Effects of Desiccation, Medium Osmolarity and Abscisic acid on the Maturation of Hevea brasiliensis Somatic Embryos

The effects of desiccation of Hevea somatic embryos and of sucroseand ABA concentrations in the maturation medium on their germinabilitywere investigated. Conversion into plant, water and histochemicalstatus of somatic embryos were compared systematically to thoseof the zygotic embryos used as reference. Slow desiccation ormaturation on 351 mol m^–3 sucrose supplemented with 1mmol m^–3 ABA strongly improved germinability and conversionof embryos into plants. The combination of the two treatmentswas the most effective, increasing the germination frequencyby 3·7 and plant conversion by 6·6 in clone PR107. Each of these two treatments increased the vigour of somaticembryos, stimulated the formation of root and shoot meristemsand the synthesis and accumulation of starch and protein reserves.At the end of maturation, the Hevea somatic embryos bore ananatomical and histochemical resemblance to mature zygotic embryos.Likewise, the two treatments brought the water status of somaticembryos closer to that of the mature zygotic embryos, but withoutachieving a perfect match. Optimization of the successful conversioninto plants may require full acquisition of this water status. Key words: ABA, embryo maturation, Hevea, somatic embryogenesis, water status     

Enhanced Maturation and Desiccation Tolerance of White Spruce [Picea glauca (Moench) Voss] Somatic Embryos: Effects of a Non-plasmolysing Water Stress and Abscisic Acid

A non-plasmolysing moisture stress effected by polyethyleneglycol (PEG) was beneficial when applied to maturing white spruce(Picea glauca) somatic embryos for the following reasons. Anosmotic treatment of 5.0–7.5% PEG stimulated a threefoldincrease in the maturation frequency. The osmotically treatedsomatic embryos displayed higher dry weights and lower moisturecontents than the controls, indicating a greater accumulationof storage reserves. Moisture contents of mature, osmotically-treated,hydrated somatic embryos were 40–45%, in contrast to 57%for the non-osmotically treated controls. Desiccation was achievedby placing the somatic embryos in a range of relative-humidityenvironments. No clear trend for the effect of PEG on survivalof desiccated somatic embryos was observed; mean survival valuesranged from 34 to 62% when somatic embryos from all osmotictreatments were desiccated for 14 d at 81% relative humidity.Following this desiccation treatment, somatic embryos from allosmotic concentrations had moisture contents of 26–31%,similar to the 32% recorded for unimbibed zygotic embryos. Afterimbibition, moisture contents for these zygotic and somaticembryos were in the order of 60%. Somatic embryos matured withPEG remained quiescent during desiccation due to their low initialmoisture contents, and gave rise to plantlets of normal appearance.Gradual desiccation of the somatic embryos directly followingmaturation with abscisic acid (ABA) was crucial to survivalduring desiccation. A plasmolysing water stress effected bysucrose at osmotic potentials similar to PEG was detrimentalto somatic embryo maturation, thereby emphasizing the importanceof the choice of osmoticum. Desiccation, maturation, osmotic potential, Picea glauca, polyethylene glycol, somatic embryo, water stress, white spruce     

Gene expression patterns, and uptake and fate of fed ABA in white spruce somatic embryo tissues2

Changes in cellular protein accumulation and in in vivo andin vitro protein synthesis, in somatic embryo tissues of whitespruce during a 42 d maturation period were followed by two-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-DSDS-PAGE). These investigations were complemented by an analysisof uptake and fate of fed abscisic acid (ABA) in somatic embryotissues grown on maturation medium. When Stage 1 somatic embryoswere cultured on ABA-containing maturation medium, many changeswere observed in patterns of gene expression and in proteinsynthesis and accumulation which could be associated with embryodevelopment. The polypeptides observed could be categorizedas constitutive, embryo-abundant, embryo maturation-relatedand embryo stage-related, as well as those with non-specificchanges. Accumulation of label from fed ³H-(+)-ABA in embryotissues reached a plateau 3 d after Stage 1 somatic embryoswere placed on maturation medium. ABA taken into tissues wasrapidly metabolized, and 40% of radioactivity in tissues after1 d of culture resulted from ABA metabolites. This value increasedto 90% after 3 weeks culture. Conjugated ABA and oxidized ABA(phaseic acid and dihydrophaseic acid) were major forms of ABAmetabolites in spruce embryo tissues. Using a single 42 d cultureperiod following transfer to medium with ABA, the conditionsthat stimulate the sequence of developmental changes of somaticembryo maturation during the first 21 d do not reoccur duringthe second 21 d. Unless greater synchronization of Stage 1 culturescan be achieved, it is therefore unlikely that yields of maturesomatic embryos will be increased by this method. Key words: Abscisic acid, gene expression, Picea glauca (Moench) Voss, protein synthesis, somatic embryo maturation     

Effect of Abscisic Acid, Osmoticum, and Desiccation on Synthesis of Storage Proteins during the Development of White Spruce Somatic Embryos

The effect of abscisic acid (ABA), non-permeating osmoticumand desiccation treatment on storage protein synthesis duringmaturation of somatic embryos of Picea glauca (Moench) Voss.was examined. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis demonstrated that someof the major crystalloid and matrix polypeptides were absentfrom somatic embryos maturing on medium containing ABA and lowosmoticum. However, treatment with polyethylene glycol-4000(PEG) in combination with ABA resulted in the synthesis of aspectrum of storage polypeptides resembling that of mature zygoticembryos. These storage proteins accumulated throughout an 8-weekculture period, resulting in a threefold higher protein contentthan somatic embryos maturing for the same time in the absenceof PEG. The structure and distribution of protein bodies incells of these osmotically treated somatic embryos was similarto that in cells of mature zygotic embryos. Treatment with 5·0-7·5%PEG prevented catabolism of the accumulated storage polypeptidesduring desiccation. The optimal culture conditions for somaticembryo maturation and storage protein deposition was 16 µMABA and 7·5% PEG for 8 weeks followed by desiccation.Analysis of mRNAs by in vitro translation and immunoprecipitationof translated products showed that the crystalloid protein mRNAprofiles of zygotic and those of somatic embryos maturing on16 µM ABA in the absence of PEG were similar. The differencesobserved in the pattern of accumulated polypeptides in thesesomatic embryos and those of mature zygotic embryos, therefore,indicates that storage-protein synthesis in response to osmoticumis in part regulated at the translational level. During regenerationof somatic embryos to plantlets the storage polypeptides wererapidly utilized in a manner similar to that in zygotic seedlings.Copyright1993, 1999 Academic Press Desiccation, osmotic stress, storage proteins, Picea, embryogenesis—somatic, mRNA (crystalloid protein)     

Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage

The limit of permeability of white spruce (Picea glauca [Moench.]Voss) somatic embryo cell walls to molecules was in the orderof 30 . Polyethylene glycols (PEGs) and dextrans of molecularweights greater than 1000 and 6000, respectively, produced anonpermeating (non-plasmolysing) water stress which improvedembryo development. Somatic embryos converted to plantlets atfrequencies of 76–84% following slow drying and storageat –20 C for 1 year, which was similar to the 77% recordedfor control somatic embryos slowly dried then germinated withoutfreezing or storage. Culture for 7–8 weeks with mediumcontaining abscisic acid, 3% sucrose, and 7.5% PEG 4000 yieldedsomatic embryos with five times the embryo storage lipid contentrecorded for zygotic embryos. During culture with PEG the moisturecontent of the somatic embryos decreased from 96% for immaturesuspension-cultured somatic embryos, to 47% for mature embryos.Somatic embryos cultured for 7–8 weeks survived rapiddrying to 5% moisture content, and converted to plantlets atfrequencies of 60–70%, but no somatic embryos survivedrapid drying when cultured for only 4 weeks; however, slow dryingdid induce desiccation tolerance in 3-week cultured somaticembryos. Abscisic acid was important to maintain embryos ina developmental state, but ABA alone did not induce desiccationtolerance. In order to induce desiccation tolerance a waterstress treatment was required. Tolerance of rapid drying coincidedwith moisture contents below 55%, which occurred after 5 weeksof culture in the presence of PEG 4000 and abscisic acid. Key words: Dextran, molecular weight, polyethylene glycol, triacylglycerol, water stress     

A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Non-freezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4–8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.     

Secretion of Specific Extracellular Proteins by Somatic Embryos of Picea abies is Dependent on Embryo Morphology

Embryogenic cell lines ofPicea abieswere categorized into twogroups, A and B, based on the morphology of the somatic embryosand the ability of the somatic embryos to proceed through amaturation process when treated with ABA. Group A embryos hada distinct, densely-packed embryonic region whereas group Bembryos had loosely packed cells in their embryonic region.Embryo morphology was shown to be regulated by changes in theplant growth regulators in the culture medium. Treatment withN⁶-benzyladenine stimulated embryos to develop large embryonicregions. The morphology of somatic embryos and especially thatof the embryonic regions was correlated with the presence ofspecific extracellular proteins. Only somatic embryos with denselypacked cells in the embryonic regions secreted proteins withrelative molecular weights of 28, 66 and 85kD. The extracellularprotein of 28kD was isolated and the first 21 amino acids inthe N-terminus were identified. These showed 52–57% identitywith the N-terminal sequence conserved among members of a proteinfamily which includes zeamatin and which have been shown tobe involved in plant anti-fungal mechanisms. Immunological studiesof extracellular chitinases and zeamatin-like proteins, as wellas of activity of extracellular peroxidase, revealed a closecorrelation between the presence of specific chitinases andembryo morphology. Auxin; cytokinin; embryogenic cell lines; embryo morphology; extracellular proteins; Norway spruce; Picea abies; somatic embryos     

Polyethylene glycol promotes maturation but inhibits further development of Picea abies somatic embryos

A combined application of abscisic acid (ABA) and high molecular mass osmoticum, polyethylene glycol (PEG) has become a routine method for stimulating somatic embryo maturation in some genera of Coniferales. The goals of the present study were to clarify how the PEG 4000-attributed low osmotic potential (ψ_s) of the maturation medium affects the yield and morphology of mature somatic embryos as well as subsequent developmental processes during germination and ex vitro plantlet growth in different genotypes of Picea abies belonging to 3 full sib seed families. Despite high within- and among-family variation, a stimulatory effect of 7.5% PEG (ψ_s=?0.645 MPa) on somatic embryo maturation was recorded for 13 out of 17 cell lines (F= 2.83, P= 0.1). PEG-treated somatic embryos were more dehydrated than embryos matured in the absence of PEG. Subsequently, embryos were partially desiccated using a high relative humidity treatment (HRH-treatment). The dynamics of embryo water content (WC) during HRH-treatment differed between embryos developed on maturation medium for 5 or 7 weeks. These two patterns remained unchanged irrespective of the ψ_s of the maturation medium. In 5-week somatic embryos, the WC decreased to the lowest level (in the range 25-35%) within the first 8 days of HRH-treatment and was not further substantially changed. Seven-week embryos also lost water within 8 to 16 days (decrease to 15-25% WC), but this drop was followed by rehydration of embryonic tissues by 24th day of HRH-treatment up to nearly the initial WC. Thus, 7-week embryos experienced both desiccation and slow imbibition in the course of the 24-day HRH-treatment. This could account for their increased germinability compared to 5-week somatic embryos found in the present study. Addition of 7.5% PEG to the maturation medium significantly inhibited somatic embryo germination for the vast majority of genotypes (F= 7.35; P= 0.01). Moreover, even after ex vitro transfer, both radicle elongation and lateral root formation were substantially suppressed (F= 3.8; P= 0.03) in those plantlets produced from PEG-treated somatic embryos. Alterations both in the organization of the root meristem and in the structure of the root cap were found by histomorphological analysis of PEG-treated somatic embryos. All those embryos possessed massive root caps with numerous intercellular spaces in the pericolumn tissue. Cells of the quiescent center exhibited clear symptoms of degradation manifested in shrinkage and collapse of the protoplasm. In addition, PEG-treated embryos were of smaller size compared to embryos matured without osmoticum. When grown in artificial substrate (up to 5 months) the PEG-induced inhibitory post-effect gradually decreased. At this stage, the duration of maturation was the only factor separating plantlets into slow- and fast-growing categories. Somatic embryos matured for 5 weeks produced plantlets twice the size of those produced by 7-week embryos (F= 37.8; P < 0.0001). This trend did not depend on ψ_s of the maturation medium, nor on the genotype.     

Somatic Embryogenesis in Abies Alba × Abies Alba and Abies Alba × Abies Nordmanniana Hybrids

Maturation and germination of somatic embryos of hybrids A. alba × A. alba and A. alba × A. nordmanniana were followed by protein analysis of single embryogenic -suspensor masses (ESM) and analysis of storage protein accumulation during somatic embryo development. Very important step was one week pre-cultivation of ESM on medium with polyethylene glycol (PEG) and abscisic acid (ABA). Low osmotic potential of maturation medium and addition of ABA supported development of somatic embryo. Also partial drying of somatic embryo during following three weeks was needed for its normal development. In spite of morphologically fully developed, the somatic embryos were not physiologically ready for germination at least in terms of storage protein accumulation.     

Changes in Abscisic Acid Content in Axis and Cotyledons of Developing Phaseolus vulgaris Embryos and their Physiological Consequences

Prevost, I. and Le Page–Degivry, M. Th. 1985. Changesin absicisic acid content in axis and cotyledons of developingPhaseolus vulgaris embryos and their physiological consequences.—J.exp. Bot. 36: 1900–1905.Changes in abscisic acid (ABA)content with time were measured in embryonic axes and in cotyledonsof Phaseolus vulgaris embryos using a radio–immunoassay.During embryogenesis, a similar pattern was observed in bothtissues: ABA increased to a maximum 29 d after an thesis, followedby a decrease as the seed matured. The level of ABA in the cotyledonswas always much higher than that in the axes. In in vitro cultures,the duration of the lag phase before germination of isolatedembryonic axes increased with ABA content. The presence of cotyledonsalways lengthened the lag phase; longer lag phases were associatedwith greater concentrations of ABA in the cotyledons. Moreoverthe presence of cotyledons stimulated the growth of seedlings. Key words: ABA distribution, embryo maturation, axis and embryo germinability     

Effect of exogenous ABA on embryo maturation and quantification of endogenous levels of ABA and IAA in <Emphasis Type="Italic">Quercus suber</Emphasis> somatic embryos

Knowledge of the relationship between indole-3-acetic acid (IAA) and abscisic acid (ABA) is relevant to control the development and the maturation of cork oak (Quercus suber L.) somatic embryos. The addition of 1 M ABA to the culture medium significantly promoted somatic embryo maturation and increased both fresh and dry matter without affecting the relative water content. This effect was parallel to the pattern of variation observed in the endogenous ABA level, which increased from the immature to the mature stage. Endogenous ABA content during the occurrence of secondary embryogenesis was similar to that of the immature stage, showing that embryos with lower ABA levels produced secondary embryos. In contrast, IAA showed the highest concentration during early embryo development and decreased afterwards. Only in somatic embryos subjected to 1-week desiccation followed by stratification at 4 °C for 2 weeks, was a moderate increment of endogenous IAA content observed. IAA and ABA showed opposite levels during the development and maturation of cork oak somatic embryos and characterised specific stages of the embryonic development.     

The Influence of Auxin and Cytokinin on Proliferation and Morphology of Somatic Embryos of Picea abies (L.) Karst

Embryogenic cell-lines of Picea abies were initiated from maturezygotic embryos and cultured on medium containing 2,4-D andBA The cell-lines were categorized into two main groups (solar/polarand undeveloped embryos), based on the morphology of the somaticembryos and their ability to go through a maturation processwhen treated with ABA The cell-lines were transferred to mediacontaining (1) 2,4-D and BA, (2) only BA, (3) only 2,4-D or(4) no growth regulators When cultured on a medium containingboth 2,4-D and BA new somatic embryos were continually formedIn contrast, when they were cultured without one or both ofthe growth regulators no new somatic embryos were formed Solar/polarand undeveloped embryos responded in the same way On a mediumcontaining only BA the somatic embryos already present increasedin size and developed an extremely large embryonic region Ona medium containing only 2,4-D the embryos already present becamedisorganized into loose aggregates When transferred from a mediumcontaining both 2,4-D and BA to one containing ABA, mature somaticembryos developed from the solar/polar type but not from theundeveloped type The ability of the solar/polar somatic embryosto go through a maturation process decreased when they wereprecultured on a medium lacking auxin and was lost when theywere precultured on medium lacking cytokinin The cell-linescontaining undeveloped somatic embryos produced mature somaticembryos in one cell-line out of three that had been culturedon the medium containing only BA Auxin, cytokinin, embryogenic cultures, Norway spruce, somatic embryos, Picea abies (L.) Karst     

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.     

Abscisic acid and sucrose increase the protein content in date palm somatic embryos,causing changes in 2-DE profile

Various supplements (abscisic acid (ABA) or sucrose) were added to the initial embryo culture medium (M3) with the aim of improving the vigour of vitroplants deriving from date palm somatic embryogenesis. ABA (20 and 40 μM) and sucrose (90 g/l) applied for 4 and 2 weeks respectively increased embryo thickness, with no apparent difference in length. ABA (5–40 μM) increased embryo proliferation rate. Somatic embryos maintained in modified M3 (M3 supplemented with ABA and an increased sucrose concentration) contained a higher amount of protein than those maintained in initial M3 (no ABA, 30 g/l of sucrose), with a 1.5–1.7-fold increase depending on the compound and concentration assayed. The 1-D and 2-DE protein profiles showed qualitative and quantitative differences between the somatic embryos cultured in initial M3 (control) and in modified M3. Statistical analysis of spot intensity was performed by principal component analysis, yielding two accurate groups of samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with an average linkage algorithm. Thirty-four variable spots were identified using mass spectrometry analysis. Identified proteins were classified into the following functional categories: energy metabolism (five proteins); protein translation, folding and degradation (9); redox maintenance (5); cytoskeleton (3); storage protein (2); and with no assigned function as (10). While “up-regulation” of stress-related proteins and “down-regulation” of energy metabolism proteins were observed in somatic embryos matured in M3 supplemented with ABA, storage proteins (legumin) were “up-regulated” in somatic embryos matured in M3 supplemented with increased sucrose.     

Temporal and nutritional factors modulate responses to abscisic acid and osmoticum in their regulation of storage protein synthesis in developing seeds of alfalfa (Medicago sativa L.)

Storage protein synthesis during alfalfa (Medicago sativa L.)seed development displays a temporal pattern and is regulatedboth by ABA and low osmotic potentials. Deposition of the 2S,7S and 11S storage proteins occurs maximally at the mid–to late stages of development. Osmoticum, but not ABA alonecould effectively induce storage protein synthesis at earlystages of development in isolated alfalfa embryos placed onwater for several days. Neither ABA nor osmoticum alone couldmaintain storage protein synthesis in isolated late-stage embryosplaced on water. On the other hand, isolated developing embryoscultured on a nutrient (Murashige and Skoog) medium could maintainstorage protein synthesis in the presence of ABA and osmoticum,alone or in combination, for up to 14 d after being dissectedfrom the pod. Of the components of this medium, the inorganicsalts appeared to be the most important. The response to ABAand osmoticum varied with the time of isolation during developmentwith the greatest enhancement of storage protein synthesis inisolated embryos coinciding with the time of maximum synthesiswithin the seed when in planta. Addition of ABA and osmoticumafter the time of maximum storage protein synthesis did notelevate the amount of synthesis, but rather prolonged the timeover which it could take place. Thus, while the amplitude ofstorage protein synthesis could be modified by ABA or osmoticum,the inherent temporal pattern, with maximum synthesis occurringonly at mid– to late stages of embryo development, couldnot. Key words: Embryogenesis, Medicago sativa L., alfalfa, abscisic acid, osmotic potential, storage protein synthesis, nutrient supply     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L.

Summary Maturation of somatic embryos of Pinus strobus L. was evaluated on media containing various types (agars and gellan gum), brands and concentrations of gelling agents in the presence of 80 μM ABA and 0.09 M sucrose. The media were characterized with respect to gel strength, water potential and water availability. Embryogenic tissue and somatic embryos cultured on medium with various concentrations of gellan gum were used to determine their water potential (Ψ). Regardless of the type of gelling agent used, gel strength increased with gelling agent concentration and was critical to the maturation response. High gel strength was associated with reduced water availability from the medium to the cultures. The water potential of gelled maturation medium remained constant between 0.4 and 1.0% gellan gum. It is concluded that the embryogenic tissue was exposed to varying amounts of water at the onset of and during the culture period, and that the amount of water in the culture environment in turn influenced the maturation response. Cotyledonary somatic embryos derived from gellan gum medium of high gel strength had a lower Ψ than somatic embryos matured on medium of lower gel strength. Once somatic embryos developed to the cotyledonary stage on the maturation medium, they were transferred to the germination medium. The germination frequency and the number of morphologically normal germinants were higher for somatic embryos matured on medium of high gel strength. Raising the concentration of the gelling agent in the maturation medium may be an alternative to the use of solutes to restrict water available to the embryogenic cultures.     

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.