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In woody species such as cocoa and mulberry, the development of somatic embryogenesis protocols constitutes a necessary and useful tool for the propagation, improvement and in vitro conservation of plant genetic resources. In the ...
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In woody species such as cocoa and mulberry, the development of somatic embryogenesis protocols constitutes a necessary and useful tool for the propagation, improvement and in vitro conservation of plant genetic resources. In the species Theobroma cacao, cotyledons and flower buds were used as explants. Experiments were performed for differentiation, maturation and conversion of somatic embryos to plants. A histological characterization of the somatic embryogenesis process was also carried out. Somatic embryos were obtained from staminoids in differentiation media with kinetin (9.32 μM) and NAA (2.69 μM) in the clone 'UF-613' and ' Pound-7'. These were observed in globular, heart-shaped, torpedo and cotyledonal stages. Somatic embryo maturation was achieved in DKW medium with 6% sucrose. The conversion rate of somatic embryos was 20.8% for 'UF-613' and ' Pound-7'. In Morus alba 'Acorazonada' different types of explants and culture media were evaluated to obtain callus and embryogenic structures. A 94% formation of embryogenic structures was achieved in calli from culture media with 4.54 μM TDZ subcultured in cocoa differentiation media.
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Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-...
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Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-derived plants be tested in field conditions. Therefore, we established a field test at Union Vale Estate, Saint Lucia. Thirty- to 50-yr-old trees were selected for clonal propagation as potentially high yielding based on local farmers observations. Clonal plants were propagated in vitro from immature flowers by embryogenesis and micropropagation. Multiple plants from nine genotypes were acclimated to greenhouse conditions then returned to Saint Lucia and planted in a field. Orthotropic rooted cuttings and locally propagated open pollinated seedlings were also planted for a total of 214 trees. Growth data were collected every 4-6 mo. including: stem diameter, stem height, length of the longest jorquette branch, number of jorquette branches, and dates of first flowering and fruiting. At 4.5 yr after planting in the field there were no major differences in all growth parameters among the propagation methods evaluated with exception of the orthotropic rooted cuttings. Trees grown from seeds were slightly taller then trees propagated by the other methods. Trees propagated as orthotropic rooted cuttings exhibited smaller average stem diameters, shorter stem heights to the jorquette, and shorter jorquette branches. We concluded that somatic embryo-derived plants demonstrated normal phenotypes in field conditions and have growth parameters similar to plants propagated by traditional methods.
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The development of efficient tissue culture systems for cacao holds the potential to contribute to the improvement of this tropical crop by providing a rapid and efficient vegetative propagation system for multiplication of elite ...
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The development of efficient tissue culture systems for cacao holds the potential to contribute to the improvement of this tropical crop by providing a rapid and efficient vegetative propagation system for multiplication of elite genotypes. It may also find application in facilitation of germplasm movement across quarantine borders, enhancement of germplasm conservation via cryo-preservation. and development of genetic transformation systems. Somatic embryogenesis using floral tissue explants was previously the only tissue culture procedure for regeneration of cacao. We report the development of a secondary embryogenesis system utilizing primary somatic embryo cotyledon explants, which results in up to a 30-fold increase in somatic embryo production compared to primary somatic embryogenesis. The influence of genotype on the efficiency of the system was evaluated. To understand the cellular origins and developmental pathways operative in this system, we investigated the morphological changes occurring over time using light and scanning electron microscopy. While primary embryos arise from clusters of cells forming embryonic nodules, secondary embryos arise predominantly front the division of single cells, in a pathway reminiscent of zygotic embryogenesis. These results have important significance to the application of tissue culture to cacao improvement programs. [References: 26]
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Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enh...
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Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5%) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.
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Induction of somatic embryogenesis from tissues in unopened flower buds of cocoa was studied with respect to physiological age, type of floral explant, genotype, and medium composition and phytohormones. Two-to-three-week-old stam...
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Induction of somatic embryogenesis from tissues in unopened flower buds of cocoa was studied with respect to physiological age, type of floral explant, genotype, and medium composition and phytohormones. Two-to-three-week-old staminodes were found to be the best explants for embryogenesis. Embryogenesis was affected by genotype and sugars. Two main types of abnormalities of the embryos were observed: fusion of the hypocotyls and multiple cotyledons. These embryos have a lower rate of conversion into plantlets. Cytological analyses of somatic embryo-regenerated plants revealed a somatic chromosome number of 2n = 2x = 20, similar to seed-derived plants. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved. [References: 24]
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The inability to conserve cocoa (Theobroma cacao L.) germplasm via seed storage and the vulnerability of field collections make the establishment of cryopreserved genebanks for the crop a priority. An effective encapsulation-dehyd...
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The inability to conserve cocoa (Theobroma cacao L.) germplasm via seed storage and the vulnerability of field collections make the establishment of cryopreserved genebanks for the crop a priority. An effective encapsulation-dehydration based cryopreservation system has been developed for cocoa but because the somatic embryos used for freezing arise after a protracted period of callus culture there is concern about maintenance of genetic fidelity during the process. Microsatellite markers for seven of the 10 cocoa linkage groups were used to screen a population of 189 primary somatic embryo-derived emblings and the 43 secondary somatic embryos they gave rise to. Of the primary somatic embryos, 38.1% exhibited polymorphic microsatellite profiles while for secondary somatic embryos the frequency was 23.3%. The same microsatellite markers used to screen another population of 44 secondary somatic embryos cryopreserved through encapsulation-dehydration revealed no polymorphisms. Scanning electron microscopy showed the secondary somatic embryos were derived from cotyledonary epidermal cells rather than callus. The influence of embryo ontogeny on somaclonal variation is discussed.
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The effects of five carbon sources (glucose, fructose, maltose, sorbitol, and sucrose) and two explant types (petals and staminodes) on cacao somatic embryogenesis was studied. No growth was observed on both types of explants cult...
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The effects of five carbon sources (glucose, fructose, maltose, sorbitol, and sucrose) and two explant types (petals and staminodes) on cacao somatic embryogenesis was studied. No growth was observed on both types of explants cultured on sorbitol containing media and slow growth was obtained on media supplemented with maltose. Depending on the genotype, the percentage of explants producing one or more embryos ranged from 6% to 99%, 18% to 98%, and 3% to 82% on media containing glucose, fructose and sucrose respectively. Explants cultured continuously on maltose or sorbitol-containing media failed to produce embryos. Staminode explants produced 3 to 10 times more somatic embryos than petals. A strong genotypic effect on somatic embryogenesis was observed. Staminode explants of the Forastero clones Laranja and PSUSca 6 produced 2 to 30 times more somatic embryos than the Trinitarios UF 613 and ICS 16. During embryo maturation and conversion, no significant differences were observed among glucose, fructose, maltose, or sucrose for embryo weight, total shoot and root production. However, we found that all plantlets produced on glucose had shoots with normal cacao leaves while the other carbon sources sometimes produced plantlets with cotyledon-like leaves..
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The establishment of cocoa embryogenic cell lines in liquid medium starting from high frequency somatic embryogenesis (HFSE) callus is described. The growth kinetics of the cultures during the multiplication and the expression ste...
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The establishment of cocoa embryogenic cell lines in liquid medium starting from high frequency somatic embryogenesis (HFSE) callus is described. The growth kinetics of the cultures during the multiplication and the expression steps conducted in 250?mL Erlenmeyer flasks were described for three genotypes selected for their agronomical traits (EET95, EET96, and EET103). The glucose and dissolved oxygen concentrations and the absorption of Murashige and Skoog medium macronutrients (nitrate, ammonium, potassium, sulfate, calcium, phosphorus, and magnesium) were monitored. The multiplication of the embryogenic calluses in a medium containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1?mg?L_(?1), initiated with an inoculation density of 20?g?L_(?1)of callus, was achieved. The growth rate was characterized by two phases, with the second being concomitant with a depletion of phosphorus and magnesium, and a decrease in the embryogenic potential of the callus. The expression of the callus embryogenic capacity was conducted in an auxin-free medium. The embryo production starting from 1 and 5?g?L_(?1)inoculation densities was compared. When placed in the optimal expression conditions in flasks, 1?g of callus produced 1000 to 1500 embryos within 5 to 7?wk. Finally, two paths for improving the plantlet regenerative capacities of cocoa SE produced in liquid medium were identified. Supplementing the expression medium with myo-inositol used as an osmotic agent at a concentration of 50?g?L_(?1)increased the embryo-to-plantlet conversion rate from 13–16% to 40–48%. A 6-wk culture of the embryos on a maturation medium in Petri dishes optimized their subsequent development into plantlets.
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Investigations were undertaken on the effect of liquid nitrogen (LN) storage time on survival and regeneration of somatic embryos of cocoa (Theobroma cacao sp. l.). Somatic embryos from different cocoa genotypes (AMAZ 3-2, AMAZ 10...
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Investigations were undertaken on the effect of liquid nitrogen (LN) storage time on survival and regeneration of somatic embryos of cocoa (Theobroma cacao sp. l.). Somatic embryos from different cocoa genotypes (AMAZ 3-2, AMAZ 10-1, AMAZ 12, SIAL 93, and IMC 14) at 15.45% moisture content were cryopreserved in LN for one h, four and eight weeks. Somatic embryos of the genotypes emerged from the alginate beads at different periods 4 to 12 weeks post-cryopreservation. Individual genotypes subjected to low temperature storage time did not show significant differences in post-cryopreservation survival, although different genotypes responded differently with AMAZ 12 and IMC 14 recording the highest and lowest mean survival rates of 58% and 35%, respectively. Plantlets originating from five genotypes had been weaned and developed normally comparable to non-cryopreserved somatic embryo-derived plantlets in the glasshouse. The effects of low temperature storage time on survival and genetic stability of somatic embryos using genotypes from the same parental and distinct lines may contribute to long-term storage of cocoa germplasm.
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Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacaoL.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was...
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Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacaoL.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was achieved through optimization of cryoprotectants (abscisic acid (ABA) and sugar), duration of osmotic and evaporative dehydration, and embryo development stage. Up to 63% of the genotype SPA4 early-cotyledonary somatic embryos survived cryopreservation following 7 days preculture with 1 M sucrose and 4 h silica exposure (16% moisture content in bead). This optimized protocol was successfully applied to three other genotypes, e.g. EET272, IMC 14 and AMAZ12, with recovery frequencies of 25, 40 and 72%, respectively (but the latter two genotypes using 0.75 M sucrose). Recovered SPA4 somatic embryos converted to plants at a rate of 33% and the regenerated plants were phenotypically comparable to non-cryopreserved somatic embryo-derived plants
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