Encapsulation Of Somatic Embryos Research Articles

A research on the production, storage and germination of synthetic seeds in tea plant (Camellia sinensis [L.] O. Kuntze)

Tea plant, is one of the most popular beverages consumed worldwide because of its rich and pleasant flavors and numerous health benefits. In this study, we performed production, storage and germination of synthetic seeds in the tea plant by encapsulation of somatic embryos. In our research, after the encapsulation of the mass-produced embryogenic calli with different doses of sodium alginate (NaAlg) and CaCl2, they were stored at different temperatures and at different times, and then transferred to different nutrient media after the expiry of the different storage period to determine the most suitable nutrient composition for germination. The resulting embryogenic calli were stored after encapsulation and then transferred to germination media. Although there was very little germination in long-term storage, the data obtained were found to be statistically insignificant. Although there was very little germination in long-term storage, the data obtained were found to be statistically insignificant. The germination rate of the beads, which were transferred directly to the germination medium without storage and encapsulated using 3% Na-Alg and 50 mM CaCl2.2H2O, was determined as 44.44% in the MS medium containing 3 mg/L BAP and 1 mg/L IBA. In addition, it was observed in the study that increasing storage time increased the darkening of the beads, while increasing NaAlg and CaCl2 doses caused obtaining harder and more nontransparent beads. Hyperhydricity problem was not encountered in any trial in the study. This study, carried out with our local tea variety, is a pioneer in its field with the scope of encapsulating somatic embryos.

Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don)

Vanda tessellata (Roxb.) Hook. ex G.Don (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. The present study investigates asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos (SEs), and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months. Seeds cultured on full strength Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP) had the highest frequency of asymbiotic seed germination (94%) as compared with half- and one-fourth strength MS media supplemented with various concentrations and combinations of 6-furfurylaminopurine (kinetin) and BAP. The protocorm like bodies (PLBs) gained dedifferentiation and proliferated into embryogenic calli on MS medium containing 2.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid (IAA). Somatic embryos (SEs) were differentiated from the callus when cultured on MS medium in combination with 1.0 mg L−1 BAP and 0.5 mg L−1 IAA. The morpho-anatomical elucidations authenticated somatic embryogenesis and various developmental stages of SEs from embryogenic calli. Short-term cold storage of SEs was carried out by encapsulating with 2% sodium alginate and 100 mM calcium chloride and stored under dark at − 4 °C for 12 months to conserve this valuable germplasm. The stored artificial seeds were germinated (91%) on MS medium incorporated with additives and 0.5 mg L−1 each of BAP, kinetin, and IAA. Complete retrieval of plantlets was achieved in 8 week with formation of shoots (4.2 cm average length) and roots (2.5 cm). The well developed plantlets were acclimatized in the greenhouse with 90% survival rate. Vanda tessellata (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. A reproducible protocol has been successfully established for asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos, and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months.

Studies on single and double layered biocompatible encapsulation of somatic embryos in Albizia lebbeck and genetic homogeneity appraisal among synseed derived lines through ISSR markers

Embryogenic synseeds were prepared in Albizia lebbeck by encapsulating cotyledon stage somatic embryos derived from in vitro maintained embryogenic cultures in different types of Ca-alginate beads. The germination rate of somatic embryos was affected significantly by the bead type, matrix composition and germination substrate. A matrix made of 3% Na2-alginate complexed with 100 mM CaCl2·2H2O for a hardening period of 20 min provided uniform encapsulation of somatic embryo. Among different types of synseeds, type IIA, wherein somatic embryos encapsulated in a single layer of Ca-alginate matrix composed of MS medium supplemented with 2 g L−1 activated charcoal and 1.0 µM gibberellic acid (GA3) as reconstituted endosperm, was found to be the most efficient type having maximum germination rates (88.6 ± 0.51%). Incorporation of GA3 in the alginate beads stimulated greater germination of somatic embryos as against GA3 supplementation in the germination substrate. Further, viability studies on short term cold (4 °C) storage of different types of embryogenic synseeds revealed that double layered synseeds (DLS) were found comparatively more robust to withstand longer storage durations than single layered synseeds as evident by greater germination rates of the former after 4–8 weeks of refrigerated storage. Also, the elevated levels of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and leaf proline content in the plantlets derived from DLS reveals the possible role of alginate coatings in conferring alleviation to low temperature stress generated during different storage durations. Similar Inter simple sequence repeat profiles of embryogenic synseeds derived plantlets and mother tree nullifies the possible occurrence of somaclones, thereby establishing the efficacy of synseed technology for clonal propagation of A. lebbeck germplasm. Encapsulation of cotyledonary stage somatic embryos combined with high in vitro germination rates provides a feasible tool for interim storage and exchange of A. lebbeck germplasm.

Encapsulation of Date Palm Somatic Embryos: Synthetic Seeds.

Synthetic seed or encapsulated somatic embryos may be used for propagation, storage, and exchange of plant germplasm and have many diverse applications in date palm cultivation. They have advantages over conventional use of offshoot material for germplasm propagation, maintenance, exchange, and transportation. This chapter describes a protocol for date palm synthetic seed production by encapsulation of somatic embryos with sodium alginate. Among three concentrations used, 3% sodium alginate followed by dropping into 2.5% calcium chloride (CaCl2) solution shows the best concentration of gel matrix for both maintenance and recovery. In addition, storage of the encapsulated date palm somatic embryos at 5°C improves the survival and conversion into plantlets; otherwise, 20g/L sucrose in the culture medium enhances conversion of the recovered somatic embryos to plantlets. This protocol is promising for in vitro conservation and international exchange of date palm germplasm.

Synthetic Seed Preparation, Germination and Plantlet Regeneration of Litchi (<i>Litchi chinensis</i> Sonn.)

Litchi chinensis sonn.) ranks second after mango amongst the most important fruit crops cultivated worldwide. Litchi is a very valuable crop throughout the world because it is a table fruit and wines are also produced from it. The existing cultivars are highly polyploidy and heterozygous in nature. It is propagated through air layering and marcottage methods and storability is very low. Synthetic seeds can be stored for a long time and its genetic constitution could remain the same. For germplasm maintenance and clonal propagation, synthetic seeds can be used. Somatic embryogenesis has been reported from anther or embryogenic suspension culture in various species of litchi. Regeneration via organogenesis and somatic embryogenesis from zygotic embryos has also been reported in certain species. Developing a methodology for getting somatic embryogenesis with a high frequency from zygotic embryos which is available once in a year, would be particularly useful for genetic improvement of litchi. Cotyledonary stage somatic embryos developed from zygotic embryos were encapsulated in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.7% agar medium containing 3% sucrose concentration in NN basal medium (half strength of major and minor salts) with 1 mg·l-1 of gibbrellic acid. Percentage germination and plantlet development for ESEs was higher than that of non encapsulated embryos (NSEs). In comparison to different hormones, gibberellic acid has a significant influence on the germination rate of ESEs after one week of dehydration was seen maximum at 9% sucrose and abscisic acid (1 mg·l-1) in half strength of major and minor salts in Nitsch and Nitsch medium resulting in extended storage up to 90 days without loss in germination potential and capability to regenerate into plantlets. Normally developed plantlets regenerated from ESEs were successfully adapted to soil to obtain a full grown plant.

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava ( Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava ( Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l −1) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l −1) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l −1 ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l −1) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.

Synthetic seed as a potential direct delivery system of mass produced somatic embryos in the coastal marsh plant smooth cordgrass (Spartina alterniflora)

A suspension culture capable of producing a high quantity of proembryogenic masses (PEMs) was evaluated to provide adequate support for synthetic seed production in saltmarsh species Spartina alterniflora. Using immature inflorescences, callus was induced on MS medium supplemented with 2 mg 1−1 2,4-dichlorophenoxyacetic acid (2,4-D). Prior to initiation of suspension culture, calli were proliferated on a modified R4 medium (R4+) for 2–4 weeks. Suspension culture was carried out in a liquid General Medium modified by adding FeSO4 · 7H20, myo-inositol, thiamine · HCl, pyridoxine · HCl, and casein acid hydrolysate. Suspension cells were subcultured weekly by replacing the spent medium with fresh medium. The amount of PEMs tripled in 1 week. Cells from suspension culture had an average regeneration rate of 90% for 6 months of culture. About 40 g of PEMs can be harvested from 1 l of suspension culture in a weekly basis. Plated on R4+ medium, these PEMs produced somatic embryos (SEs) that gave rise to 2,000 plantlets. Encapsulation of SEs or microplantlets (MPs) derived from suspension cells may facilitate direct delivery of micro-propagated S. alterniflora to field planting. Evaluated under in vitro conditions, both SEs and MP-derived synthetic seeds had high conversion rates.

Somatic embryogenesis and embryo encapsulation in Fraxinus angustifolia Vhal.

SummaryThis is the first paper on somatic embryogenesis in F. angustifolia, an important tree in the Mediterranean Basin for landscaping and wood production. The induction of somatic embryogenesis from immature zygotic embyros and the subsequent development, maturation, encapsulation and conversion of somatic embryos into plantlets, as well as the long-term maintenance of embryogénie capacity, are reported. The developmental stage of immature zygotic embryos affected morphogenic response. An auxin-cytokinin ratio of 2:1 (2,4-dichlorophenoxyacetic acid: benzyladenine) was essential for induction. A combination of 2,4-dichlorphenoxyacetic acid (0.44 |j,M) and benzyladenine (4.4 |j,M) has made it possible to maintain embryogénie capacity for over three years. Mannitol (30 g 1_1) and activated charcoal (2 g l”1) positively affected somatic embryo maturation. The encapsulation of somatic embryos in sodium-alginate beads adversely affected plant conversion, regardless of nutrient additions to the alginate. Sixty-five per cent of non-encapsulated somatic embryos were successfully converted into whole plantlets on hormone-free MS medium in comparison to 23% of the encapsulated ones.

Germination and plantlet regeneration from encapsulated somatic embryos of mango (Mangifera indica L.).

Cotyledonary-stage somatic embryos (3-5 mm in length) originating from nucellar explants of Mangifera indica L. cv. Amrapali were encapsulated individually in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.6% agar-gelled medium containing B5 macrosalts (half strength), Murashige and Skoog microsalts (full strength), 3% sucrose and 2.9 μM gibberellic acid. The percentage of germination of ESEs was higher than that of naked somatic embryos of the same size on the same medium. The germinability of ESEs was increased (73.61±7.08%) when the medium was supplemented with full-strength B5 macrosalts. Of the germinating ESEs, 45.83±3.40% developed into plantlets. Abscisic acid at 0.004 and 0.02 μM had no significant influence on germination and plantlet development, but caused a 3-week delay in germination. Well-developed plantlets regenerated from ESEs have been successfully established in soil.

Artificial endosperm of Cleopatra tangerine zygotic embryos: a model for somatic embryo encapsulation

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype-derived plants in a short time. In this work, a range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. Different complexing ions in the form of alginate capsules, zeolite as an ion exchanger and the relationship between capsule-nutrient gel on germination of zygotic embryos, were evaluated. Artificial endosperm assays showed that abscisic acid (1 μM) and mannitol (0.25 M) delayed germination and conversion of zygotic embryos, whereas amino acid supplements (proline, glutamic acid and arginine) accelerated the conversion process. An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants.

In vitro response of encapsulated somatic embryos of camellia

Somatic embryos of Camellia japonica were hydrogel encapsulated using 3% sodium alginate and 0.1 M calcium chloride to produce synthetic seeds. Both germinability and repetitive embryogenesis capacity of the encapsulated embryos were investigated. The frequency of in vitro germination into plants of artificial seeds was affected by various nutrient additives included in the encapsulating matrix. The addition of Ca-free Murashige and Skoog basal medium plus 3% sucrose plus 14.4 µM gibberellic acid and 28.5 µM indole-3-acetic acid to the alginate capsule resulted in 63% plant recovery rate, which was similar to that of non-encapsulated embryos. Encapsulation of somatic embryos did not negatively affect the maintenance of their embryogenic competence, the mean number of secondary embryos being significantly increased when the alginate beads included the growth regulators of the secondary embryogenesis medium (4.44 µM 6-benzyladenine and 0.41 µM indole-3-butyric acid). Storage at 4°C significantly reduced the survival and germination into plants frequencies of both encapsulated and non-encapsulated embryos, but the reduction was much greater for non-encapsulated embryos. Plant recovery of encapsulated embryos was 40% and 30% following storage for 30 and 60 days, respectively.

Germination and growth of encapsulated somatic embryos of carrot for mass propagation

Shoot growth and root development of plantlets germinated from encapsulated somatic embryos of carrot were promoted by transferring the embryos from a culture medium containing sucrose (1.5%) to a culture vessel with a similar medium without sucrose, which was kept under a constant relative humidity condition (90%) during the culture period. The length and dry weight of plantlet shoots placed on medium supports (ceramic wool) containing a liquid culture medium without sucrose from three weeks after placing encapsulated somatic embryos were approximately two times greater than those placed on medium supports containing a culture medium with sucrose throughout the five-week culture period.

Artificial seeds: A method for the encapsulation of somatic embryos

A detailed description of alfalfa (Medicago sativa L.) somatic embryogenesis and methods to produce alfalfa artificial seeds are described. These protocols provide directions for producing alfalfa artificial seeds that will convert into whole plants when planted in vitro or directly into soil.

Encapsulation of Somatic Embryos in Synthetic Seed Coats

Abstract The importance of tissue culture for clonal propagation in agriculture continues to increase each year. In general, commercial use of tissue culture propagation has been limited to crops that have a high per-unit value, such as ornamentals and fruit and nut trees. A lowcost, high-volume propagation system is not available, but could be of significant value to medium per-unit value crops such as lettuce, celery, and many others (Table 1). For these crops, highvolume propagation potential of somatic embryogenesis combined with formation of synthetic seeds for low-cost delivery would open a new field for clonal propagation. Candidate crops for synthetic seed production can be classified into two categories: 1) those that have a strong technological basis, such that high quality somatic embryos can currently be produced, and 2) those with a strong commercial basis. The latter category of crops are those in which seed costs are high because of fertility problems, gamete instability, labor-intensive hybrid seed formation, or a number of other reasons. Currently, there are few crops (Table 1) that meet both requirements and are suitable for synthetic seed technology.