Epigenetics of long-term somatic embryogenesis in <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.: DNA methylation and recovery of embryogenic potential

In Theobroma cacao L., declined embryogenic potential was observed in regenerated somatic embryos from long-term secondary somatic embryogenesis (SE). In order to explore the relationship between DNA methylation and the long-term secondary SE, the embryogenic potential and global DNA methylation levels in young (12 months-old), aged (36 months-old) and extra somatic embryogenesis (39 months-old) subjected to different 5-Azacytidine (5-azaC) treatments were comparatively assessed. Global DNA methylation levels increased in aged somatic embryos with long-term in vitro culture, but 5-azaC-supplemented treatments resulted in unaltered levels. In addition, DNA methylation pattern during SE was not affected by 5-azaC. DNA methylation increased during SE expression. Interestingly, the extra SE induction showed that aged somatic embryos can recovery the embryogenic potential in treatment supplemented with 5-azaC at specific concentration. The outcome of this study suggested that the long-term SE in cacao induced the decline on embryogenic potential, which can be reversible trough 5-azaC supplementation. Besides, increased DNA methylation levels might be a response to the stress conditions that plant cells were exposed to during SE.     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Somatic embryogenesis in Araucaria angustifolia

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.     

Clonal plant production from self- and cross-pollinated seed families of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

Somatic embryogenesis of <Emphasis Type="Italic">Myrciaria aureana</Emphasis> (Brazilian grape tree)

The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process.     

Efficient somatic embryogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N⁶-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.     

Regeneration of plantlets in Sapindus trifoiatus L

Continuous production of healthy plantlets of Sapindus trifoliatus L. was achieved via somatic embryos from long term cultures of an embryogenic mass (EM). A highly embryogenic culture of S. trifoliatus L. was obtained by recurrent embryogenesis from somatic embryos cultured on Murashige and Skoog's (MS) medium supplemented with kinetin (2.3 microM) and benzyladenine (8.8 microM). The cultures could be maintained without reduction of embryogenic competence for more than 20 months by subculture at 4 week intervals. About 90% mature somatic embryos on transfer to basal MS medium, germinated to plantlets, of which more than 70% survived when transferred to a sand and soil mixture in green house.     

Genetic variation in somatic embryogenic response in open-pollinated families of black spruce

Summary Zygotic embryos from open-pollinated seeds of 20 black spruce (Picea mariana) families were used to investigate the proportion of genotypes that would give rise to embryogenic tissue (ET) and mature somatic embryos. Eighty-five percent of the maternal genotypes gave rise to embryogenic tissue. Within-family rates of ET induction ranged from 0 to 17%, with an average of 8%. The largest proportion of variation was among families, indicating the additive nature of the genetic variation. On a medium with 6% sucrose and 3.7 M ABA, 90% of the embryogenic lines gave rise to abundant (>100/100 mg of ET), well-formed, mature somatic embryos. A medium with 2% sucrose, without 2,4-D, was used to germinate the mature somatic embryos. These were grown in the greenhouse and have now been established in field trials.     

Maturation of somatic embryos in two embryogenic cultures of <Emphasis Type="Italic">Picea morrisonicola</Emphasis> Hayata as affected by alternation of endogenous IAA content

Embryogenic culture lines T4 and T2 were initiated from two mature zygotic embryos of Picea morrisonicola Hay. Mature somatic embryos (SEs) were produced in culture line T2 but not in line T4 after 8-week abscisic acid (ABA) treatment. High performance liquid chromatography (HPLC) analysis of the endogenous indole-3-acetic acid (IAA) content has shown 7.5 times higher IAA production in T4 line than in T2 line during the proliferation phase. However, after ABA incubation the line T4 produced much less IAA than line T2. The application of an anti-auxin, 2,3,5-triiodobenzoic acid (TIBA) or 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB) induced culture line T4 to produce mature SEs. Both 1 μM TIBA and 5 μM PCIB increased the production of stage 2 SEs in T4 culture line when cultures were treated during the proliferation stage for 8 weeks. Occasionally cotyledonary (stage 3) SEs were even produced from treated T4 culture line. Both chemicals have also been demonstrated to significantly decrease the amount of IAA in the treated T4 and T2 embryogenic lines. However the decrease of the IAA level was not beneficial for SE production in the T2 embryogenic line. These results indicated the importance of endogenous IAA level in manipulating the process of SE maturation in spruce embryogenic cultures.     

High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature,dry seed

Peanut (Arachis hypogaea L.) somatic embryos were produced from the embryo axes of mature, dry seeds of cultivar GK-7. Percent embryogenic explants ranged from 88–100% using 10–40 mg/1 of 2,4-D in the induction medium. Neither 2,4-D concentration nor photoperiod during the induction period had a large effect on percent embryogenesis, mean number of embryos per explant, or embryo morphology. However, embryos obtained from cultures grown in the dark were easier to remove from the explant than those under a 16-h photoperiod. Somatic embryos developed on the epicotyl portion of the embryo axis, primarily on the young, expanding leaves. A survey of 14 genotypes indicated that genotype had a large influence on embryogenic capacity, with all genotypes being embryogenic to some extent. The ability to recover somatic embryos from axes of harvested, stored seeds represents significant advantages for the establishment of peanut embryogenic cultures, including the use of simple sterilization procedures and a constant source of explant tissue.Abbreviations B5 medium of Gamborget al. (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts medium     

Plant regeneration <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis in <Emphasis Type="Italic">Pinus kesiya</Emphasis> (Rolye ex. Gord.) influenced by triacontanol

Embryogenic cultures were initiated and established for the first time in 3 different genotypes of Pinus kesiya using mature zygotic embryos and triacontanol. Mature zygotic embryos produced white-mucilaginous embryogenic callus when cultured on half strength MSG (Becwar et al. 1990) basal medium supplemented with 90 mM maltose, 2.0 g l⁻¹ Gellan gum, 9.0 M 2, 4-D and 10 g l⁻¹triacontanol. On subculture of such embryogenic callus on the maintenance medium (II) containing 2.0 M 2,4-D and 2.0 g l⁻¹ triacontanol induced cleavage polyembryogenesis with proembryos. The percentage of somatic embryogenesis was not similar in all the three genotypes. The highest percentage of somatic embryogenesis (88.5 %) was recorded in PK04 genotype. Somatic embryos were successfully germinated on half strength MSG basal medium without growth regulators. Somatic seedlings showed fast growth and a survival rate of 95%. This work for the first time reveals that triacontanol can be used as an effective growth regulator for inducing somatic embryogenesis in conifers.     

Optimized somatic embryogenesis in Pinus strobus L.

Summary Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.     

Somatic embryogenesis of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.     

<Emphasis Type="Italic">Arabidopsis tanmei/emb2757</Emphasis> embryo mutant is defective for in vitro plant morphogenesis

A mutation in the Arabidopsis TANMEI/EMB2757 (TAN) gene with an embryo defective phenotype was analysed for its effect on the morphogenic potential of somatic tissue cultured under in vitro conditions. The capacity for in vitro morphogenesis was evaluated using cultures of immature zygotic embryos, and seedling explants of the tan mutant and the parental Col-0 genotype. The explants were cultured on media supplemented with different plant growth regulators, and the capacity for two alternative pathways of morphogenesis, somatic embryogenesis (SE) and shoot organogenesis, was evaluated. Reporter genes (GUS, GFP) were used to monitor auxin and LEC2 and FUS3 gene activity in the tan explants. Moreover, the expression pattern of the TAN gene was analyzed during SE and in callus tissue of Col-0. It was indicated that the tan mutation resulted in a total lost of embryogenic and organogenic capacity of cultured tissues, suggesting the involvement of the TAN gene in basic cellular processes related to cell growth and differentiation. However, differential expression of the TAN gene during SE, and its increased activity at advanced stages of embryogenesis, implicate a specific role for the gene in the development of somatic embryos.     

Somatic embryogenesis in holm oak male catkins

Somatic embryogenesis (SE) of tree species is the most promising method for the implementation of multivarietal forestry and for biotechnological approaches. To date, however, the application of this technology to mature trees is restricted to a few species. This is the first report on the induction of SE from male catkins of 100-year-old holm oaks (Quercus ilex L.). Embryogenic competence was mainly dependent on genotype and restricted to the most advanced catkin developmental stage with distinguishable closed flowers along the axis. Following a three-stage treatment procedure, embryogenic response (frequencies up 3.3 %) was obtained in three [Remedio, Villar del Arzobispo (VA) and Hunde (HU)] out of the five genotypes evaluated. In the culture conditions tested, the preferred protocol to induce SE in holm oak catkins should include: induction on MS medium with 6-benzyladenine and naphthaleneacetic acid, subculture onto medium with a reduced concentration of both plant growth regulators and a final transference to medium without growth regulators. Under these conditions, cotyledonary-stage somatic embryos developed from brown calli with or without nodular structures. Secondary SE, favored by the addition of sorbitol to the manifestation medium, allowed the establishment of 14 embryogenic lines belonging to VA and HU genotypes. Histological observations of the proliferating cultures revealed the presence of globular, torpedo and cotyledonary somatic embryos. Somatic embryos were diploid as verified by flow cytometry analysis, suggesting that they originated from the perianthic tissue of the male flower.     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

A clonal propagation system for Atlantic white cedar (<Emphasis Type="Italic">Chamaecyparis thyoides</Emphasis>) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l^?1 producing more embryos than 0 g l^?1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l^?1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.     

In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1‐week proliferation) to 61.5% mC for immature somatic embryos (1‐week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8‐weeks maturation). The presence of 5‐azacytidine (hypo‐methylating agent) or hydroxyurea (hyper‐methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin‐ and vicilin‐like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two‐dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper‐ and hypo‐treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.