An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.     

Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

Regulation of starch and protein accumulation in alfalfa (Medicago sativa L.) somatic embryos

Compared to seeds, somatic embryos accumulated relatively low levels and different types of storage carbohydrates. The regulation of starch accumulation was studied to determine its effects on desiccation tolerance and vigor of dry somatic embryos. Somatic embryos of Medicago sativa are routinely matured through three phases: 7 days of development; 10 days of phase I maturation, a rapid growth phase; and 10 days of phase II maturation, a phase leading to the acquisition of desiccation tolerance. The control of starch deposition was investigated in alfalfa somatic embryos by manipulating the composition of the phase I maturation medium with different levels of sucrose, abscisic acid, glutamine and different types of carbohydrates and amino acids. After phase II maturation, mature somatic embryos were collected for desiccation and subsequent conversion, or for biochemical analyses. Starch deposition occurred primarily during phase I maturation, and variations in the composition of this medium influenced embryo quality, storage protein and starch accumulation. A factorial experiment with two levels of glutamine × three levels of sucrose showed that increasing the sucrose concentration from 30 to 80 g/l increased embryo size and starch content, but had minimal effect on accumulation of storage proteins; glutamine also increased embryo size, but decreased starch content and increased accumulation of the high salt soluble S-2 (medicagin) storage proteins. ABA did not influence any of the parameters tested when included in phase I maturation at concentration up to 10 μM. Replicating sucrose with maltose, glucose, or glucose and fructose did not alter embryo size or starch accumulation (mg/g fresh weight), but replacement with fructose alone reduced embryo size, and replacement with glucose alone reduced germination. Suplementation with the amino acids, asparagine, aspartic acid and glutamine increased seedling vigor, but decreased the starch content of embryos. The data indicate that starch accumulation in somatic embryos is regulated by the relative availability of carbon versus nitrogen nutrients in the maturation medium. The quality of mature somatic embryos, determined by the rate of seedling development (conversion and vigor), correlated with embryo size, storage protein and free amino acid but not with starch. Therefore, further improvements in the quality of somatic embryo may be achieved through manipulation of the maturation medium in order to increase storage protein, but not starch deposition.     

In vitro culture of embryos of Cucumis spp.: heart-stage embryos have a higher ability of direct plant formation than advanced-stage embryos

Summary The ability of embryos at different developmental stages to form plants in vitro has been studied in cultivated Cucumis sativus L. and in the wild species C. zeyheri 2 x Sond. and C. metuliferus Naud. On MS medium containing 3.5% sucrose, 0.1 mg 1^–1 kinetin (Kn) and 0.01 mg 1^–1 indoleacetic acid (IAA), proembryos (0.03–0.05 mm) and early globular embryos (0.05–0.08 mm) from the wild species developed into plants in low frequencies of 8% and 21%, respectively. These embryos should be surrounded by the embryo sac tissue. On the same medium late globular (0.08–0.1 mm) and early heart-stage embryos (0.1–0.3 mm) developed into plants in moderately high and high frequencies of 48% and 83%, respectively. The presence of the embryo sac at these stages was still beneficial, but no longer a prerequisite. Late heart-stage embryos (0.3–0.8 mm) also showed high frequencies of plant formation, 63%, if Kn was applied at a concentration of 1 mg 1^–1. From the early cotyledon stage onwards, the frequency of plant formation gradually decreased, reaching a minimum at the late cotyledon stage. Subsequently it began to increase again up to the late maturation stage. The poor plant formation shown by the intermediate-aged embryos could be improved slightly by lowering the sucrose concentration to 0.5% and by increasing the Kn concentration to 10 mg 1^–1. Relative to the wild species, embryos of C. sativus showed lower percentages of plant formation. The optimum sucrose concentration was 2% for the heart-stage C. sativus embryos. In all three species the ability to form plants strongly decreased with increasing embryo age, from early to late cotyledon. This is thought to be caused by the increasing tendency of the embryos at these stages to continue in vitro the normal embryo development.     

Manipulating the desiccation tolerance and vigor of dry somatic embryos of Medicago sativa L. with sucrose,heat shock and abscisic acid

Summary The effects of sucrose concentration in the maturation medium in combination with a heat shock treatment at 36°C were investigated in an attempt to improve the vigor of seedlings grown from dry somatic embryos of alfalfa (Medicago sativa L.). Callus was formed from petiole expiants and dispersed in liquid suspension medium in the presence of 5 M 2,4-D. The cell suspension was sieved to synchronize embryo development. The 200 – 500 m fraction was plated on embryo development medium without 2,4-D, grown for 14 days, and transferred to maturation medium. With 3% sucrose in the maturation medium, the somatic embryos germinated precociously and were unable to survive desiccation. At higher sucrose concentrations, germination was delayed and the embryos continued to accumulate dry matter. After 13 days on 6% sucrose medium (27 days after sieving), the somatic embryos were tolerant of drying to 12% moisture without exposure to exogenous ABA. Heat shock, which presumably stimulates endogenous ABA synthesis, improved the desiccation tolerance of somatic embryos if applied prior to day 27 after sieving, but its effects were minimal after day 27. High sucrose concentrations up to 9% in the maturation medium were optimal during the first 8 days on maturation medium (days 14 to 22 after sieving), but a lower concentration (6%) was optimal during the later stages of embryo maturation (days 22 to 30 after sieving). The inclusion of 10^–5 M ABA in the maturation medium with 6% sucrose further improved embryo quality if applied approximately 20 days after sieving.     

From callus to embryo: a proteomic view on the development and maturation of somatic embryos in <Emphasis Type="Italic">Cyclamen persicum</Emphasis>

In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

Control of hyperhydricity of mango somatic embryos

Hyperhydricity of immature somatic embryos has been a limiting factor for the development of highly embryogenic suspension cultures of many important mango cultivars. Reversion of hyperhydricity was achieved in two ways: 1) heart-stage somatic embryos (2–3 mm length) were partially dehydrated under controlled conditions at high relative humidity (RH) for 24–48 h and 2) the gelling agent (Gel-Gro) concentration of the plant growth medium was increased from 2.0 to 6.0 g l^-1. Partially dehydrated immature somatic embryos were normal in appearance. Somatic embryos that were partially dehydrated germinated precociously when cultured on maturation medium. Although abscisic acid (ABA) did not reverse hyperhydricity of primary somatic embryos, ABA did stimulate the reversal of this abnormal pattern of development among secondary embryos. ABA (500 M) inhibited precocious germination and permitted somatic embryo maturation. Partially dehydrated, immature somatic embryos (4–7 mm long) remained viable for up to 32 days in the absence of maturation medium under high RH.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - BA 6-benzyladenine - RH relative humidity     

Development of haploid asparagus embryos from liquid cultures of anther-derived calli is enhanced by ancymidol

Summary The effect of ancymidol concentration on the development of haploid asparagus embryos was determined. Liquid cultures from anther-derived calli were grown for three weeks in MS medium plus 1.0 mg l^–1 2,4-D, 0.1 mg l^–1 NAA, 0.2 mg l^–1 kinetin, 800 mg l^–1 glutamine, 500 mg l^–1 casein hydrolysate, 2% sucrose and 0.0–1.0 mg l^–1 ancymidol. Cell clumps (224–500 m) were plated on solid embryo maturation medium (MS medium plus 3% sucrose, 0.1 mg l^–1 NAA, 0.5 mg l^–1 kinetin and 0.0–1.0 mg l^–1 ancymidol) and grown for eight weeks. Ancymidol enhanced embryo maturation and germination and was more critical in the solid than liquid medium. Total embryo number did not vary among most treatments. The best response was observed when ancymidol concentrations were 0.1 and 0.5 mg l^–1 in the liquid and solid media, respectively; two-thirds of the embryos produced were bipolar and 35% of bipolar embryos germinated. Seven to 82% of plants recovered from different ancymidol treatments were haploid; the others were diploid, triploid or chimeric for ploidy level.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962)     

Response of Abies alba embryonal-suspensor mass to various carbohydrate treatments

Cultures of embryonal-suspensor mass of European silver fir (Abies alba) were screened for their response to various sucrose concentrations (29–175 mM) and to equimolar (100 or 200 mM) concentrations of glucose, fructose, galactose, sucrose, lactose and soluble starch. The higher concentrations were combined with 3.78 M abscisic acid. While fructose was ineffective in promoting any somatic embryo maturation glucose, sucrose and galactose promoted early maturation stages. Somatic embryo development up to the torpedo stage was observed only on media with soluble starch or lactose. Lactose was superior to other carbohydrate treatments and led to more synchronized maturation and more normally structured embryos. In comparison with the pronounced carbohydrate effect abscisic acid at this concentration proved to be of less importance. A sequence of culture steps was established in order to stimulate differentiation and maturation.Abbreviations ABA (±) 2-cis-4-trans-abscisic acid - BAP N6-benzylaminopurine - ESM embryonal-suspensor mass - L.S.D. Least Significant Difference - SE somatic embryo(s) - SH Schenk and Hildebrandt     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

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     

Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure

The prophenoloxidase (proPO) system catalyzing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true PO activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 μg L⁻¹) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new “juvenile” form of the PO enzyme. In similar conditions as for Ag, Cu (230 μg L⁻¹) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.     

Evaluation of somatic embryos of interior spruce. Characterization and developmental regulation of storage proteins

Storage proteins of interior spruce ( Picea glauca engelmanii complex) somatic embryos were compared to those of zygotic embryos by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Somatic embryos contain the same storage proteins as zygotic embryos based on similarities of molecular weight, isoelectric variants, solubility characteristics and disulfide linkages. Storage protein levels varied among different somatic embryo genotypes; however, all genotypes tested accumulated significant amounts of storage proteins. Zygotic and somatic embryos display a similar developmental accumulation of storage proteins. The 22, 24, 33 and 35 kDa proteins appear in early stage embryos, while the 41 kDa protein begins to accumulate during mid cotyledon development. The 22, 24 and 41 kDa proteins accumulate continuously during cotyledon development in somatic embryos cultured on abscisic acid. In contrast, zygotic embryos display a more rapid and transient accumulation of these proteins.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

Comparison between somatic and zygotic embryo development in Quercus robur L.

ABSTRACT

Somatic embryogenesis from juvenile explants as an efficient way for oak clonal propagation is drastically limited by the low rate of embryo germination. A comparison of the development of immature somatic and zygotic embryos, and a study of the changes in sugar content and lignin accumulation during somatic versus zygotic embryo development were conducted in view of understanding the effect of reserve substance deficiency upon somatic embryo maturation. A morphological comparison of somatic and zygotic embryos led to the identification of 4 to 7 similar developmental stages in both types of embryos, thus indicating that the accumulation phase in both zygotic and somatic embryos occurs at the same stage, when the cotyledons became thicker and opaque. Carbohydrate analysis showed the presence of glycerol, inositol, mannitol, galactose, trehalose, xylose, arabinose, glucose, fructose and sucrose in all stages of zygotic and somatic embryo development, but in different amounts. The amount of glycerol, inositol, glucose and sucrose during the early stages is larger in zygotic embryos than in somatic ones, but the time course of their accumulation is similar in both types of embryos. Lignin content, which increased continuously during development, showed a similar behaviour in zygotic and somatic embryos. In somatic embryos which were able to germinate, lignin content was higher than in nongerminating embryos at the same stage.     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.     

Somatic embryogenesis and plant regeneration in Gymnema sylvestre

Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from hypocotyl, cotyledon and leaf explants excised from seedlings of Gymnema sylvestre. Embryogenic callus was induced on Murashige and Skoog (MS) medium containing 2,4-D (0.5–5.0 M) +BA (0.5–2.0 M) and 2% (w/v) sucrose in 6–8 weeks of culture. Globular/heart stage embryos developed on induction medium. These embryos produced torpedo and cotyledon stage embryos upon sub-culturing on embryo maturation medium EM8 (medium containing MS salts, B5 vitamins, 0.5 M BA and 2% sucrose). Embryo germination and plantlet formation was achieved by sub-culturing mature embryos on fresh EM8 medium. The plantlets were acclimatized in the greenhouse.     

Comparing carbohydrate status during norway spruce seed development and somatic embryo formation

Summary The carbohydrate status of developing seeds of Picea abies was examined in order to provide a frame of reference for the evaluation of changes in carbohydrate content in maturing somatic embryos of the same species. Samples were taken at weekly intervals from 12 May 1998 (estimated time of pollination) until 20 October 1998. The total non-structural carbohydrate content was high (≈150–180 μg mg⁻¹ dry weight) at the time of the first samples and the carbohydrate spectrum consisted of sucrose, glucose, fructose, and pinitol. A dramatic decrease in carbohydrate content took place from June 6 onwards, that was accompanied by changes in carbohydrate partitioning to favor sucrose over hexoses and the disappearance of pinitol. Raffinose and stachyose were first detected on July 28, and their content gradually increased thereafter. Isolated embryos and remaining megagametophytes were analyzed starting with September 1. Carbohydrate content was higher in isolated zygotic embryo than in the rest of the seed, with a slowly increasing fraction of raffinose and stachyose. Comparisons of presented data with the results of our previous study of somatic embryo carbohydrate status (Lipavská et al., 2000) revealed the following common features: (1) a decrease in total carbohydrate content and (2) an increase in sucrose:hexose ratios in developing seeds and embryonal suspensor mass. Marked differences were observed in carbohydrate spectra: (1) somatic embryo development was not accompanied by pinitol accumulation in any phase; (2) mature zygotic embryos, in contrast to mature somatic embryos, contained raffinose and stachyose. These observations will provide a solid basis for improvement of protocols for somatic embryogenesis in Picea.