Oxygen influences benzyladenine and 2,4-dichlorophenoxyacetic acid levels in cultured embryogenic tissue of Norway spruce

It is known that reducing the partial pressure of O₂ influences the induction of somatic embryogenesis. We tested the hypothesis that O₂ causes changes in the endogenous levels of exogenously supplied benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic tissue of Picea abies was incubated under reduced (2.5, 5 kPa) and ambient (21 kPa) levels of O₂ for 1, 3, 7 and 11 days and the endogenous concentrations of BA and 2,4-D were measured. For all treatments the concentration of BA in the tissue increased until the third day. At day 3, the ratio of BA in the tissue relative to the initial concentration in the medium, was 3.9, 2.8 and 1.9 for tissue incubated under 2.5, 5 and 21 kPa O₂, respectively. The BA concentration then declined gradually. Uptake of 2,4-D was inhibited at low O₂ levels. However, 2,4-D gradually accumulated in tissue grown under hypoxia, so that high levels were reached by day 11. These shifts in the BA and 2,4-D levels also caused a transient increase in the BA to 2,4-D ratio in tissue incubated under hypoxia. Although relevant for the previously reported effects of oxygen on induction of embryogenic tissue, it is unlikely that oxygen-induced alterations in BA and 2,4-D levels alone suffice to explain these findings.     

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.     

Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings

Cuttings were taken from 4-week-old seedlings of Norway spruce ( Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL).
Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Embryology in Norway spruce (Picea abies). Immunochemical studies on transport of a seed storage protein

One of the main seed storage proteins of Norway spruce ( Picea abies ), is a salt-soluble protein with an average molecular mass of 42 kDa. This protein was localized by immunocytochemical methods in ultrathin sections of megagametophytes active in storage protein synthesis, as analyzed by SDS-PAGE. The megagametophyte in spruce starts accumulating storage materials, proteins and lipids, as the young embryo grows into the gametophytic tissue. It then continues to accumulate these storage products throughout seed development (Hakman 1993). Megagametophytes at an early stage of storage protein accumulation were chosen in this study for analysing the likely transport pathway of the proteins, since only a small amount of lipid had yet accumulated in the cells, and cell organelles were still easy to distinguish. An antibody against the 42 kDa storage protein showed very good reactivity with the 42 kDa protein in immunoblot experiments with total protein extracts from megagametophytes and embryos. In ultrathin sections of the megagametophyte, the antibodies were preferentially localized in the lumen of Golgi cisterna, in Golgi-associated vesicles, protein deposits close to the vacuolar membrane and in protein storage vacuoles (protein bodies). These observations indicate that the transport is mediated by the Golgi apparatus.
Also, proteins present in storage vacuoles in mature zygotic and somatic embryos showed intense labelling with these antibodies in ultrathin sections.     

Distribution of lead in mycorrhizal and non-mycorrhizal Norway spruce seedlings

Non-mycorrhizal spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus (Scop.) Fr. or two strains of Paxillits involutus (Batsch) Fr. were grown in an axenic silica sand culture system with frequently renewed nutrient solution. After successful mycorrhizal colonization, the seedlings were exposed to 1 μM PbCI₂ for 19 weeks. The degree of infection in all of the mycorrhizal treatments approached 100% during the experiment and was not affected by exposure to Pb. However, the number of root tips per root dry weight and the shoot: root ratio, both in the non-mycorrhizal and the mycorrhizal seedlings, had decreased after the 19 week treatment with PbCl₂ Using X-ray microanalysis, the distribution and concentration of Pb in the tissues of mycorrhizal and non-mycorrhizal root tips were compared. In the mycorrhizae of seedlings exposed to Pb no significant accumulation of Pb in the hyphal mantle or in fungal cell walls of the Hartig net were detected. Lead accumulated primarily in the cortex cell walls both of non-mycorrhizal and mycorrhizal root tips. No significant difference of Pb concentrations in root cortex cell walls of non-mycorrhizal and mycorrhizal seedlings was found; except for seedlings colonized with Paxillus involutus strain 537. However, at the endodermis no effect of mycorrhizal fungal colonization on the Pb tissue concentration was detected. The presence of the fungal sheath did not prevent Pb from reaching the root cortex. The endodermis acted as a barrier to Pb radial transport in both mycorrhizal and non-mycorrhizal seedling roots.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Variable growth and reproduction response of the spruce shoot aphid, Cinara pilicornis, to increasing ozone concentrations

Spruce shoot aphid, Cinara pilicornis Hartig (Homoptera: Lachnidae), is an aphid species that has shown enhanced performance on trees exposed to SO₂ or mixtures of air pollutants, whereas results with ozone have been contradictory. Using a 4-week chamber fumigation experiment, we tested how different population dynamic parameters of aphids are affected by ozone (O₃). Mean relative growth rate (MRGR), development time and reproduction rate of C. pilicornis were determined using 7h day-time O₃ concentrations of 0, 40, 80 and 160 ppb. Development period from birth to reproductive stage was fastest at 80 ppb during early shoot elongation, while reproduction and intrinsic rate of population increase was not significantly affected by ozone concentration. There was a significant negative correlation between ozone concentration and the MRGR of first instar nymphs in the third fumigation week. In feeding test performed on seedlings after fumigation, the MRGR was reduced on shoots which had been exposed to 80 ppb O₃. The results suggest that elevated O₃ concentration during early shoot elongation period may stimulate population development of C. pilicornis, but on maturing shoots, high O₃ concentration has a negative effect on aphid performance. This might be due to accelerated ageing of O₃ exposed shoots.     

Effects of 1-aminocyclopropane-1-carboxylic acid,aminoethoxyvinylglycine, methylglyoxal bis-(guanylhydrazone) and dicyclohexylammonium sulfate on induction of embryogenic competence of mango nucellar explants

The effects of aminoethoxyvinylglycine (AVG),1-aminocyclopropane- 1 -carboxylic acid (ACC), dicycyclohexylammonium sulfate (DCHA) and methylglyoxal bis-(guanylhydrazone) (MGBG) on induction of embryogenic competence from the nucellus of two mango genotypes, ‘Tutehau’ (polyembryonic) and ‘Tommy Atkins’ (monoembryonic), were compared. Induction of embryogenic competence in the explanted nucellus of ‘Tommy Atkins’ was more sensitive to AVG and DCHA than ‘Tutehau’. MGBG had no effect on induction of embryogenic competence of either genotype, and ACC suppressed somatic embryogenesis with both cultivars. Ethylene biosynthesis was greater from explanted ‘Tommy Atkins’ cultures. Reducing ethylene biosynthesis with AVG marginally increased the number of embryogenic cultures of ‘Tutehau’, but completely inhibited somatic embryogenesis in ‘Tommy Atkins’ cultures. Ethylene biosynthesis was stimulated by ACC in nonembryogenic cultures of both genotypes and in ‘Tommy Atkins’ embryogenic cultures, in which ethylene biosynthesis was ca 7 X greater than in ‘Tutegau’ embryogenic cultures. The greater sensitivity of ‘Tommy Atkins’ to the ethylene antagonist, AVG, may be due to its greater sensitivity to disturbance of ethylene biosynthesis and spermidine synthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seasonal profiles of sulphur, phosphorus, and potassium in Norway spruce wood

Seasonal profiles of sulphur, phosphorus, and potassium content in the wood of trees have been established for the first time. This became possible by using a novel laser ablation system coupled to HR-ICP-MS for measuring these elements in Norway spruce drill cores. This technique combines excellent spatial resolution with superior detection power, and makes it possible to measure low element concentrations even in relatively narrow annual rings. Despite its low quantity in wood, sulphur is an important macronutrient for plants and seems to display seasonal variations of its concentration, which correspond to actual theories of sulphur metabolism in plants. A similar seasonal pattern was also found for phosphorus, another crucial element in tree nutrition. This was unexpected, because it was previously assumed that the distribution of phosphorus remains constant throughout the year. Potassium, the third element measured, seems to be especially accumulated in the latewood. The profiles presented in this article suggest a seasonal variation, revealing some new aspects of Norway spruce (PICEA ABIES) metabolism.     

The rate of protein synthesis in needles of Norway spruce (Picea abies): Light stimulation, regulation through photophosphorylation, stress enhancement

The incorporation of ¹⁴C-leucine into the total-protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer-winter) followed the Michaelis-Menten algorithm, exhibiting marked differences with regard to light saturation (V_max) and the half-saturation constant (K_{5. 2}). The light saturation curves of ATP level (mg g^?1 fresh weight) and of leucine incorporation into protein (nmol mg^?1 h^?1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of non-cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum. Furmigation of 5 years old spruce trees for 3 months with SO₂ in combination with O₃ and/or NO₂ caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun-exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased ¹⁴C-leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress-physiological mechanisms, which could possibly explain this stimulation, are discussed.     

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo 14N and 15N NMR spectroscopy

In vivo ¹⁵N and ¹⁴N nuclear magnetic resonance spectroscopy was used to investigate the assimilation of nitrate and ammonium in seedlings of Norway spruce (Picea abies [L.] Karst.). The main objective was to study accumulation of free NH+₄ and examine to what extent the nitrogen source affects the composition of the free amino acid pools in roots, stems and needles. NH+₄ concentrations in plants growing in the presence of 0.5–50 mM ammonium were quantified using ¹⁴N NMR. The NH+₄ values in tissues ranged from 6 to 46 μmol (g fresh weight)^?1. with highest concentrations in roots and needles. The tissue NH+₄ peaked at 5.0 mM NH+₄ in the medium. and failed to increase when NH+₄ in the medium was increased to 50 mM, indicating metabolic control of the concentration of this cation in tissues. The ¹⁴N NMR spectra were used to estimate pH of the NH+₄ storage pools. Based on the pH sensitivity of the quintet of ¹⁴NH+₄ resonance, we suggest that the pH of the ammonium storage compartments in the roots and stems should be 3.7–3.8, and in needles 3.4–3.5, representing extremely low pH values of the tissue. ¹⁵N from nitrate or ammonium was first incorporated into the amide group of glutamine and then into α-amino groups, confirming that the glutamine synthetase/ glutamate synthase cycle is the major route of nitrogen assimilation into amino acids and thus plays a role in lowering the levels of NH+₄ in the cytoplasm. NH+₄ can also be assimilated in roots in plants growing in darkness. The main ¹⁵N-labelled amino acids were glutamine. arginine and alanine. Almost no ¹⁵N signals from needles were observed. Double labelling (δN + w, wN) of arginine is consistent with the operation of the ornithine cycle, and enrichment indicates that this cycle is a major sink of newly assimilated nitrogen. Nitrogen assimilation in roots in the presence of added methionine sulphoximine and glutamate indicated the catabolic action of glutamate dehydrogenase. The ¹⁵N NMR spectra of plants grown on ¹⁵N-urea showed a marked increase in the labelling of ammonium and glutamine. indicating high urease activity. Amino acids were also quantified using high pressure liquid chromatography. Arginine was found to be an important transport form of nitrogen in the stem.     

Regeneration of transgenic Picea glauca, P. Mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.     

Genotype dependent blue and red light inhibition of the proliferation of the embryogenic tissue of Norway spruce

Summary The influence of light quality on the proliferation of embryogenic tissue of three genotypes of Norway spruce (Picea abies [L.] Karst), with different capacities for mature somatic embryo production, was studied. The proliferating tissues were subjected to light from commercially available light sources: Philips TLD Warm White 36W/29, Philips TLD Blue 18W/18, Philips TLD Red 36W/15, Osram L Fluora 36W/77 and Sylvania Far Red 7080, for 18 h a day with the photon flux (PAR) at 30 μmol m⁻²s⁻¹. The effect of light quality on the growth of embryogenic tissue was strongly genotype dependent. In genotype 164-4 tissue proliferation was strongly inhibited by blue and red light. Genotype 86∶52 reacted in a similar way, but not as strongly as 164-4, whereas the tissue of genotype 186-3 was almost insensitive to light quality and grew fast in all light conditions.     

NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce ( Picea abies [L.] Karst.) by nitrogen dioxide (NO₂) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO₂ treatment. During the first 2 days with 100 µg NO₂ m⁻³, NRA reached a constant level and did not change during the following 4 days. At the same level of NO₂, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO₂ was provided. The NO₂ gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO₂ on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO₂ concentrations were above 20 µg m⁻³. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO₂ levels nor in the decreasing NRA of spruce needles with the distance from the highway.     

Aluminium accumulation in root cell walls coincides with inhibition of root growth but not with inhibition of magnesium uptake in Norway spruce

High levels of aluminium in the soil solution of forest soils cause stress to forest trees. Within the soil profile, pH and aluminium concentration in the soil solution vary considerably with soil depth. pH strongly influences the speciation of A1 in solution, and is a factor when considering toxicity of A1 to roots. Norway spruce ( Picea abies [L.] Karst.) seedlings were grown for 7 weeks in nutrient solutions at pH 3.2, 4.0 or 5.0 containing 0, 100 or 400 µ M A1. At the end of this period, seedling growth, the cation exchange capacity of the roots and the amount of exchangeable Ca and Mg in roots were determined. A1 concentrations in whole roots, root segments, and in needles were measured. Using X‐ray microanalysis, the concentrations of Al, Ca, Mg and P were determined in cortical cell walls. We wanted to test the hypotheses that (1) the amount of Al bound to cation exchange sites can be used as a marker for Al toxicity and (2) the Mg concentration of needles is controlled by the amount of Mg bound to cation exchange sites. Low pH reduced the inhibition of Al on root growth and shoot length. Both low pH and Al lowered the concentration of Ca and Mg in needles. Al concentrations in the roots decreased as the pH decreased. In the roots, Al displaced Mg and Ca from binding sites at the root cortical cell walls. A comparison of the effects of Al at the different pH values on root growth and Mg concentration in the needles, suggests that, at pH 5.0, an Al fraction in the apoplast inhibits root growth, but does not affect Mg uptake. This fraction of Al is not available for transport to the shoots. In contrast, Mg uptake is strongly affected by Al at pH 3.2, although only very low levels of Al were detected in the roots. Thus, Al accumulation in the apoplast is a positive marker for Al effects on root growth, but not Mg uptake. The Mg concentration of needles is not controlled by the amount of Mg bound to cation exchange sites.     

The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway Spruce)

Embryos of Picea abies at various developmental stages were cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (10^?5 M) and N⁶-benzyladenine (BA) (5×10^?6 M). The immature embryos gave rise to a highly friable and embryogenic callus which could be maintained by subculture and contained polarized and organized structures (somatic embryos) consisting of long highly vacuolated cells at one end (suspensor) and a group of small meristematic cells at the other (embryonal end). These structures closely resembled the early stages of normal zygotic embryogeny. Upon further culture these structures formed a bipolar shoot-root axis with an independent and closed vascular system. In many instances either the shoot or the root meristems failed to differentiate. Embryogenic tissues obtained on agar media could be transferred to liquid media and maintained by subculture for at least 6 months. The development of somatic embryos was observed in the liquid cultures also.