Cacao Somatic Embryogenesis Research Articles

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

<p class="abstrakinggris">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<sup>-1 </sup>+ kinetin 0.5 mg l<sup>-1</sup> or 2,4-D 2 mg l<sup>-1</sup> + kinetin 0.125 – 0.250 mg l<sup>-1</sup> + thidiazuron (TDZ) 2.5 – 5 µg l<sup>-1</sup> or 2,4-D 2 mg l<sup>-1</sup> + TDZ 10 µg l<sup>-1</sup>. 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<sup>-1</sup> + kinetin 0.5 mg l<sup>-1</sup> (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<sup>-1</sup> + kinetin 0.5 mg l<sup>-1</sup>. The formation of SSEs could increase the multiplication rate of MCC 01 clone by 7 times.</p>

Effect of Container Culture Types on The Success of Cacao Somatic Embryos Formation

<p><em>Container culture have an important role in determining the success of in vitro culture since it will affect the development of culture, such as the formation of embryonic structures. The study aimed to determine the effect of culture container types on cacao somatic embryogenesis. The study was conducted at Tissue Culture Laboratory, Superior Seed Development Unit of IAARD, Bogor, from April to September 2016. The tests were conducted on the effect of container and explant types as well as the effect of container types and genotypes. The effects of container and explant types were tested using callus induced from petal and staminoid explants of Sca 6, whereas the effects of container types and genotypes were tested using callus induced from petal explants of Sca 6 and ICCRI 4. Afterwards, the somatic embryos were induced using petri dishes or culture bottles according to treatment. The results showed no significant interaction between container and explant types on the average percentage of the formation and number of somatic embryos (10.28% embryos/explants in culture bottles and 7.89% embryos/explants in petri dishes). Meanwhile, there was significant interaction between genotypes and container types in the initial period of somatic embryos formation (15 and 18 weeks after culture), but the effect was not significant in the final period of observation (21 weeks after culture). The results indicate that culture bottles, which have lower prices, can be used to replace petri dishes to induce the formation of somatic embryos in cacao.</em></p>

Inducible somatic embryogenesis in Theobroma cacao achieved using the DEX-activatable transcription factor-glucocorticoid receptor fusion

ObjectivesTo carry out mass propagation of superior plants to improve agricultural and silvicultural production though advancements in plant cell totipotency, or the ability of differentiated somatic plant cells to regenerate an entire plant.ResultsThe first demonstration of a titratable control over somatic embryo formation in a commercially relevant plant, Theobroma cacao (Chocolate tree), was achieved using a dexamethasone activatable chimeric transcription factor. This four-fold enhancement in embryo production rate utilized a glucocorticoid receptor fused to an embryogenic transcription factor LEAFY COTYLEDON 2. Where previous T. cacao somatic embryogenesis has been restricted to dissected flower parts, this construct confers an unprecedented embryogenic potential to leaves.ConclusionsActivatable chimeric transcription factors provide a means for elucidating the regulatory cascade associated with plant somatic embryogenesis towards improving its use for somatic regeneration of transgenics and plant propagation.

Pengaruh Sitokinin, Jenis Eksplan, dan Genotipe terhadap Embriogenesis Somatik Kakao

<p><em>Information on the effect of cytokinins on cacao (</em>Theobroma cacao<em> L.) primary somatic embryogenesis and its interaction with explant types and genotypes is not yet known. This study aimed to evaluate the effect of cytokinins and its interaction with explant types and genotypes on cacao somatic embryogenesis. The study was conducted at tissue culture laboratory of IAARD, Bogor from April until December 2012 and October 2014 until February 2016. Three types of cytokinins i.e. kinetin (0.58, 1.16, and 2.32 </em><em>μ</em><em>M), thidiazuron (0.01, 0.02, and 0.04 </em><em>μ</em><em>M) and benzylaminopurine (0.55, 1.11, and 2.22 </em><em>μ</em><em>M) in combination with 9 </em><em>μ</em><em>M 2,4-D were tested for their effectiveness in inducing somatic embryogenesis from petals and staminoid explants of Cimanggu 1 genotype. Furthermore, three levels of kinetin (0.58, 1.16, and 2.32 </em><em>μ</em><em>M</em><em>) also in combination with 9 </em><em>μ</em><em>M 2,4-D were evaluated for their influences on the somatic embryogenesis from petals and staminoid explants of three cacao genotypes i.e. Sulawesi 02, ICCRI 04 and Cimanggu 3. The result demonstrated that 2.32 </em><em>μ</em><em>M kinetin and staminoids explant were more effective to induce cacao somatic embryogenesis of Cimanggu 1 genotype (7%, 0.23 embryos/explant). Additionally, there were interaction effects between the level of kinetin with explant types and genotype on the percentage of explants forming embryo at 12 weeks after culture. The highest percentage of somatic embryo formation was shown by ICCRI 04 genotype with the use of petals explant and a kinetin level of 1.16 </em><em>μ</em><em>M (31.85%), but not significantly different from the level of kinetin 2.23 </em><em>μ</em><em>M (25.55%). The formation of primary somatic embryos of cacao is largely determined by the type and level of cytokinins, type of explant, and genotype.</em></p>

EFFECTIVE CACAO SOMATIC EMBRYO REGENERATION ON KINETIN SUPPLEMENTED DKW MEDIUM AND SOMACLONAL VARIATION ASSESSMENT USING SSRs MARKERS

This study aimed to develop the cacao ( Theobroma cacao L.) in vitro regeneration system through somatic embryogenesis on kinetin supplemented DKW medium and somaclonal variation assessment using SSR markers. Callus were initiated from basal petal and staminoid explants cultured on callus induction (CI) medium contained DKW basalt salts and kinetin:2,4-D ratios of 1:15.5, 1:7.8 or 1:3.9 and then transferred onto secondary callus growth (SCG) medium contained WPM basalt salts and kinetin:2,4-D ratios of 1:7.8 or 1:3.9. The calli were then subsequently transferred onto embryo development medium contained DKW basal salts with or without the addition of amino acids, adenine or activated charcoal for the formation of somatic embryos. Nine cacao genotypes were tested for their ability to develop somatic embryos. Results of this study indicated DKW medium supplemented with Kinetin in combination with 2,4-D effectively induced cacao somatic embryogenesis. The highest somatic embryos formation was abtained from kinetin:2,4-D ratio of 1:3.9 and 1:7.8 in CI and SCG medium respectively. Cacao genotype responses were highly explant type dependent. The developed method resulted in a high percentage of somatic embryo formation (5.6-66.7%), germination (50%) and plantlet conversion (65%) and a medium percentage of somaclonal variations based on SSRs marker analysis.

Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree.

BackgroundTheobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray.ResultsThe expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos.ConclusionsLarge numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis.

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation.

BackgroundThe Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network.ResultsAn ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction.ConclusionsOur study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm.

Sulphur depletion altered somatic embryogenesis in Theobroma cacao L. Biochemical difference related to sulphur metabolism between embryogenic and non embryogenic calli

Somatic embryogenesis is a useful tool for Theobroma cacao improvement and propagation. Depending on culture medium composition, different morphogenetic structures (including somatic embryo) occur in response to alteration of genes expression patterns and biochemical changes. The effect of SO 4 2 - ion deficiency in culture media on somatic embryogenesis was studied through sequential replacement of MgSO 4 and K 2 SO 4 by MgCl 2 and KCl, respectively, at different steps of somatic embryogenesis. It appears that explants gradually lost their embryogenic competence as the period of exposition to sulphur free medium increases. These results suggest that, sulphur availability and the duration to sulphur exposition might modulate the expression of genes involved in somatic embryo differentiation in T. cacao . Cysteine, glutathione, reducing sugars, cysteine synthase and cysteine desulfurase activities were analysed in different morphogenetic structures obtained in vitro . Cysteine and reducing sugars contents appeared to be higher in embryogenic calli than their nonembryogenic homologues, whereas glutathione content appears to be lower in embryogenic calli. Cysteine synthase activities also discriminate the embryogenic calli from non embryogenic calli. In the embryogenic calli, the ratio cysteine synthase/cysteine desulfurase activities were above unit. The assimilation of exogenous sulphur (sulphate) for the synthesis of cysteine might hence be crucial for somatic embryogenesis in T. cacao . This explains the reduction and the absence of somatic embryo response observed during sulphur depletion in culture media. Sulphur nutrition is therefore critical in cacao somatic embryogenesis. Keyswords: Cacao, embryo, sulphate, cysteine synthase, glutathione, deficiency African Journal of Biotechnology Vol. 9(35), pp. 5665-5675, 30 August, 2010

Effects of Carbon Source and Explant Type on Somatic Embryogenesis of Four Cacao Genotypes

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.

Characterisation of the cacao somatic embryogenesis receptor-like kinase ( SERK) gene expressed during somatic embryogenesis

Somatic embryogenesis is a useful tool for plant propagation and consists of a good model for embryo development studies. During somatic embryogenesis, some morphological and biochemical changes occur in response to alterations in gene expression patterns. The somatic embryogenesis receptor kinase ( SERK) gene is an important element related to this process. In this work we have focused on the function of SERK in Theobroma cacao. Our main questions were whether this gene exists in this plant with another arrangement; if it is functional and, in a positive case, in which tissues it acts. We have found only one copy of SERK that is functional and analyzed its expression in a series of tissues. The phylogenetic tree showed that the available sequences of SERK are highly conserved and a considerable number of species contain different sequences of SERK, confirming it as a gene family with variant repeats.

Moxalactam as a counter-selection antibiotic for Agrobacterium-mediated transformation and its positive effects on Theobroma cacao somatic embryogenesis

Previous work in a number of laboratories has led to the development of a highly efficient cacao somatic embryogenesis (SE) protocol, establishing the basis for an Agrobacterium tumefaciens-mediated genetic transformation system. Such a system will be valuable as a basic research tool, and possibly in the future as a means to introduce specific disease resistance or other genes into cacao. However, during our initial attempts at transformation, cacao tissue was frequently destroyed due to Agrobacterium overgrowth following co-cultivation. Furthermore, the addition of the antibiotic cefotaxime, commonly applied to tissue culture media to eliminate Agrobacterium post infection, decreased cacao somatic embryo production by 86%. To define antibiotic conditions sufficient to suppress Agrobacterium while not interfering with cacao SE, we evaluated the efficacy of four antibiotics. Two cephalosporins, cefotaxime and moxalactam; and two penicillins, amoxicillin and carbenicillin. Moxalactam, a β-Lactam antibiotic, was proven to effectively suppress Agrobacterium growth. Furthermore, at certain concentrations, it also significantly enhanced the efficiency of cacao SE. A possible mechanism for this enhancement is proposed.