Sugars as a metabolic regulator of storage protein mobilization in germinating seeds of yellow lupine (Lupinus luteus L.)

The intensity of protein reserve activation in yellow lupine (Lupinus luteus L.) organs cultured in vitro in the presence of saccharose and without sugar in the medium was studied. Isolated embryo axes, excised cotyledons and seeds deprived of their testae were grown on Heller medium: a) with 60 mM saccharose (+S), b) without sugar (−S) and c) for 72 hours without saccharose + for 24 hours in the presence of saccharose (−S→+S). Using nitroanilide substrates, exo- and endopeptidase proteolytic activity was investigated in enzymatic extracts. Proteolytic activity was examined in organs isolated from swollen seeds and also in organs cultured for 24, 48, 72 and 96 hours. The proteolytic activity was higher in organs cultured on medium without saccharose. Stimulation of the proteolytic activity on the first day of culture was not significant, but intensified in the successive days of culture. The organ subcultured onto a medium with saccharose (−S→+S) caused a significant limitation of proteolytic activity, even to a markedly lower level in comparison to that activity level in the material continuously supplemented with saccharose. Observations of ultrastructure in Transmission Electron Microscopy revealed increased protein body degradation in the absence of saccharose in the medium and an increased degree of cell vacuolization, which may be indicative of intensifying autophagic processes under conditions of carbohydrate deficit.     

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]

Summary Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1⁻¹ (45.2 μM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1⁻¹ (90.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1⁻¹ (11.3 μM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

Turnover of cell-wall polysaccharides during somatic embryogenesis and development of celery (Apium graveolens L.)

Non-embryogenic cells (NEC) and embryogenc cells (EC) were separated from cell clusters derived from the hypocotyl segments of celery seedlings, which had been suspension-cultured in MS medium supplemented with 10⁵ M 2,4-D. The EC formed globular embryos in medium without 2,4-D. The globular embryo developed through heart-shaped, torpedo to cotyledonary embryos within 10 days. The EC and developing embryos were fractionated into symplastic [MeOH, hot water (HW), starch (S)] and apoplastic [pectin, hemicellulose, TFA (trifluoroacetic acid)-soluble and cellulose] fractions. The EC contained lower levels of sugar in the MeOH fraction and higher levels of starch than NEC. In the apoplastic fractions, there were no differences of total sugar amounts between NEC and EC. Cellulose contents were about 10% of the wall polysaccharides. During somatic embryogenesis, total sugar contents of the MeOH and HW fractions increased till the heart-shaped embryo stage, and then decreased during the torpedo and cotyledonary embryo stages. The sugar contents of the starch, pectin, TFA-soluble, and cellulose fractions did not change during the stages mentioned above. However, the hemicellulose substances remarkably increased during embryogenesis, and then decreased as the development proceeded. The neutral sugar components of the hemicellulosic fractions were analyzed. Arabinose increased markedly in EC to the globular embryo stage, but decreased as the development proceeded. Galactose increased only at the torpedo and cotyledonary embryo stages. Xylose was present at lower levels in all stages of embryogenesis than in the differentiated hypocotyl cell walls. These results suggest that there was a high turnover of arabinogalactan polysaccharides during embryogenesis, and that xylan accumulated in the cell walls of differentiated cells     

Regeneration through organogenesis in rattan

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l⁻¹ glutamine, 100 mg l⁻¹ arginine, 100 mg l⁻¹ asparagine, 60 g l⁻¹ sucrose, 8 g l⁻¹ Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxidative events during in vitro regeneration of sunflower

The changes in the activity of some antioxidant enzymes and endogenous H₂O₂ level in zygotic sunflower embryos during organogenesis and somatic embryogenesis were monitored. Pathways of regeneration were induced on media differing with sucrose concentration 87 mmol dm⁻³ for shoot [shoot induction medium (SIM) medium] and 350 mmol dm⁻³ [embryo induction medium (EIM) medium] for somatic embryo induction. Water potential of the explants cultured on SIM increased, while the embryos maintained on EIM showed middle water deficit stress. The pattern of superoxide dismutase (SOD) isoforms was similar in organogenic and embryogenic culture; however, the intensity of MnSOD bands was higher on SIM than on EIM. Differences in catalase activity were observed: high activity on SIM predominated, whereas on EIM it was reduced. The activity of guaiacol peroxidase in the explants producing shoots and somatic embryos differed at the beginning of culture, but became comparable at the time of shoot and somatic embryo formation (day 5). H₂O₂ content was unchanged in organogenic culture, but on EIM it increased on day 1 followed by significant decrease. The results indicate that sugar concentration per se, or via induction of different developmental pathways influences the activity of antioxidant enzymes and also H₂O₂ level in cultured sunflower embryos.     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Somatic embryo proliferation, maturation and germination in Catharanthus roseus

In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l⁻¹). However, only NAA (1.0 mg l⁻¹) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l⁻¹) added medium following a treatment with gibberellic acid (1.0 mg l⁻¹) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.     

Abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato

Summary Somatic embryos of sweet potato have potential as synthetic seeds. The effects of abscisic acid (ABA) (0,0,0.1, 1.0, 10.0 and 50.0 μM) were examined to improve synchrony and proliferation of somatic embryos. Transferring embryos compared to those cultures transferred at day 0. The development of embryos in suspension culture supplemented with ABA was poor. However, when calli proliferation cultures were in gelled medium and pulsed with 0.1 μM ABA for 14 d, the number of somatic embryos increased. Proembryonic masses cultured in mannitol-containing medium (Y=−1.5 MPa) increased embryo development and synchrony of embryo development. Thus, in this work ABA and mannitol have been shown to improve both the total number and the synchrony of sweet potato somatic embryos.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Effects of mutagens on somatic embryogenesis and plant regeneration in groundnut

The embryogenic calli (EC) were obtained from hypocotyl explants of groundnut (Arachis hypogaea L.) cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg dm⁻³ 6-benzylaminopurine (BAP). The EC were exposed to γ-radiation (10–50 Gy) or treated with 1–5 mM of ethyl methane sulphonate (EMS) or sodium azide (SA). The mutated EC were subcultured on embryo induction medium containing 20 mg dm⁻³ 2,4-D. Somatic embryos (SE) developed from these calli were transferred to MS medium supplemented with BAP (2.0 mg dm⁻³) and 0.5 mg dm⁻³ 2,4-D for maturation. The well-developed embryos were cultured on germination medium consisting of MS salts with 2.0 mg dm⁻³ BAP and 0.25 mg dm⁻³ naphthaleneacetic acid (NAA). Well-developed plantlets were transferred for hardening and hardened plants produced normal flowers and set viable seeds. The fresh mass of the EC, mean number of SE per explant and regeneration percentage were higher at lower concentrations of mutagens (up to 30 Gy/3 mM). Some abnormalities in regenerated plants were observed, especially variations in leaf shape.     

Somatic embryogenesis and plant regeneration through leaf culture in <Emphasis Type="Italic">Arachis glabrata (Leguminosae)</Emphasis>

Plants of two accessions of Arachis glabrata were regenerated via somatic embryogenesis. Embryogenic calli were initiated from leaflet explants on Murashige and Skoog medium supplemented with picloram alone or picloram in combination with 6-benzylaminopurine. Leaflets of accession A6138 induced the highest percentage of somatic embryos in media composed of 10 mg dm⁻³ and 15 mg dm⁻³ picloram. In contrast, 5 mg dm⁻³ picloram with 0.1 mg dm⁻³ 6-benzylaminopurine was one of the most effective combinations in accession AF385. MS medium supplemented with 2 g dm⁻³ activated charcoal (AC) used for 30 days was the most effective for embryo maturation. After 20 days of culture on MS medium devoid of growth regulators, 6 % of embryos converted into plantlets in accession A6138.     

Influence of Boron on Somatic Embryogenesis in Papaya

Influence of boron on somatic embryogenesis in papaya (Carica papaya L.) cv. Honey Dew was investigated. Immature zygotic embryos were grown in the induction medium containing Murashige and Skoog basal salts, with B₅ vitamins, picloram (1 mg dm⁻³) or 2,4-dichlorophenoxy acetic acid (2 mg dm⁻³) and different concentrations of boric acid (30 to 500 mg dm⁻³). Maximum somatic embryo initiation was observed at 62 mg dm⁻³ boric acid irrespective of the growth regulator used. The cotyledonary stage somatic embryos were germinated on MS basal medium devoid of growth regulators. The regenerated plantlets were hardened under greenhouse conditions and transferred to field. This revised version was published online in July 2006 with corrections to the Cover Date.     

Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) somatic embryogenesis from isolated scutellum: Days post anthesis,days of spike storage,and sucrose concentration affect efficiency

Summary To improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l⁻¹ casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.     

Induction,development and maturation of somatic embryos in Bunge’s pine (<Emphasis Type="Italic">Pinus bungeana</Emphasis> Zucc. ex Endl.)

Embryogenic tissue was induced from developing immature zygotic embryos in Bunge’s pine (Pinus bungeana Zucc. ex Endl.). Induction rate reached 84.4% with our best treatment. Zygotic embryos were dissected from megagametophytes and inoculated on different induction media, DCR₁ (Gupta PK, Durzan DJ (1985) Plant Cell Rep 4:177–179), BM₁ (Gupta PK, Pullman G (1991) U.S. Patent No. 5,036,00) and MSG (Becwar MR, et al. (1988) Somatic cell genetic of woody plants. Kluwer Academic Publishers, Dordrecht, pp. 1–18), supplemented with 2,4-dichlorophenoxyacetic acid (2, 4-D) and 6-benzylaminopurine (BA). DCR₁ was the best medium for initiating embryogenic tissue. Induction rates were affected significantly by developmental stages of explants. The highest induction rate was obtained with embryos collected on either June 20 or June 30 with 10 mg l⁻¹ 2, 4-D and 4 mg l⁻¹ BA. Embryogenic tissue was subcultured monthly on DCR₁ medium supplemented with 0.3 mg l⁻¹ 2, 4-D and 0.2 mg l⁻¹ α-naphthaleneacetic acid. In order to enhance embryo maturation, embryogenic tissue was transferred onto DCR₁ medium for two weeks, in which 1,000 mg l⁻¹ myo-inositol was included and all plant growth regulators were eliminated. This pretreated tissue was then transferred onto a maturation medium that was DCR₁ medium containing 50 g l⁻¹ sucrose and 0.1 mg l⁻¹ indolebutyric acid. In this study, benefits of embryo maturation were not observed when abscisic acid and polyethylene glycol were applied in the culture.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.     

Influence of freezable/non-freezable water and sucrose on the viability of <Emphasis Type="Italic">Theobroma cacao</Emphasis> somatic embryos following desiccation and freezing

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08–1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60–95% was recorded for embryos exposed to 0.5–1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H₂O g⁻¹dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.     

Applications of DL-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of dl-buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg⁻¹ tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell–cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.