enhanced to 720 U ml⁻¹ in 36 h. High cell density (2.12 g l⁻¹) and flocculating activity (820 U ml⁻¹) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l⁻¹, and residual urea decreased to 0.03 g l⁻¹. Even higher flocculating activity of 920 U ml⁻¹ and biomass of 3.26 g l⁻¹ were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.     

Clonal micropropagation of <Emphasis Type="Italic">Crataeva adansonii</Emphasis> (DC.) Prodr.: A multipurpose tree

Summary A method of micropropagation through multiple shoot formation from axillary buds of mature tree and rootstock growths of Crataeva adansonii (DC.) Prodr. (a multipurpose tree) has been developed. Factors affecting multiplication rate included season, age of explant source, explant type, type of bud, position of bud on the foliage twig, type of medium, various additives, and explant implantation on the medium. The maximum number of buds was produced from the sixth to 10th axillary buds taken from foliage twigs of 40–50-d-old rootstock growth in the months of October to December. At this time of the year the contamination was minimum. Optimum response was recorded on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 3 mgl⁻¹, 13.3μM) and 1-naphthaleneacetic acid (NAA; 0.05 mgl⁻¹, 0.27 μM) after 21 d of culture. Shoot buds were further multiplied and maintained on BA (1–1.5 mgl⁻¹, 4.4–6.7 μM) while shoots elongated on BA (0.1–0.5 mgl⁻¹, 0.44–2.2 μM) supplemented medium. The number of shoots was further multiplied by using nodal segments of in vitro-regenerated shoots as microcuttings and repeated subculturing of stumps after excising the microshoots. In vitro rooting on growth regulator-free MS medium was possible with 70% of microshoots after 4 wk. From one nodal segment 150 plantlets were produced within 14 wk.     

High efficiency organogenesis and analysis of genetic stability of the regenerants in <Emphasis Type="Italic">Solanum melongena</Emphasis>

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ zeatin, 0.1 mg dm⁻³ indoleacetic acid and 10 g dm⁻³ sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0–2.0 mg dm⁻³ gibberellic acid and 4.0–8.0 mg dm⁻³ AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

The Antarctic notothenioid fish <Emphasis Type="Italic">Pagothenia borchgrevinki</Emphasis> is thermally flexible: acclimation changes oxygen consumption

Antarctic marine organisms are considered to have extremely limited ability to respond to environmental temperature change. However, here we show that the Antarctic notothenioid fish Pagothenia borchgrevinki is an exception to this theory. P. borchgrevinki was able to acclimate its resting metabolic rate and resting ventilation frequency after a 5°C rise in temperature. Acute exposure to 4°C resulted in an elevation in metabolic rate (57.8 ± 4.79 mg O₂ kg⁻¹ h⁻¹) and resting ventilation rate (40.38 ± 1.61 breaths min⁻¹) compared with fish at −1°C (metabolic rate 34.45 ± 3.12 mg O₂ kg⁻¹ h⁻¹; ventilation rate 29.88 ± 3.72 breaths min⁻¹). However, after a 1-month acclimation period, there was no significant difference in the metabolic rate (cold fish 29.52 ± 3.01; warm fish 31.13 ± 2.30 mg O₂ kg⁻¹ h⁻¹), or the resting ventilation rate (cold fish 28.75 ± 0.98; warm fish 34.25 ± 2.28 breaths min⁻¹) of cold and warm acclimated fish. Acclimation changes to the rate of oxygen consumption following exhaustive exercise were complex. The pattern of oxygen consumption during recovery from exhaustive exercise was not significantly different in either cold or warm acclimated fish.     

Mutation rate and pattern of microsatellites in common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56×10⁻⁴/locus/generation (95% confidence interval 1.52×10⁻⁴ and 1.63×10⁻³). The change of allele size was between +2 to −5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.     

Mechanisms of vasodilation in the dorsal aorta of the elephant fish, <Emphasis Type="Italic">Callorhinchus milii</Emphasis> (Chimaeriformes: Holocephali)

This study investigated vasodilator mechanisms in the dorsal aorta of the elephant fish, Callorhinchus milii, using anatomical and physiological approaches. Nitric oxide synthase could only be located in the perivascular nerve fibres and not the endothelium of the dorsal aorta, using NADPH histochemistry and immunohistochemistry. In vitro organ bath experiments demonstrated that a NO/soluble guanylyl cyclase (GC) system appeared to be absent in the vascular smooth muscle, since the NO donors SNP (10⁻⁴ mol l⁻¹) and SIN-1 (10⁻⁵ mol l⁻¹) were without effect. Nicotine (3 × 10⁻⁴ mol l⁻¹) mediated a vasodilation that was not affected by ODQ (10⁻⁵ mol l⁻¹), l-NNA (10⁻⁴ mol l⁻¹), indomethacin (10⁻⁵ mol l⁻¹), or removal of the endothelium. In contrast, the voltage-gated sodium channel inhibitor, tetrodotoxin (10⁻⁵ mol l⁻¹), significantly decreased the dilation induced by nicotine, suggesting that it contained a neural component. Pre-incubation of the dorsal aorta with the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP_8–37 (10⁻⁶ mol l⁻¹) also caused a significant decrease in the nicotine-induced dilation. We propose that nicotine is mediating a neurally-derived vasodilation in the dorsal aorta that is independent of NO, prostaglandins and the endothelium, and partly mediated by CGRP.     

Olfactory sensitivity to amino acids in the blackspot sea bream (<Emphasis Type="Italic">Pagellus bogaraveo</Emphasis>): a comparison between olfactory receptor recording techniques in seawater

The current study investigated the olfactory sensitivity of the blackspot sea bream to amino acids, odorants associated with food detection in fish, and compared the efficacy of two different experimental methods: multi-unit recording from the olfactory nerve and the electro-olfactogram (EOG). Twenty essential amino acids plus l-DOPA evoked clear, concentration-dependent olfactory responses using both methods, with estimated thresholds of 10^−8.5–10^−6.2 M (nerve recording) and 10^−7.5–10^−4.8 M (EOG). The most potent amino acids were l-cysteine, l-methionine (both sulphur-containing), l-alanine, l-leucine (both neutral), l-glutamine (amide-containing) and l-serine (hydroxyl-containing). The least potent were l-proline (secondary α-amino group), the aromatic amino acids and glycine (simplest). Although the rank order of olfactory potency was similar for the two methods used, and the calculated thresholds given by the two methods were positively correlated, the sensitivity of the EOG was consistently lower than multi-unit recording by approximately one order of magnitude, presumably due to the electrical shunting effect of seawater. As in freshwater, the EOG could be a valid method for comparing olfactory potency of different odorants in stenohaline marine fish; however, for absolute ‘biological’ thresholds, a more invasive recording technique, such as multi-unit recording from the olfactory nerve, should be used.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.