The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.     

Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos

Different carbohydrates were investigated for somatic embryo development of black spruce and red spruce. They were tested in a basal maturation medium consisting of Litvay's salts at half-strength containing 1 g l^-1 glutamine, 1 g l^-1 casein hydrolysate, 7.5 M abscisic acid, and 0.9% Difco Bacto-agar. A comparison of different sucrose concentrations showed that 6% was optimal for embryo development. Among the nine carbohydrates tested, sucrose, fructose, glucose, maltose, and cellobiose supported embryo development while arabinose, mannitol, myo-inositol, and sorbitol did not. A comparison of sucrose, glucose, and fructose at three concentrations showed that the general pattern of response for both species followed concentration expressed as a percentage, independent of the molarity of carbohydrate in the medium. Interspecific differences were observed concerning carbohydrate requirements. For red spruce, 6% fructose was found best for embryo development, while no such preference was observed for black spruce. No significant difference was observed in the number of embryos produced with 6% sucrose or 3% sucrose plus an equimolar concentration of either mannitol, sorbitol, or myo-inositol in the maturation medium, suggesting that the effect of the carbohydrate on the maturation was partly osmotic.     

Carbohydrate status during somatic embryo maturation in Norway spruce

Summary The development of Norway spruce (Picea abies (L.) Karst.) somatic embryos on a maturation medium was accompanied by changes in nonstructural carbohydrate status. During embryo maturation, the content of total soluble sugars in the embryonal suspensor mass decreased and the partitioning between sucrose and hexoses changed considerably in favor of sucrose. Developing somatic embryos were mainly responsible for these changes. Osmotic stress caused by the presence of 3.75% polyethylene glycol (PEG) in the maturation medium (decrease in osmotic potential by 52.5 kPa) resulted in dramatic changes in the content of endogenous saccharides. There was a lower total carbohydrate content in the embryonal suspensor mass grown on the medium containing PEG in comparison with the untreated control. Isolated embryos from later stages of embryo development contained mainly sucrose with a small amount (20%) of fructose and nearly no glucose. A further increase in PEG concentration in the medium (7.5%; decrease in osmotic potential by 112.5 kPa compared to the maturation medium) led to a large increase in the total endogenous sugar content. This increase in sugars was a result of the enhanced content of sucrose, fructose, and glucose. The increased glucose content was in contrast to embryos grown on the medium with lower or no PEG content.     

Cowpea embryo rescue. 1. Influence of culture media composition on plant recovery from isolated immature embryos

Factors responsible for successful rescue of immature embryos of cowpea [Vigna unguiculata (L.) Walp.] and V. vexillata (L.) and for in vitro embryo development were studied. A new basal medium for embryo development in vitro was formulated on the basis of the mineral composition of embryos. Sucrose, fructose and glucose were compared as carbohydrate sources. The highest frequency of embryos developing into plants was obtained with sucrose. Adding casein hydrolysate to the medium increased plant recovery by 30%. Among the plant growth factors used, cytokinins, zeatin, 6-benzylaminopurine and kinetin were the most effective in promoting embryo maturation and development. A method that can routinely ensure high plant recovery from cultured immature cowpea embryos is proposed. Received: 4 June 1996 / Revision received: 28 October 1996 / Accepted: 22 November 1996     

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.     

THE PHYSICAL AND CHEMICAL ENVIRONMENT OF THE DEVELOPING EMBRYO OF PINUS RESINOSA

The relationship between changes in soluble protein, hexose sugar, total lipid concentration, and osmotic potential occurring in gametophytic supernatant of Pinus resinosa Ait. during in vivo embryogenesis was measured. The effects of varying sucrose levels of culture medium on in vitro embryo and gametophyte development were examined. Increases in embryo volume, and fresh and dry weight of the female gametophyte during in vivo embryogenesis coincide with increasing levels of soluble protein, hexose sugar, and total lipid in the gametophytic supernatant. In contrast, osmotic potential of the supernatant increased only slightly between the zygote and proembryo stages of embryo development, and remained constant thereafter. Gametophytes plus embryos grown in vitro achieved dry weights approaching those of in ovulo gametophytes on media containing levels of sucrose up to 21%. Gametophytes on media with sucrose concentrations up to 21% also resembled normal in ovulo gametophytes in appearance. However, embryo development appeared to be suspended on treatment media containing from 9% to 21% sucrose, while embryos degenerated on media with constant sucrose levels of 3% and 6%. A treatment medium containing approximately 12% sucrose would provide an osmotic environment that duplicates that found in ovulo. While greater sucrose levels promoted more normal gametophyte development in Pinus resinosa, we failed to achieve complete development of the embryo in vitro. Conclusions and implications drawn from these results are discussed.     

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.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

The effect of sugars on niger embryogenesis and plant regeneration in anther culture

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05–0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg’s B5 medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 μM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 μM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 μM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.     

Optimization of horse chestnut (<Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.) somatic embryo conversion

Factors affecting conversion of horse chestnut (A. hippocastanum L.) somatic embryos into plantlets were evaluated. Anther filament derived embryogenic tissue developed bipolar structures with two cotyledons and a well-developed shoot and root apical meristem upon auxin omittance from the culturing medium. The impact of carbohydrate type (glucose, fructose, sucrose and maltose) and concentration (3 and 6%) on somatic embryo maturation and conversion were evaluated. Although conversion frequencies were high for all treatments, overall quality of regenerated plantlets was poor. Increasing the carbohydrate concentration in the maturation medium did not increase conversion of somatic embryos or quality of regenerated plantlets in terms of shoot height. On the contrary, addition of PEG (polyethylene glycol) in maturation media had a beneficial effect on shoot quality of regenerated plantlets. Sucrose was a superior carbon source when PEG was included in the maturation medium, in terms of conversion rate (65.7%) as well as of shoot quality of plantlets (43.8% of plantlets had shoots >2 cm). Clonal fidelity of the different development stages of somatic embryogenesis and of converted plantlets was assessed by flow cytometry and no major ploidy changes were found.     

Effect of abscisic acid, osmolarity and temperature onin vitro development of recalcitrant mango nucellar embryos

Development of cotyledonary-stage nucellar embryos of mango was arrestedin vitro by exposure to 750–1750 M ABA. The enlargement and germination of nucellar embryos was inhibited for as long as 4 weeks after subculture from ABA-containing medium. Mannitol at concentrations between 7.5 and 12.5% inhibited nucellar embryo development, presumably due to osmotic effects; however, there was no residual effect after subculture of somatic embryos onto medium without mannitol. Temperatures between 22.5 and 37.5°C stimulated embryo development, whereas lower temperatures (7.5 and 15°C) delayed germination. There was no germination 1 month after somatic embryos, pulsed for 8 weeks at 7.5°C, were transferred to 22.5°C; however, after 2 months, 86% of these somatic embryos germinated. These results indicate that it is possible to induce developmental arrest in recalcitrant mango embryos with high concentrations of ABA, mannitol or low temperature (7.5°C).Abbreviations ABA Abscisic acid - MM1 Mango maturation medium     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Effect of abscisic acid and stratification on somatic embryo maturation and germination of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.)

Summary The effect of abscisic acid (ABA) was evaluated during the maturation and germination of holm oak (Quercus ilex L.) somatic embryos. The addition of ABA to the culture medium significantly reduced unwanted recurrent embryogenesis in mature somatic embryos without affecting the germination of embryos subjected to stratification at 4°C. Stratification at 4°C for 2 mo. was the most efficient for stimulating somatic embryo germination of holm oak. The addition of 90 and 450 mM sucrose also improved germination, while higher sucrose concentrations were inhibitory.     

Nutrient uptake and growth of an embryogenic and a non-embryogenic cell line of birch (Betula pendula Roth.) in suspension culture

The nutrient uptake of an embryogenic and of a non-embryogenic cell line of birch (Betula pendula Roth.) during cell growth and embryo production was studied in suspension culture. The embryogenic and non-embryogenic cell suspensions grew differently in the same medium. The non-embryogenic cell line started to grow without any lag period after the inoculation. It rapidly hydrolyzed sucrose in the medium to glucose and fructose and consumed the glucose as carbon source. The concentration of fructose in the medium decreased only after the depletion of glucose. The embryogenic cell line also rapidly hydrolyzed the sucrose to glucose and fructose, but the monosaccharides were consumed only after the embryos started to germinate after three weeks of culture. Both monosaccharides were then taken up at the same rate.     

Effect of exogenous ABA on somatic embryo maturation and germination in Persian walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of embryo maturation, germination and conversion to plantlets is a major problem in many species including Persian walnut. We studied the effects of abscisic acid (ABA) and sucrose, on the maturation and germination of Persian walnut (Juglans regia) somatic embryos. Individual globular somatic embryos were grown on a maturation medium supplemented with different combinations of ABA and sucrose for ca. 1 month, until shoot meristems and radicles had developed. White and opaque embryos in late cotyledonary stage were subjected to desiccation after the culture period on maturation media. The number of germinated somatic embryos was influenced by the concentrations of ABA in the maturation medium. The best treatment for germination, in which both shoot and root were developed contained 2 mg l⁻¹ ABA and resulted in 41% conversion of embryos into plantlets. Regeneration was reduced at higher levels of ABA. While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos. On the other hand, sucrose had little influence on maturation. Normal and abnormal embryos were verified anatomically.     

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.     

Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses

The effect of various abiotic stresses on germination rate, growth and soluble sugar content in Sorghum bicolor (L.) Moench cv. CSH 6 seed embryos and endosperm during early germination was investigated. Under stress conditions germination, water potential and tissue water content decreased markedly. Subsequently, this reduction resulted in marked decreases in fresh weight both in embryos and endosperm. Conversely, a substantial increase in dry weight was observed. Furthermore, a considerable increase in the sugar contents in both embryo and endosperm was detected. The fructose level was always higher than glucose and sucrose in response to various stresses. However, as compared to the control the level of glucose and sucrose was higher in embryos and endosperm after stress treatments. Based upon these results a possible physiological role of sugars in the germination of sorghum seeds is discussed.     

Analysis of the effects of maturation treatments on the probabilities of somatic embryo germination and plantlet regeneration in pistachio using a linear logistic method

Embryogenic masses (EMSes) of pistachio (Pistacia vera L.) were proliferated in liquid Murashige and Skoog (MS) medium without growth regulators. To determine the effects of benzylaminopurine (BAP), racemic (±) abscisic acid (ABA) and sucrose treatments during maturation on the subsequent germination and plantlet regeneration, clusters of mature somatic embryos were transferred from maturation medium onto the surface of 0.7% agar-solidified Murashige and Skoog medium. Neither germination nor plantlet development medium contained BAP or ABA. Germination studies were carried out using 80 somatic embryos at every combination of four sucrose concentrations, three maturation periods and either five concentrations of BAP or four of ABA, and the numbers germinating were recorded after four durations of culture. A similar experimental plan was used to study plantlet regeneration. The number of germinated somatic embryos increased markedly with duration of the culture on germination medium, and was influenced by the concentrations of BAP or ABA in the maturation medium; the concentration of sucrose in this medium had little influence. Plantlet regeneration also increased with culture duration and was reduced at the highest levels of BAP or ABA; with ABA, the probability of plantlet regeneration was lower for longer maturation periods. ABA and BAP have similar effects on somatic embryo germination (except at the highest levels used), but BAP is superior to ABA for promoting subsequent plantlet regeneration. Linear logistic models were used to investigate the significance of the treatments, and to estimate the optimum conditions for germination and plantlet regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

Dormancy induction of somatic embryos of Siberian ginseng by high sucrose concentrations enhances the conservation of hydrated artificial seeds and dehydration resistance

. In most plants, somatic embryos tend to germinate prematurely, a process that is detrimental to controlled plant production and the conservation of artificial seeds. We investigated the dormancy characteristics of Siberian ginseng somatic embryos induced simply by a high sucrose treatment, a treatment that enables the long-term conservation of artificial seeds following encapsulation and provides embryos with an enhanced resistance to dehydration. Early-cotyledonary stage somatic embryos were mass-produced by means of bioreactor culture. These embryos were then plated on medium supplemented with various levels of sucrose (1%, 3%, 6% or 9%) and allowed to mature. Subsequent germination of these embryos following the maturation period depended significantly on the sucrose level. At concentrations of 9% sucrose, none of the somatic embryos germinated after maturation, and none were recovered after being transferred to half-strength MS medium containing 2% sucrose. Gibberellic acid treatment was necessary to induce germination; other growth regulators such as auxins and cytokinins did not induce a response. Endogenous abscisic acid content in somatic embryos matured at 9% sucrose (487.8 ng/g FW) was approximately double that found in those matured at 3% sucrose (258.4 ng/g FW). This indicates induced dormancy in embryos under high osmotic stress. Alginate encapsulation of embryos facilitated the artificial induction of dormancy to extend the conservation period without germination. The induction of dormancy strengthened resistance to dehydration after the embryos were desiccated to 15% of their normal water content. Reduced chances of embryo survival during long-term desiccation were distinctly delayed in dormant embryos. These results indicate that the induction of dormancy in embryos is a promising application for synthetic seed production.