Somatic Embryogenesis in Theobroma cacao L.

Theobroma cacao L. is a tropical tree originating in the Amazon, where it grows naturally in the shade of tropical rainforests. Cacao sub-products, such as butter and powder, are produced as principal components of chocolate and contain important nutritional compounds such as polyphenols and flavonoids. However, bean production is decreasing because plantations are antiquated and unproductive. Cacao propagation has been traditionally performed through classical propagation methods, such as grafting or rooted cuttings, but those methods are not sufficient to obtain large quantities of planting material with the desired genetic quality and optimal plant health. In the search for solutions to this problem, somatic embryogenesis (SE) is a vegetative method used for cacao propagation that has the potential to be explored. SE is a type of clonal propagation by which totipotent cells in the somatic tissue can develop into embryos and subsequently convert into plants.This method offers significant technological advantages because it is possible to obtain a large quantity of disease-free planting material with good agronomic characteristics and genetic stability. In T. cacao, tow techniques of in vitro micropropagation have been reported as direct and indirect SE. Indirect SE requires the additional step of cell dedifferentiation, unlike direct SE, which does not require this step. Here, we report a protocol using direct and indirect SE techniques using two types of culture methodologies-solid and liquid culture media.

Transcription Factors: Their Role in the Regulation of Somatic Embryogenesis in Theobroma cacao L. and Other Species.

Transcription factors are proteins that help with the control and regulation in the transcription of the DNA to mRNA by binding to special DNA sequences. With the aim to understand more about gene transcription regulation in Theobroma cacao L., this review outlines the principal transcription factors that were reported in other plants especially Arabidopsis thaliana and attempts at looking for the homologies with transcription factors in T. cacao. The information cited in this work is about the initiation, development, and maturation of the cacao somatic embryos and other crops. It is important to underline that there are very few publications in T. cacao discussing transcription factors that control the somatic embryogenesis process, but there is some information about transcription factors in other crops that we have used as a guide to try to understand this process.

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

OBJECTIVES: To 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. RESULTS: The 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. CONCLUSIONS: Activatable 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.

Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor.

BACKGROUND: Theobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state. RESULTS: An ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development. CONCLUSION: Our work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.

Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao.

Two dimensional electrophoresis and nano-LC-MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L(-1)) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses.

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

BACKGROUND: Theobroma 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. RESULTS: The 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. CONCLUSIONS: Large 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.

BACKGROUND: The 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. RESULTS: An 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. CONCLUSIONS: Our 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.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification.

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 percent) 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.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants.

*Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. *For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. *Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. *Statistical analysis confirmed this rather counterintuitive finding that leaves of 'late regenerants' exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

Influence of freezable/non-freezable water and sucrose on the viability of Theobroma cacao somatic embryos following desiccation and freezing.

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08-1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60-95% was recorded for embryos exposed to 0.5-1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H(2)O g(-1)dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.

The effectiveness of somatic embryogenesis in eliminating the cocoa swollen shoot virus from infected cocoa trees.

Investigations were undertaken on the use of somatic embryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from the infected callus tissues at the rate of 10 (19%), 18 (14%) and 16 (15%) for T1, T2 and T4, respectively. Virus free primary somatic embryos from the infected callus tissues converted into plantlets tested CSSV negative by PCR/capillary electrophoresis 2 years after weaning. Secondary somatic embryos induced from the CSSV-infected primary somatic embryos revealed the presence of viral fragments at the rate of 4 (4%) and 9 (9%) for T2 and T4, respectively. Real-time PCR revealed 23 of the 24 secondary somatic embryos contained no detectable virus. Based on these findings, it is proposed that progressive elimination of the CSSV in infected cocoa trees occurred from primary embryogenesis to secondary embryogenesis.

Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

Headspace volatile markers for sensitivity of cocoa (Theobroma cacao L.) somatic embryos to cryopreservation.

The mechanisms that reduce the viability of plant somatic embryos following cryopreservation are not known. The objective of the present study was to evaluate the sensitivity of cocoa (Theobroma cacao L.) somatic embryos at different stages of an encapsulation-dehydration protocol using stress-related volatile hydrocarbons as markers of injury and recovery. The plant stress hormone ethylene and volatile hydrocarbons derived from hydroxyl radicals (methane) and lipid peroxidation (ethane) were determined using gas chromatography headspace analysis. Ethylene and methane were the only volatiles detected, with both being produced after each step of the cryogenic protocol. Ethylene production was significantly reduced following exposure to liquid nitrogen, but then increased in parallel with embryo recovery. In contrast, the production of methane was cyclic during recovery, with the first cycle occurring earlier for embryos recovered from liquid nitrogen and desiccation than those recovered from earlier steps in the protocol. These results suggest that loss of somatic embryo viability during cryopreservation may be related to the oxidative status of the tissue, and its capacity to produce ethylene. This study has demonstrated that headspace volatile analysis provides a robust non-destructive analytical approach for assessing the survival and recovery of plant somatic embryos following cryopreservation.

Determination of theobromine, theophylline, and caffeine in by-products of cupuacu and cacao seeds by high-performance liquid chromatography.

Theobromine, theophylline, and caffeine are determined simultaneously by a rapid and selective reversed-phase high-performance liquid chromatography (HPLC) method with UV detection in by-products of cupuacu and cacao seeds. The determination is carried out in the raw and roasted ground cupuacu seeds and in the corresponding powders obtained after pressure treatment. The by-products of both cupuacu seeds and cacao seeds are obtained under the same technological conditions. The HPLC method uses isocratic elution with a mobile phase of methanol-water-acetic acid (80:19:1) (v/v) at a flow rate of 1 mL/min and UV absorbance detection at 275 nm. Total elution time for these analytes is less than 10 min, and the detection limit for all analytes is 0.1 mg/g. The amounts of theobromine and caffeine found in all the cupuacu samples are one or more orders of magnitude lower than those from cacao. Theophylline is found in all cacao samples except for the roasted ground paste, and it is only found in the roasted ground paste in the cupuacu samples.

Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.).

Mutation rates are often elevated in plants regenerated from in vitro culture, giving rise to so-called 'somaclonal variation'. Detailed characterisation of mutation profiles that arise during culture should improve our understanding of processes influencing mutation and allow the selection of protocols yielding the fewest/least severe changes. Somatic mutations will usually produce genetic chimeras where unchanged alleles are retained by some cells. Such chimeras are difficult to detect but likely to form a significant proportion of any regenerant population. We present a simple protocol that enables the provisional diagnosis of both homogenous and chimeric mutants among large regenerant populations, together with a semi-quantitative means of estimating the proportion of mutant cells. The assay exploits consistent differential amplification of alternate simple sequence repeat alleles at heterozygous loci. Calibration of the relative amplification of alleles from two genotypes-and the synthetic chimeras created from them-revealed a strong linear relationship between 'peak heights' representing alternate alleles following capillary electrophoresis. The assay predicts chimeric composition to a reasonable level of confidence (+/-5%) so long as the infrequent allele exceeds 15% of the template. The system was applied to 233 regenerants of cocoa somatic embryogenesis and identified 72 (31%) putative chimeric mutants for slippage mutation or allele loss across two loci.

Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis.

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues.

Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

Developmental variation of sugars, carboxylic acids, purine alkaloids, fatty acids, and endoproteinase activity during maturation of Theobroma cacao L. seeds.

The changes of mono- and oligosaccharides, carboxylic acids, purine alkaloids, and fatty acid composition, and of aspartic endoproteinase activity, were analyzed during seed development in two varieties of cacao (Theobroma cacao). The majority of the components examined either decreased or accumulated steadily in concentration during the second half of bean development. Sucrose is the major sugar in the mature embryo, whereas fructose and glucose are at higher concentrations in the endosperm tissue. Considerable amounts of malate are found in the endosperm, whereas citrate is the dominant carboxylic acid in the embryo. A major change in the fatty acid composition occurs in the young embryo when the proportion of stearic acid increases rapidly at the expense of linoleic acid, which is reduced from about 18 to 3%. Theobromine is the dominant purine alkaloid (ca. 80%), and caffeine appears only toward the end of seed maturity. Aspartic endoproteinase activity increases rapidly during embryo expansion, reaching a maximal activity before final maturity. The results are discussed in conjunction with physiological changes in developing seeds, and the potential contributions of the compounds analyzed for cocoa quality.

Somatic embryogenesis from flower explants of cocoa (Theobroma cacao L.).

Two types of flower explants, staminoides and petals, were used for in vitro induction of somatic embryos in cocoa. After 14 days in culture, we observed globular structures and callus formation on both types of explants. However, the better results were obtained on staminoides: 98.3% formed callus and 86.2% somatic embryos on Murashige and Skoog (1962) medium supplemented with sucrose, coconut water, 2,4-D, kinetin and agar.