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.     

Development and germination of Abies alba somatic embryos

Mature zygotic embryos of Abies alba mull were placed on a modified MCM medium (basal medium-BM) with 2.2 M benzyladenine and 2.3 M kinetin to induce embryogenic suspensor masses (ESM). These ESM proliferated on induction medium supplemented with 0.2 M 2,4-dichlorophenoxyacetic acid. From 61 ESM lines induced, 36 are still in culture after 2 years, of which 18 show embryogenic potential indicated by spontaneous formation of globular somatic embryos on the proliferation medium supplemented with 500–1000 mg l^-1 casein hydrolysate and 500 mg l^-1 l-glutamine. ESMs from cell line 2/56 were conditioned 1 week on BM with 58 mM sucrose and 10 g l^-1 activated charcoal for maturation of somatic embryos. Maturation was achieved on BM containing 20 M (±)cis-trans-abscisic acid in combination with 111 mM maltose. Organic nitrogen supplements improved the proliferation rate of cell line 2/56 as well as the maturation and vitality of the somatic embryos. Partial drying was necessary for subsequent root development. Plantlets with a root, primary needles and a terminal bud developed on BM when a combination of 30 mM sucrose and 50 mM maltose was provided as carbon source.Abbreviations BM basal medium - BA benzyladenine - ESM embryogenic suspensor mass - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - l-gln l-glutamine - ABA (±) cis-trans-abscisic acid     

Some features of somatic embryo maturation of algerian fir

Summary Maturation of Abies numidica De Lann. somatic embryos was tested on media with abseisic acid and various maltose and polyethylene glycol-4000 concentrations. The effect of basal medium and subculture period on maturation was also examined. The maturation of somatic embryos was promoted by polyethylene glycol-4000, at 7.5–10%. Three to 6% maltose significantly enhanced the yield of mature embryos. The most effective somatic embryo maturation occurred when embryogenictissue was transferred to maturation medium after 14–21 d cultivation on proliferation medium. The ability for A. numidica cultures to form cotyledonary somatic embryos was assessed over a period of 3 yr. Plantlets germinated on half-strength SH (Schenk and Hildebrandt) medium with charcoal and survived transfer to the soil.     

Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk.

Summary Abies nordmanniana Lk. is used in short intensive rotations for Christmas tree production. Thus there is a high demand for development of advanced propagation and breeding methods. Somatic embryogenesis was easily induced from immature (precotyledonary) embryos collected in July 1989 with cytokinin as the sole plant growth regulator. The proliferating embryogenic cell masses were characteristic of conifer somatic embryogenesis and could be maintained on a simple basal medium containing 5 M benzylaminopurine. Auxin inhibited induction as well as proliferation. Proliferation was improved by up to 30 % by addition of L-glutamine and/or casein hydrolysate. Neither cytokinin concentration nor culture on 3 different basal media, differing markedly in their nitrogen composition, affected the proliferation rate. Embryos matured using a 4 week subculture on medium containing 10 M abscisic acid and subsequent transfer to medium devoid of plant growth regulators.Abbreviations TDZ thidiazuron (Schering) - BAP benzylaminopurine (Sigma) - KIN kinetin (Sigma) - 2,4-D 2,4-dichlorophenoxyacetic acid (Sigma) - ABA +/2-cis-4-trans-abscisic acid (Sigma) - CH casein hydrolysate (Sigma Type 1, acidic) - L-gln L-glutamine (Sigma) - EDTA ethylene diamine tetra acetic acid (Sigma)     

Somatic embryogenesis from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Maturation of black spruce somatic embryos. Part I. Effect of l-glutamine on the number and germinability of somatic embryos

Different concentrations of l-glutamine and different nitrogen sources in the medium were compared during maturation of black spruce (Picea mariana (Mill.) B.S.P.) somatic embryos. l-glutamine can be used as the sole nitrogen source for the maturation of Picea mariana somatic embryos at 2 to 3 gl^-1. A significantly lower number of somatic embryos was obtained on a medium prepared with only inorganic nitrogen. Compared with a medium supplement to inorganic nitrogen resulted in a twofold increase in the number of embryos for six genotypes. The nitrogen source and concentration in the maturation medium significantly affected the germination sensus stricto of somatic embryos (radicle appearance), but not their development into plantlets; at the time of epicotyl appearance, an effect of the nitrogen source was no longer found. A comparison of the development of somatic embryos into plantlets from seven genotypes showed that the genotype had more effect in terms of epicotyl appearance and in conversion rate than the nitrogen source present in the maturation medium.Abbreviations HLM-1 half-Litvays's medium with 10 M 2,4-D and 5 M BA - i only inorganic nitrogen in the medium - i+1 gG inorganic nitrogen plus 1 g l^-1 glutamine in the medium - SMM standard maturation medium - 2.5gG only 2.5 g l^-1 glutamine in the medium     

Maturation and germination of somatic embryos as affected by sucrose and plant growth regulators in soybeans Glycine gracilis Skvortz and Glycine max (L.) Merr.

The effects of sucrose on maturation and of plant growth regulators on germination of soybean somatic embryos were investigated for the purpose of developing an efficient culture method for plant recovery. Somatic embryos produced on medium with a low sucrose concentration (5 gl^-1), less than 1 mm in length, 0.6 mg in fresh weight, and green in color, were grown for 2 weeks on MS medium containing 5 gl^-1 or 30 gl^-1 sucrose and then for another 5 weeks on MS medium containing 5–90 gl^-1 sucrose. The highest increase in fresh weight of somatic embryos was obtained in the treatment of transferring from 30 gl^-1 sucrose (2 weeks) to 60 gl^-1 (5 weeks). With the increase in fresh weight, the somatic embryos gradually changed color from green to yellow, and finally to white, when they stopped growth. Soybean seed storage proteins (-conglycinin and glycinin) were accumulated in somatic embryos under tissue specific and stage specific control analogous to that in zygotic embryos. Exogenous gibberellic acid was effective in promoting precocious germination of premature soybean somatic embryos, but was not necessary for the germination of mature somatic embryos. The efficiency of somatic embryo germination was as high as 77% from semi-wild soybean and 60–64% from cultivated soybeans, showing that the plant regeneration system developed in this study was efficient and practical.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BR brassinolide - GA₃ gibberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS Sodium Lauryl Sulfate     

Peroxidase activity in non-embryogenic and embryogenic calli and in developing somatic embryos of white fir (Abies concolor Gord. et Glend)

ABSTRACT

Peroxidase activity was monitored during somatic embryogenesis of white fir (Abies concolor Gord. et Glend) starting from a non-embryogenic callus. Results revealed profound differences between non-embryogenic and embryogenic calli with an elevated level of enzyme activity in non-embryogenic ones. Precotyledonary, early cotyledonary and late cotyledonary stages of somatic embryogenesis were characterized by a substantially reduced peroxidase activity compared to callus tissues and regenerated plantlets. Changes in peroxidase activity are as a rule paralleled by variation in isoenzyme composition. The utility of the enzyme in the induction stage of somatic embryogenesis in white fir is proposed.     

Improvement of somatic embryogenesis and plant recovery in cassava

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA Benzyl aminopurine - GA Giberellic acid - MS Murashige and Skoog - NAA Naphthalene acetic acid     

High frequency plant regeneration from Aralia cordata somatic embryos

An efficient method of plant regeneration from Aralia cordatasomatic embryos was developed. Somatic embryos at early stages obtained through inflorescences–derived embryogenic cell suspension cultures were matured in liquid Murashige and Skoog (MS) medium containing various concentrations of abscisic acid (ABA). For plant regeneration, mature cotyledonary embryos were transferred to solid MS basal medium for 6 weeks. Plant regeneration frequency of the embryos matured from heart-shaped embryos was proportional to the concentration of ABA from 0.76 to 3.8 M. The highest frequency (60.7%) was obtained from 3.8 M ABA pretreatment. The survival rate of the plantlets after transfer to plastic pots containing vermiculite in the growth room was 90%. All plants transferred to soil in greenhouse survived. The results indicate that micropropagation procedure can be applied for an efficient mass propagation of Aralia cordata.     

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.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantitative analysis of abscisic acid in needles of Abies alba Mill. by electron capture gas chromatography

Summary The amount of abscisic acid (ABA) in needles of silver fir from a natural location was investigated with regard to position in the crown, damage, seasonal variation, and needle age. Because of problems of quantification of ABA in coniferous needles, which contain numerous secondary plant products, a method for reliable determination of both isomers cis-trans-ABA (c-ABA) and transtrans-ABA (t-ABA) was developed. By means of gas chromatography (GC) using an electron capture detector (BCD) and a programmed temperature vaporizer (PTV) injector complete separation of both compounds was achieved. Two different pairs of fir were investigated — in each case a damaged and a healthy tree. Needles from both trees from the first and the second pair collected in September contained 500–1100 ng c-ABA/g fresh weight (FW), and the concentrations of t-ABA varied from 400 to 700 ng/g FW. Investigations from the second pair show highest amounts of 2900 ng/g Fw c-ABA and 1800 ng/g FW of t-ABA in May and June. For the first pair a higher c-ABA content was found in needles from the top of the crown than in those from the middle and the base. This difference could not be confirmed in the analysis of the second pair. Because of the strong natural deviation no statistically significant difference between the healthy and the damaged tree was found. The first pair of firs examined showed a higher t-ABA concentration than the second one. In this case the highest amount was found in the top of the crown. Methodical mistakes during the clean-up procedure and in quantification by gas chromatography could be excluded. The presence of c- and t-ABA in the purified extract was corroborated by mass spectrometry. With regard to the seasonal variation both isomers of ABA show an unequivocal trend. The maximum concentration is achieved in May to June, whereas the content is minimal in August/September. In any case the level of t-ABA is lower than that of c-ABA. No correlation between the amount of ABA and the needle age could be established.     

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

Storage protein dynamics in zygotic and somatic embryos of white fir

Composition and accumulation patterns of storage proteins in female gametophyte and embryos of the white fir (Abies concolor) were investigated during embryogenesis and germination of mature seeds using SDS-PAGE and immunological approach. Altogether 9 major and minor protein components with molecular masses of 14, 16, 22, 24, 27, 30, 35, 38, and 43 kDa were detected in female gametophytes and 9 protein bands in the embryos with the molecular sizes of 14, 16, 22, 24, 25, 27, 34, 38, and 43 kDa. The species seems to deviate in this respect from other representatives of Pinaceae. A conspicuous increase of storage protein synthesis was observed at the stage of fully cellularized female gametophytes and at the cotyledonary stage of embryo development. There exists a high degree of similarity between storage protein profiles of white fir zygotic and somatic embryos. Successive stages of somatic embryogenesis exhibited a high degree of similarity of storage proteins except for cotyledonary stage when a noticeable increase in storage protein synthesis was registered. Conversely, during germination of somatic embryos, an overwhelming majority of storage proteins was depleted.     

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.     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Effect of plant growth regulators on the cellular growth and levels of intracellular protein,starch and polyamines in embryogenic suspension cultures of Pinus taeda

Somatic embryogenesis is the most important in vitro culture system for conifer propagation. However, Pinus taeda has been considered recalcitrant to somatic embryogenesis in commercial scale-up. The study of biochemical and physiological aspects of cell growth could lead to a better understanding of somatic embryogenesis in this species. In the present work, we investigated the cell growth dynamics, intracellular levels of proteins, starch and polyamines in suspension cultures of Pinus taeda established in plant growth regulator-free medium (BM0) and in medium supplemented with 2 M 2,4-dichlorophenoxyacetic acid, 0.5 M 6-benzylaminopurine and 0.5 M Kinetin (BM2). Cell cultures growing in BM0 medium showed an increase in the sedimented cell volume from 3.77 to 17.73 ml after 24 days of culture. Those cultured in BM2 medium showed an increase in the sedimented cell volume from 4.23 to 25.17 ml after 20 days of culture. Intracellular proteins levels increased during the exponential growth phase and starch levels decreased until the exponential phase, followed by a synthesis up to the stationary phase, in both BM0 and BM2 media. Highest putrescine levels occurred in cultures growing in BM0 medium and this was associated with the low cellular growth.