Secondary Somatic Embryogenesis Research Articles

Influence of exogenous polyamines on the secondary somatic embryogenesis of cork oak (Quercus suber L.)

ABSTRACT Quercus suber L. is the main woody tree species in the Mediterranean basin. The in vitro regeneration from adult material, through primary somatic embryogenesis, is a well-known process, but the use of secondary somatic embryos for plant regeneration remains a very sparsely studied process. The main objective of this work is to explore the cork oak regeneration potential by using the secondary somatic embryogenesis process. Mainly, in this work, we report the polyamine effect. Explants used consisted on primary mature embryos, derived from leaves rejuvenated by epicormic shoot of the Moroccan Quercus suber. Three different polyamines were added to the basal medium, which was composed by macronutrients of N 30 K, 30 g/l glucose, and 7 g/l agar. Three polyamines, Putrescine, Spermine, and Spermidine, were added to the basal medium at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/l. Explants were tested after 8 weeks. Morphological analysis showed that the medium with 0.4 mg/l Spermidine provided the best result for secondary embryos, which corresponds to a very significant (p < 0.05) increase of 375%. The number of secondary embryos directly formed was 2.70 ± 0.51. Similarly, the optimum concentrations for high number of clusters (0.50 ± 0.11) and embryo clusters (1.43 ± 0.35) were increased by 145% and 158%. The addition of the polyamine also acted on the quality of embryos formed. A very significant (p < 0.05) increase in the size of secondary embryos was observed compared to the medium without polyamines. Spermidine showed the greatest increase (about 38%).

An efficient embryogenic cell suspension culture system through secondary somatic embryogenesis and regeneration of true-to-type plants in banana cv. Sabri (silk subgroup AAB)

An efficient embryogenic cell suspension culture system through secondary somatic embryogenesis and regeneration of true-to-type plants in banana cv. Sabri (silk subgroup AAB)

In Vitro Growth and Regeneration of Brassica oleracea var. gongylodes: A Decade of Research

Kohlrabi (Brassica oleracea var. gongylodes) is a vegetable cultivated for its edible stem tuber. Although valued for its nutritional properties and tolerance to abiotic stress, kohlrabi is one of the least studied brassicas. In this review, we summarize the results of our decade-long research on in vitro morphogenesis of kohlrabi, starting from 2013. Protocols for efficient in vitro regeneration with minimal requirements for external application of plant growth regulators (PGRs) have been developed, both by somatic embryogenesis (SE) and by de novo shoot organogenesis (DNSO). Efficient regeneration by direct SE was achieved using immature zygotic embryos as explants incubated on PGR-free media, and the production process was maintained in culture thanks to highly efficient secondary SE. Conversely, efficient regeneration by indirect DNSO was achieved using entire seedlings as explants and adding only cytokinin (CK) without the need for exogenous auxin. Comprehensive phytohormone analyses revealed that different exogenously applied CKs differentially affected the composition of endogenous phytohormones and induced changes in the expression of cell cycle-related genes and other genes involved in the organogenic response. The addition of high sucrose concentrations to the nutrient media failed to induce the formation of stem tubers in in vitro culture, but revealed a complex interaction with exogenously applied CKs, interfering with both the endogenous phytohormonome and the expression of organogenesis-related genes. Our work has provided substantial biotechnological advances in the field of in vitro regeneration of kohlrabi, as well as in understanding the underlying phytohormonal regulation. The review aims to introduce kohlrabi to the scientific community as a model system for both basic and applied research, while we continue seeking answers to the outstanding questions and trying to pave the way for the development of more resistant varieties.

Secondary somatic embryogenesis in Cork oak: influence of plant growth regulators

Cork oak (Quercus suber L.) is one of the most important Mediterranean forest tree species, and the Maâmora forest-Morocco is considered to be the world largest cork oak stand. Clonal propagation of Quercus suber via somatic embryogenesis is an alternative to conventional tree propagation methods. However, complete maturation of somatic embryos is considered the major bottleneck for mass propagation of Quercus suber during somatic embryogenesis. This study focuses on influences of cytokinins on secondary somatic embryogenesis. Cytokinins showed a negative effect on the induction of secondary somatic embryogenesis. For the various concentrations tested, the number of secondary somatic embryos was significantly reduced except in the case with low dose of 2iP, which showed a slight increase. Abscisic acid (ABA) may be beneficial at low concentrations between 0.3 and 0.5 mg/l. The best number of secondary embryos was given at 0.5 mg/l ABA with an average number of 5 embryos from each primary embryo, which represents a slight increase below 10% compared to control medium. Also, we found that the number of responsive embryos presenting secondary somatic embryos based on their cotyledons decreased once adding ABA. Phenylurea derivatives TDZ and DPU do not promoted the process of secondary somatic embryogenesis. DPU showed no significant effect with a similar response to that of the control medium and TDZ showed an inhibitory effect. Gibberellic acid (GA3) partially blocked the process of somatic embryos multiplication, but promoted remarkably the germination of cotyledonary embryos without requesting a particular protocol. Our results provide a new insight into embryo development, establishing the basis for further research toward improvement of secondary somatic embryogenesis in Moroccan cork oak.

Tissue Culture Response of Ornamental and Medicinal Aesculus Species-A Review.

Species of the genus Aesculus are very attractive woody ornamentals. Their organs contain numerous health-promoting phytochemicals. The most valuable of them—aescin—is used in commercial preparations for the treatment of venous insufficiency. The industrial source of aescin is horse chestnut seeds because the zygotic embryos are the main site of its accumulation. Horse chestnut somatic and zygotic embryos contain similar amount of aescin, hence somatic embryos could be exploited as an alternative source of aescin. Somatic embryogenesis, androgenesis and de novo shoot organogenesis were successfully achieved in several Aesculus species, as well as secondary somatic embryogenesis and shoot organogenesis, which enables mass production of embryos and shoots. In addition, an efficient method for cryopreservation of embryogenic tissue was established, assuring constant availability of the plant material. The developed methods are suitable for clonal propagation of elite specimens selected as the best aescin producers, the most attractive ornamentals or plants resistant to pests and diseases. These methods are also useful for molecular breeding purposes. Thus, in this review, the medicinal uses and a comprehensive survey of in vitro propagation methods established for Aesculus species, as well as the feasibility of in vitro production of aescin, are presented and discussed.

Callogenesis and Embryogenic Potential of New Superior Cocoa (Theobroma cacao L.) Genotypes Treated with Ascorbic Acid

Clonal propagation is an alternative method of maintaining the genetic purity of superior cocoa plants (Theobroma cacao L.). The somatic embryogenesis technique is likely the most effective and efficient method for plant propagation. This study aimed at investigating the callogenesis and embryogenic potential of new superior cocoa clones on culture media added with various concentrations of antioxidants and variation in the concentration of auxin hormone combined with ascorbic acid. The superior clones used in this study were MCC 01, MCC 02, Sulawesi 3, ICCRI 07, and ICCRI 09. The parameters observed in this study were percentage of embryogenic callus formation, number of secondary somatic embryos, and percentage of embryo somatic cells that develop into planlets. The addition of 2,4,5-T combined with ascorbic acid affected the initiation of cocoa secondary somatic embryogenesis. Treatment combination of ascorbic acid 100 mg.L-1 and 2,4,5-T 1 mg.L-1 had a significant effect on the initiation of secondary somatic embryogenesis of cocoa in the embryogenic callus phase (4.73%), globular embryos (2.53%), torpedo embryos (4.67%) and sprouts (0.47%). Three clones i.e ICCRI 07, Sulawesi 3 and ICCRI 09 clones showed a higher percentage (3.0%, 2.10%, and 1.80%, respectively) of embryogenic callus growth than the other clones. Meanwhile, on MCC 01 clone, all treatments did not affect the regeneration of embryogenic callus. The combination treatment of organic acid and adenine showed a low embryogenic callus response in MCC 01 and MCC 02 clones. However, MCC 02 clone also did not show a response in form of globular, torpedo, and germination phase somatic embryo regenerations. This indicates that different plant cultivars show different responses to the addition of organic acids. Treatment combinations of adenine 0.0125 mg.L-1 + ascorbic acid 100 mg.L-1 gave the best response to the regeneration of somatic embryos for the globular, torpedo, and g

Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis.

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars ‘Alpinist,’ ‘Ay-Nor,’ ‘Bal Tsvetov,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Madame Julia Correvon,’ ‘Nevesta,’ ‘Nikitsky Rosovyi,’ ‘Nikolay Rubtsov,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’ were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3–0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20–8.90 μM) and 0.049 μM NAA, or TDZ (3.0; 6.0, and 9.0 μM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 μM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 μmol m–2 s–1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 μM BAP, or 6.0 μM TDZ were optimal cytokinin concentrations for micropropagation. The explants of ‘Alpinist,’ ‘Ay-Nor,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Nevesta,’ and ‘Serenada Kryma’ cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 μmol m–2 s–1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 μM BAP with 0.09 μM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in ‘Crystal Fountain’ (100%), ‘Crimson Star’ (100%), ‘Nevesta’ (97%), and ‘Ay-Nor’ (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

Secondary Somatic Embryogenesis in Centaurium erythraea Rafn.

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU). A medium containing 0.1 mgL−1 2,4-D and 0.25 mgL−1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL−1 2,4-D and 0.5 mgL−1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury—an emerging model in developmental biology.

Enhanced Secondary Somatic Embryogenesis in Suspension Culture of Four Diploid Banana Cultivars from Kerala

ABSTRACT Secondary somatic embryogenesis was observed in the suspension culture of bract-derived banana calli in the presence of liquid Murashige and Skoog medium with additives such as glutamine (0.68–34.21 µM), biotin (0.40–20.46 µM), or ascorbic acid (0.56–28.38 µM) along with malt extract (100 mg/l). After the initiation of homogenous cell suspension, fully developed somatic embryos were observed after fifth subculture in the four cultivars. Somatic embryos were successfully regenerated in basal MS medium and were transplanted after fifth week. After fifth subculture, the frequency of somatic embryos increased in each culture flask. Maximum number of somatic embryos were observed in cv. Sannachenkadali (46.12 ± 0.85a) when cultured in liquid MS medium supplemented with biotin (8.18 µM) along with malt extract (100 mg/l), in followed by cv. Matti (44.75 ± 1.19a) having (AA) genome. SEM analysis revealed the presence of secondary somatic embryos on the surface of primary somatic embryos in each culture and it attributed a high frequency of somatic embryogenesis. The addition of biotin along with malt extract increased the frequency of secondary somatic embryogenesis in the diploid banana cultivars.

Enhanced Secondary Somatic Embryogenesis in Suspension Culture of Four Diploid Banana Cultivars from Kerala

ABSTRACT Secondary somatic embryogenesis was observed in the suspension culture of bract-derived banana calli in the presence of liquid Murashige and Skoog medium with additives such as glutamine (0.68–34.21 µM), biotin (0.40–20.46 µM), or ascorbic acid (0.56–28.38 µM) along with malt extract (100 mg/l). After the initiation of homogenous cell suspension, fully developed somatic embryos were observed after 5th subculture in the four cultivars. Somatic embryos were successfully regenerated in basal MS medium and were transplanted after fifth week. After 5th subculture, the frequency of somatic embryos increased in each culture flask. Maximum number of somatic embryos were observed in cv. Sannachenkadali (46.12 ± 0.85a) when cultured in liquid MS medium supplemented with biotin (8.18 µM) along with malt extract (100 mg/l), in followed by cv. Matti (44.75 ± 1.19a) having (AA) genome. SEM analysis revealed the presence of secondary somatic embryos on the surface of primary somatic embryos in each culture and it attributed high frequency of somatic embryogenesis. Addition of biotin along with malt extract increased the frequency of secondary somatic embryogenesis in the diploid banana cultivars.

Induction of somatic embryogenesis and evaluation of genetic stability in regenerated plants of Magnolia dealbata

The utility of plant tissue culture for the mass propagation of trees is well known, but continuous in vitro multiplication of plant material may increase the possibility of somaclonal variation; therefore, it is essential to evaluate the genetic integrity of regenerants from species-specific in vitro protocols prior to mass production and implementation. The objectives of this study were: 1) to determine the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration over two cycles of secondary somatic embryogenesis in Magnolia dealbata; and 2) to verify the genetic stability of the regenerants obtained. The embryogenic response was not significantly affected by the concentration of 2,4-D but did vary across cycles of induction. The addition of 4.52 μM 2,4-D induced the highest total number of embryos (100.5), the mean number of somatic embryos (25.1) and somatic embryos per explant (80.6). In both 2,4-D concentration (2.26 or 4.52 μM), genetic integrity between the donor and the propagated clones was 0.90, and the low genetic instability (≤ 0.10 in both PGR treatments) might be due to effect of cyclic somatic embryogenesis or the different response of the explants at stress in in vitro culture conditions. However, it is necessary to examine more cell lines and somatic embryogenesis cycles.

PRIMARY AND SECONDARY SOMATIC EMBRYOGENESIS OF CACAO: THE EFFECT OF EXPLANT TYPES AND PLANT GROWTH REGULATORS

&lt;p class="abstrakinggris"&gt;The success of cacao somatic embryogenesis is affected by many factors, including the basal salt medium, the genotype, the explant type, and the concentration and composition of plant growth regulators (PGRs). The study aimed to evaluate the effects of PGRs composition on the primary somatic embryo (PSE) response and the effect of explant type and PGRs composition used in inducing PSE on the secondary somatic embryogenesis (SSE) response. PSEs were induced from basal petal and staminoid explants of MCC 01 and MCC 02 clones on DKW medium containing 2,4-D 2 mg l&lt;sup&gt;-1 &lt;/sup&gt;+ kinetin 0.5 mg l&lt;sup&gt;-1&lt;/sup&gt; or 2,4-D 2 mg l&lt;sup&gt;-1&lt;/sup&gt; + kinetin 0.125 – 0.250 mg l&lt;sup&gt;-1&lt;/sup&gt; + thidiazuron (TDZ) 2.5 – 5 µg l&lt;sup&gt;-1&lt;/sup&gt; or 2,4-D 2 mg l&lt;sup&gt;-1&lt;/sup&gt; + TDZ 10 µg l&lt;sup&gt;-1&lt;/sup&gt;. Genotype, explant type, and PGR composition dependently affected PSE response. The best PSE response was obtained from staminoid explant of MCC 02 clone on medium containing 2,4-D 2 mg l&lt;sup&gt;-1&lt;/sup&gt; + kinetin 0.5 mg l&lt;sup&gt;-1&lt;/sup&gt; (20%, 9 embryos). The explant type and PGR composition used in inducing PSEs affect the SSE response. The highest SSE response of MCC 01 clone was obtained from petal explant with medium containing 2,4-D 2 mg l&lt;sup&gt;-1&lt;/sup&gt; + kinetin 0.5 mg l&lt;sup&gt;-1&lt;/sup&gt;. The formation of SSEs could increase the multiplication rate of MCC 01 clone by 7 times.&lt;/p&gt;

Effect of Amino Acids on Secondary Somatic Embryogenesis of Moroccan Cork Oak (&amp;lt;i&amp;gt;Quercus suber&amp;lt;/i&amp;gt; L.) Tree

In the present study, we tested the effect of amino acids on secondary somatic embryogenesis of Moroccan cork oak (Quercus suber L.). Secondary mature and immature somatic embryos were obtained from primary somatic embryos cultured in N30K medium supplemented with nineteen amino acids. Stimulation of embryogenesis was dependent on the type and concentration of amino acid in the medium. Thus, L-glutamine and L-asparagine at 3.42 mM have been proved to be the most favorable for the formation of functional somatic embryos and the induction of secondary somatic embryos.

Long-Term Maintainable Somatic Embryogenesis System in Alfalfa (Medicago sativa) Using Leaf Explants: Embryogenic Sustainability Approach.

Alfalfa (Medicago sativa) is one of the most important forage legume crops because of its mass production and high feeding value. It originated in Asia and is one of the most ancient plants cultivated throughout the world as a fodder. Despite the well-studied somatic embryogenesis of alfalfa, there is a lack of a long-term maintainable somatic embryogenic system. Every time an embryogenic callus culture must be started from new explants, which is laborious, costly and time consuming. In addition to this, endogenous microorganisms present in ex vitro explants of alfalfa can often cause contamination, reducing the efficiency of callus culture. An attempt was made to establish long-term continuous somatic embryogenesis system in alfalfa using cultivar Regen-SY. Nine somatic embryogenesis pathways were studied and evaluated for embryo yield, plant conversion rate and embryogenic sustainability. Somatic embryos passed through the same stages (globular, heart-shaped, torpedo and cotyledonary) as characteristic of the zygotic embryo and secondary somatic embryogenesis was also observed. B5H-B5 system showed the highest embryo yield and plant conversion rate whereas SH4K-BOi2Y system demonstrated the highest embryogenic sustainability and maintained the embryogenic potential even after six subculture cycles. Scanning electron microscopy was applied to study the morphology of the somatic embryos and secondary somatic embryogenesis. Therefore, long-term maintainable somatic embryogenesis system protocol was developed through this study, which will help to enhance and accelerate the alfalfa biotechnology research.

Primary and secondary somatic embryogenesis in Jatropha curcas L. From leaf transverse thin cell layers

An efficient method for plant regeneration in Jatropha curcas L. via primary and secondary somatic embryogenesis culture from ex vitro leaves of 6-month-old plants was presented in this study. Leaves were cut into transverse thin cell layers (tTCLs) and cultured on MS medium supplemented with kinetin (KIN) at 0.5, 1.0, 1.5, and 2.0 mg/l in combination with indole-3-butyric acid (IBA) at 0.1, 0.5, and 1.0 mg/l or 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0, 1.5 and 2.0 mg/l . The highest embryogenic callus formation rate (89.3%) was obtained on medium supplemented with 1.0 mg/l KIN and 1.5 mg/l 2,4-D. The calli were selected for the study of primary somatic embryogenesis on MS medium containing 2,4-D (0.01, 0.03, 0.05, and 0.07 mg/l) or KIN (0.5, 1.0, 1.5, and 2.0 mg/l). The highest primary somatic embryos formation rate (76.67%) was achieved on MS medium supplemented with 1.0 mg/l KIN. The primary embryos were cultured on medium supplemented with KIN (0.1, 0.5, 1.0, 1.5, and 2.0 mg/l) combined with 0.2 mg/l indole-3-butyric acid (IBA) or 0.05 mg/l 2,4-D. The combination of 1.5 mg/l KIN and 0.05 mg/l 2,4-D was suitable for secondary embryos formation. Embryos proliferated rapidly, and the highest number of secondary embryos (77.5 embryos) wasobtained from a single primary embryos inoculated. Results also showed that the addition of proline (0.75 g/l) or spermidine (0.15 mM) to the culture medium increased the number of secondary embryos considerably. The fully developed plantlets exhibiting healthy roots and shoots were obtained when somatic embryos were sub-cultured onto B5 medium containing 1.5 mg/l IBA.

Caracterización morfo-anatómica de la embriogénesis somática secundaria en Azadirachta indica (Meliaceae)

Antecedentes y Objetivos: Las especies de Meliaceae son extremadamente recalcitrantes durante la germinación y en los procesos in vitro. Por lo tanto, este estudio está enfocado en caracterizar y optimizar un sistema altamente eficiente de propagación a través de embriogénesis somática secundaria en Azadirachta indica, paso importante para la producción de metabolitos secundarios, la propagación o regeneración de estas especies recalcitrantes.Materiales y Métodos: Las secciones de hojas y cotiledones fueron inducidos en medio MS suplementado con bencil-aminopurina (BAP) sola o combinada con ácido 1-naftalenacético (NAA) y ácido abscísico (BA) con tidiazuron (TDZ).Resultados clave: Azadirachta indica desarrolla embriones somáticos primarios con BAP. La formación de tallos y raíces ocurre en bajas concentraciones de BAP, mientras la embriogénesis somática es favorecida a altos niveles de BAP o TDZ. Los embriones somáticos primarios y secundarios fueron evidenciados continuamente y sincrónicamente o asincrónicamente. Una gran cantidad de embriones somáticos fueron obtenidos con citoquininas. Sin embargo, la concentración tiene un rol importante en la diferenciación entre embriones primarios y secundarios. Además, las auxinas son la clave para inducir la histo-diferenciación en los embriones. La inducción de tallo ocurrió después de que los embriones somáticos se transfirieron a un medio libre de hormonas (MS). Estos tallos fueron enraizados con MS1/2.Conclusiones: Los embriones somáticos secundarios fueron distinguidos y caracterizados durante todo el proceso y el sistema fue establecido con segmentos de cotiledones en corto tiempo; esto ofrece oportunidades ventajosas como la producción de metabolitos.

Insights into the conversion potential of Theobroma cacao L. somatic embryos using quantitative proteomic analysis

Abstract Somatic embryogenesis (SE) has been routinely used as mass micropropagation technique, and as a model system for investigating the structural, physiological, and molecular events occurring during somatic embryo development. Successful in vitro SE is related to the quality and yield of somatic embryos obtained. In cacao (Theobroma cacao L.) SE, the efficiency of somatic embryo production has improved with secondary SE. However, the low number of somatic embryos able to conversion into viable plantlets is still low. Then, two morphological types of normal mature somatic embryos can be identified during cacao secondary SE. The first type shows white appearance and high conversion potential (75%), while the second type shows translucent appearance and exhibit low conversion potential (15%). In order to investigate the proteins that can be associated to conversion potential in cacao somatic embryos, the mass spectrometry HDMSE proteomic approach was used. At least 60 proteins showed differences in abundance levels in cacao white somatic embryos, when compared to translucent. An increased abundance of Beta-glucosidase, NAD(P)-linked oxidoreductase and Electron transfer flavoprotein proteins were observed in white somatic embryos. Moreover, in translucent somatic embryos were observed an increased abundance of Cytochrome P450 and Pathogenesis-related proteins. Using white somatic embryos as a model, we suggest that carbohydrate metabolism process and the redox regulation are involved in the control/regulation of somatic embryo quality. These new findings may improve cacao SE protocol, as well as the understanding of the role of pivotal metabolic pathways associated to this in vitro morphogenetic route.

High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in Pseudotsuga menziesii [Mirb.

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L−1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g−1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g−1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g−1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg−1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg−1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.

Epigenetics of long-term somatic embryogenesis in Theobroma cacao 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.