Bhatt, Jishnu J.Enhancing the Efficiency of Somatic Embryogenesis and Genetic Transformation in Theobroma cacao L. Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. May 2025.
Abstract: The chocolate tree, Theobroma cacao L. or cacao, is one of the most globally important woody tree species because its seeds provide the raw ingredients to make chocolate. Global cacao productivity is threatened by diseases and pests which result in annual potential losses of around thirty percent while its demand is ever-growing. Currently, there is a global replanting requirement of over one billion trees annually that is not being met. Utilizing high efficiency propagation systems such as somatic embryogenesis (SE) and the acceleration of genetic improvements for disease resistance through biotechnology are powerful strategies to combat these problems. Both basic and applied research in cacao is currently bottlenecked by limitations in the regeneration of somatic embryos (SEs) and the genetic modification system, highlighting the importance of improving these methodologies.
The work in this dissertation begins with investigating a means to expand the tissue range by which SE can be initiated from the cacao tree. Nearly all tissues in cacao are recalcitrant to the formation of embryogenic calli, from which SEs are indirectly regenerated. Initiating embryogenic cultures has only been possible from petals and staminodes of immature cacao flowers, or zygotic tissues as starting explants and limits all SE work to adult, flowering trees. Previous work has established that the overexpression of the Leafy Cotyledon 2 (TcLEC2) gene significantly enhanced the embryogenic potential of floral explants, and its activation allowed for regeneration of SEs from cacao leaves. In Chapter 2, a tissue from transgenic tree expressing inducible TcLEC2-glucorticoid receptor fusion protein was used to evaluate embryogenic potential of additional vegetative tissues. Upon activating TcLEC2 for a 72-hour pulse, by adding dexamethasone to the regeneration medium, all tissues including peduncles, shoot apices, petioles, internodes, and leaves from development stages A-D were able to regenerate SEs. Notably, under the induction conditions, explants from fully expanded leaves (developmental stage C and D) regenerated SEs at rates similar to floral petals. In addition, the leaves regenerated abnormal SEs at lower rates compared to other vegetative tissues evaluated in this experiment. This demonstrates that various non-sexual somatic tissues from juvenile cacao trees contain cells that are competent for embryonic conversion.
Most plant genetic engineering methods are based on Agrobacterium tumefaciens-mediated transformation. A. tumefaciens excises and delivers T-DNA (transfer DNA) molecules often from a binary vector (or plasmid) to be incorporated into the genome of plant cells through a process called genetic transformation. It is well known that the compatibility of the plant host and A. tumefaciens strain are important factors for transformation success. However, it has not been well reported in the literature the influence of the binary vector and/or T-DNA design on transformation success. In Chapter 3, three binary vectors were functionally tested using a cacao leaf transient expression assay to determine whether vector backbone could aid in overcoming transformation recalcitrance. It was found that a ‘pLSU’ vector backbone outperformed the others by transiently expressing an enhanced green fluorescent protein (EGFP) reporter gene by over 18% and therefore was chosen for further optimization experiments. The T-DNA region of the pLSU binary vector was re-designed to be compatible with restriction enzyme-based, recombinase-based, PCR-based and Golden-Gate based cloning methods and additionally modified to contain translationally efficient Kozak sequences. The modified pLSU binary vector with the improved T-DNA region was used for a stable transformation of cacao SE cotyledons. Compared to the standard pCambia- based binary plasmid, pLSU resulted in a 3.49-fold increase in EGFP fluorescence in transformed cells measured six days after transformation. However, under the experimental conditions provided, transient transformation coverage and regeneration efficiency of stable transgenics was not improved, indicating that further optimization is required to overcome stable transformation recalcitrance, which is bottlenecked by other factors such as the host plants biological variability.
This work demonstrates that ectopically activating the TcLEC2-GR fusion protein contributed to in vitro regeneration of SEs from previously recalcitrant somatic tissues of cacao (Chapter 2). TcLEC2 is one of a growing number of developmental regulating genes (DRGs) that have been demonstrated to have embryogenesis-promoting effect. In Chapter 4 of this work, five additional DRGs SERK1, PLT5, WIND1, WUS, and WOX9 were chosen based on their potential to improve SE in cacao and to be used as positive selection for regeneration of transgenic plants. The ortholog genes were identified in the cacao Scavina 6 genome bioinformatically, synthesized and cloned into the improved binary vectors described in Chapter 3 under control of an estradiol-inducible system (XVE-DRGs). The estradiol-inducible system was tested using an EGFP transient expression assay in tobacco leaves and was validated to be functional and ‘non-leaky.’ The five XVE-DRG vectors were transformed into wild-type PSU-Sca6 cacao secondary SE (SSE) cotyledons using a standard cacao transformation protocol. DRG expression was induced one month after culture initiation (ACI) in vitro. Approximately 400 explants were imaged individually at 7 time points over a three-month period under a fluorescence microscope to quantify regeneration and transformation metrics and the influence of DRG expression. It was found that a two-week pulse of DRG induction one-month ACI was not effective at changing the growth rate of calli or the regeneration of both non-transgenic and transgenic tertiary SEs (TSEs). Analysis of EGFP fluorescence as a marker for successful transformation revealed that stable transformation area coverage of cacao SSE cotyledon explants averaged around 9.5% and cacao calli grew at a rate of ~4.4% per week. Additionally, the size of SSE cotyledon explants did not show correlation with their transformation coverage and TSE regeneration rate. The growth rate of calli was analyzed under increasing concentrations of geneticin antibiotic, and the results showed that increasing the concentration to 150 mg/L was inhibitory for both non-transgenic and transgenic cacao cells in vitro, establishing a previously unreported upper threshold for geneticin selection.
This study also yielded six transgenic TSEs from transformations of all the XVE-DRGs except TcWUS, which can be utilized for further investigations into their potential for overcoming cacao in vitro and transformation recalcitrance.
This work aimed to improve the efficiency of regeneration of genetically modified cacao somatic embryos for basic and applied plant science research. First, it demonstrated that nearly all differentiated non-sexual somatic cells of the juvenile cacao tree must harbor pre-competent and/or competent cells which require the correct spatiotemporal ectopic expression of totipotency- promoting genes for their conversion towards an embryogenic fate. Next, it provided evidence that the binary vector backbone is an important element of transformation recalcitrance. Finally, methodology was created to characterize transformation and in vitro regeneration in cacao to generate new insights on how the system can be optimized in the future. Altogether, these findings are synergistic towards improving the transformation and regeneration system of cacao and identified knowledge gaps that must be addressed to overcome the recalcitrance of this globally important and beloved tree. View online.
Menéndez Burns, Francisco Miguel. Genomics and Physiological Response to Cadmium and the Evolution of Heavy Metal ATPases in Theobroma cacao L. Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. May 2025.
Abstract: Exposure to toxic elements through contaminated food poses significant health risks to consumers globally. Metal toxicity, particularly cadmium (Cd), has been identified as a major health concern. Regulatory bodies like the European Food Safety Authority (EFSA) and the World Health Organization (WHO) have recognized cacao beans and chocolate as sources of Cd in the human diet, prompting the establishment of safe upper limits for Cd in these products. Additionally, California’s Proposition 65 addresses dietary exposure to Cd by establishing safe harbor levels for toxic elements, highlighting the importance of monitoring and regulating Cd levels in food. The literature reviewed on this topic is included in Chapter 1.
This thesis examines molecular mechanisms and physiology of cadmium (Cd) uptake in cacao, focusing on the Heavy Metal ATPase (HMA) gene family known for its role in Cd transport. The research provides insights that could aid development of future mitigation practices and selection of low Cd-accumulating genotypes. Chapter II describes the identification and evolutionary analysis of the HMA gene family in Theobroma cacao L. Eight genes were identified that encode for HMA proteins, including 2 IB-2, Zn²⁺, and Cd²⁺ transporters gene orthologs, TcHMA2 and TcHMA3. Synteny analysis across diverse cacao genomes revealed structural variations and positive selection within the IB-2 HMA gene family. Expression pattern analysis demonstrated a high correlation between TcHMA2 and TcHMA3, suggesting low evolutionary divergence. The comparative analysis between T. cacao and Arabidopsis thaliana HMA genes revealed evolutionary divergence and sub-functionalization A. thaliana.
Chapter III describes the functional characterization of cacao candidate genes TcHMA2 and TcHMA3 in yeast, a heterologous system, resulting in reduced yeast growth rates, an indirect effect of the import of Cd to the yeast cytosol. TcHMA2 has a truncated C-terminal domain and demonstrated lower Cd transport efficiency than the full-length TcHMA3 gene.
Chapter IV includes the results of a collaborative study between Penn State and the Colombian Agricultural Research Corporation (AGROSAVIA), Colombia. The study characterizes the transcriptional responses to Cd treatment in leaf and root tissues from two cacao genotypes: PA121 (a high-Cd accumulator) and TSH660 (a low-Cd accumulator). Seedlings from these genotypes were exposed to Cd at 10 PPM for 48 hours. Total mRNA from leaves and roots was extracted and sequenced, and differential gene expression analysis was conducted. The study identified thousands of differentially expressed genes (DEGs) after 48 hours of Cd exposure: 4185 in roots and 2396 in leaves. Key pathways were significantly regulated, including ion transport, water stress, carbohydrate metabolism, pathogen response, and the biosynthesis of plant growth hormones such as abscisic acid (ABA), ethylene, indole-3-acetic acid (IAA), and melatonin. Based on the results from the transcriptome study, a working model for the Cd response in cacao was developed. To test the predicted model, open-pollinated seedlings of PA121 were grown in greenhouse conditions and exposed to Cd. The physiological response was evaluated after 36 hours of Cd treatment. Results indicated cacao plants treated with Cd had a significant reduction in leaf stomatal conductance, correlating gene expression changes in the ABA pathway to physiological outcomes.
This study provides comprehensive insights into the evolution and functions of HMA genes in cacao. It also offers valuable information related to the molecular and physiological responses of cacao plants to Cd exposure. Together, this work contributes significantly to the body of knowledge on abiotic stress and provides significant insights on heavy metal uptake in cacao. Download PDF.
Winters, Noah P. Evolutionary and functional genetics of disease resistance in theobroma cacao and its wild relatives. A Dissertation in Ecology Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2022
Abstract: Plants have complex and dynamic immune systems that have evolved over millennia to help them resist pathogen invasion. Humans have worked to incorporate these evolved defenses into crops through breeding. However, many crop cultivars only harness a fraction of the overall genetic diversity available to a given species, or have such a long history of domestication that most diversity has been lost. Evaluating previously neglected germplasm for desirable traits, such as disease resistance, is therefore an essential step towards breeding crops that are adapted to both current and emerging threats. In this dissertation, we examine the evolution of defense response across populations of Theobroma cacao L. and wild species of Theobroma, with the goal of identifying genetic elements essential for protection against the cacao pathogen Phytophthora palmivora. Download PDF.
Knollenberg, Benjamin. Characterization of Specialized Metabolites Involved in Black Pod Rot Resistance in Theobroma Cacao. A Dissertation in Plant Biology Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy June 2020. Download PDF.
Abstract: Cacao (Theobroma cacao, chocolate tree) is an important tropical crop that provides the raw ingredients for chocolate production and provides an important source of income to over five million farmers worldwide. Production of cacao is continually limited by diseases, with global annual crop losses estimated between thirty and forty percent. Oomycete (water mold) pathogens in the genus Phytophthora are the most widespread and economically destructive of the cacao pathogens, causing a disease known as black pod rot (BPR). Increasing cacao resistance to BPR is the most promising strategy for reducing yield losses due to the disease but requires an understanding of cacao defense mechanisms of cacao against BPR. Plant specialized metabolites are well known to contribute to plant defense and have been underexploited as breeding targets in cacao. In this work, the metabolite composition of a BPR tolerant and a BPR susceptible genotype were compared by untargeted metabolomics using liquid chromatography – tandem mass spectrometry (LC-MS/MS). This analysis revealed that the tolerant genotype ‘Scavina 6’ (‘Sca6’) accumulates clovamide, a hydroxycinnamic acid amide (HCAA), and several related compounds in leaf tissue at much higher concentrations that the susceptible genotype ‘Imperial College Selection 1’ (‘ICS1’). Both genotypes tested produced equivalent amounts of clovamide in fruit peel, but sulfated HCAAs accumulated to high levels only in ‘Sca6’ in this tissue. These results implicated clovamide and HCAAs as important contributors to the difference in resistance between the two genotypes, so clovamide was synthesized and characterized for its role in defense. Clovamide was found to be a substrate for cacao polyphenol oxidase (PPO), which is involved in reactive quinone formation, protein cross linking, and oxidative browning. Furthermore, clovamide inhibited protease and pectinase in vitro, activities associated with pathogenicity of Phytophthora spp. Clovamide also inhibited growth of three Phytophthora species in vitro. Based on these activities of clovamide and the high relative accumulation of clovamide in ‘Sca6’ I determined that clovamide is an important resistance factor in cacao and a promising breeding target for enhancing resistance. Since clovamide appears important for BPR resistance in cacao, I sought to elucidate cacao genes involved in its biosynthesis by combining metabolomics (LC-MS/MS) and transcriptomics (RNA-Seq) analysis of ‘Sca6’ and ‘ICS1’ leaves of three developmental stages and functional characterization of candidate clovamide biosynthetic genes. By correlating clovamide abundance with that of over 1,000 metabolites identified in extracts by LC-MS/MS, I found a strong negative correlation between clovamide and several flavonoid glucosides. Based on this observation I hypothesized that clovamide biosynthesis directs flux of UDP-glucose away from flavonoid glucoside production and therefore proceeds through a pathway requiring UDP-glucose dependent glycosyltransferase (UGT) and serine carboxypeptidase-like acyltransferase (SCPL-AT) activities. Following this hypothesis, I identified three cacao UGT and three SCPL-AT candidate genes based on expression patterns consistent with HCAA accumulation. All possible UGT/SCPL-AT candidate combinations were co-expressed in N. benthamiana to identify only one combination that resulted in clovamide production: cacao gene IDs (Criollo v2 reference genome) Tc04v2_g000570 (UGT) and Tc02v2_g013480 (SCPL-AT). The UGT candidate, Tc04v2_g000570 was more highly expressed in ‘Sca6’ than ‘ICS1’ (~5.6-fold, p < 0.00001) and was also found to have a premature stop codon in the coding sequence of the ‘ICS1’ allele, resulting in a truncated predicted protein sequence lacking a conserved UGT domain identified by InterProScan. Taken together, these results demonstrate that differential expression and allelic variation in the UGT Tc04v2_g000570 are the likely cause of the difference in leaf clovamide content between ‘Sca6’ and ‘ICS1’. The proposed UGT/SCPLAT pathway for clovamide biosynthesis in cacao is in contrast to the 4-CL/BAHD-AT pathway reported in red clover. BAHD- and SCPL-acyltransferases are evolutionarily distinct protein families, meaning that the two species evolved clovamide biosynthesis completely independently. In order to identify other genes controlling leaf clovamide content that were not detected using the transcriptomics/metabolomics approach, a quantitative trait loci (QTL) analysis for clovamide content was performed in an F2 population derived from ‘Sca6’ and ‘ICS1’. Leaf clovamide content was quantified in 116 F2 trees by HPLC-DAD and clovamide was found to be segregating. Another hydroxycinnamic acid derivative, chlorogenic acid (CGA), was also segregating. Clovamide and CGA content had a slight but significant negative correlation in the F2 population, so CGA was included as a covariate in QTL analyses. Two QTL were identified for clovamide, including one on chromosome 1 (“QTL1”) and one on chromosome 4 (“QTL4”). A three-factor model including QTL1, QTL4, and CGA explained 20.91% of variation in leaf clovamide content (p = 8.34E-05), suggesting that environmental factors or other undetected loci are also contributing to the variation. RNA-Seq data from ‘Sca6’ and ‘ICS1’ leaves was analyzed to identify differentially expressed genes (DEGs) in the two QTL. QTL4 contained several DEGs including Tc04v2_g000570, the UDP-glucose dependent glycosyltransferase (UGT) previously identified as a clovamide biosynthetic gene using the metabolomics/transcriptomics approach described above. QTL4 also contained an uncharacterized chloroplastic ABC transporter with a suspected role in clovamide precursor transport that was more highly expressed in ‘Sca6’ than ‘ICS1’ (~4-fold higher, p < 0.05). QTL1 also contained several DEGs including one uncharacterized serine carboxypeptidase-like acyltransferase (SCPL-AT) that had slightly higher expression in ‘Sca6’ than ‘ICS1’ (~0.9-fold, p < 0.05). A different cacao SCPL-AT was previously identified as a clovamide biosynthetic gene, described above. Surprisingly, the two clovamide QTL overlapped perfectly with two previously identified self-incompatibility loci in cacao, suggesting a potential but currently tenuous connection between clovamide biosynthesis and self-incompatibility in cacao. Overall, this work identifies a new resistance factor in cacao, clovamide, that should be explored as a breeding target and metabolic selectable marker in cacao breeding programs. Furthermore, it contributes to our broader understanding of plant specialized metabolite biosynthesis by identifying an example of convergent evolution in clovamide biosynthesis, which proceeds by an SCPL-AT in cacao and a BAHD-AT in red clover. This work also generates new questions about the potential role of clovamide in self-incompatibility in cacao that warrant further exploration. The integration of metabolomics, transcriptomics, and QTL analysis presented in this work may provide a road map for future attempts to identify important defense metabolites and elucidate their biosynthetic pathways in other systems.
Fister, A. S. (2016). Genomics of the theobroma cacao L. defense response (Order No. 10300628). Available From ProQuest Dissertations & Theses A&I. (1848683680). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/1848683680?accountid=13158
Abstract: Theobroma cacao, the source of cocoa and a cash crop of global economic importance, suffers significant annual losses due to several pathogens. While study of the molecular mechanisms of defense in cacao has been limited, the recent sequencing of two cacao genomes has greatly expedited the ability to study genes and gene families with roles in defense. Here, the pathogenesis-related (PR) gene families were bioinformatically identified, and family size and gene organization were compared to other plant species, revealing significant conservation throughout higher monocots and dicots. Expression of the PR families was also analyzed using a whole genome microarray to measure transcriptomic regulation in leaves after treatment of cacao seedlings with two pathogens, identifying the induced PR genes within each family. We found significant overlap between the PR genes induced by the pathogens, and subsequent qRTPCR revealed up to 5000-fold induction of specific PR family members. Next, the regulation of the defense response in cacao by salicylic acid, a major defense hormone, was analyzed. The study focused on two genotypes, the broadly resistant Scavina 6 and the widely susceptible ICS1. First, treatment of leaves of two cacao genotypes with salicylic acid was shown to enhance resistance of both. Moreover, overexpression of TcNPR1, a master regulator of systemic acquired resistance, is also shown to enhance the defense response, supporting the importance of salicylic acid and its downstream targets in cacao immunity. Microarray analysis of the transcriptomic response to salicylic acid revealed genotype-specific responses to hormone treatment. ICS1 appeared to show a more canonical response to salicylic acid, with more PR genes induced, while Scavina 6 exhibited increased expression of chloroplastic and mitochondrial genes. It was hypothesized that this induction was linked to increased ROS production, and subsequent ROS staining experiments confirmed higher concentration of superoxide in salicylic acid-treated Scavina 6 leaf tissue. Third, a pilot study was performed to quantify genetic variability within defense genes. Using DNA samples representing three populations of cacao - Peruvian, Ecuadorian, and French Guianan - we amplified three genes involved in defense, two predicted to be more variable (cysteine-rich repeat secretory peptide 38 and a polygalacturonase inhibitor) and one predicted to harbor less polymorphism (pathogenesis-related 1). Population genetic analysis of variability suggested that the gene predicted to be more variable may be under diversifying selection, suggesting that they may directly interact with rapidly evolving pathogen proteins. The experiment validated previously described observations about the populations, in particular that the French Guianan population was less variable than the others. The study also supported the predictions regarding gene variability, indicating that our strategy for identifying genes with more variation appears to be applicable but will require further validation. The Guiltinan-Maximova lab developed a protocol for transient transformation of cacao leaf tissue, which has been applied to characterizing gene function in several published analyses. Here the highly efficient protocol is presented in full, along with data collected in a series of optimization experiments. We also use the protocol to demonstrate the effect of overexpression of a cacao chitinase after subsequent infection with Phytophthora mycelia. A preliminary study describing a strategy for selection of high-priority candidate genes for functional characterization is described. Six genes were cloned and overexpressed using the transient transformation protocol; and while the study showed the ability of our protocol to significantly increase transcript abundance of the gene of interest, it did not validate the role of any of the genes in defense by showing decreased susceptibility. This dissertation contributes to the study of genomics and molecular mechanisms of defense in four key ways: 1) 15 classes of defense genes are identified and their expression dynamics are characterized, 2) genotype-specific differences in defense response are identified, providing insight into different strategies for survival, 3) variability within defense genes is discovered, differentiating populations of cacao and providing evidence for diversifying selection, and 4) a rapid and efficient strategy for gene functional analysis, which will enhance future genetic analyses in cacao, is presented.
Zhang, Y. (2014). Functional genomics of theobroma cacao fatty acid biosynthesis: Convergence of fatty acid desaturation, embryo development, and defense signaling responses (Order No. 3690183). Available From ProQuest Dissertations & Theses A&I. (1658228179). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/1658228179?accountid=13158
Abstract: Theobroma cacao L. (chocolate tree) is an important cash crop for 40-50 million farmers and their families in its tropical growing regions worldwide. Cocoa butter and cocoa powder extracted from cacao seeds provide the main raw ingredients for chocolate manufacturing, supporting a $80 billion global business. A unique fatty acid composition of cocoa butter makes its melting temperature close to the human body temperature, which is not only of particular importance for industrial uses, but also a valuable quality trait targeted by breeding programs. My Ph.D. dissertation focused mainly on the fatty acid biosynthesis pathway in cacao seeds. I identified a key desaturase gene TcSAD1 from a large stearoyl-acyl carrier protein-desaturase gene family in cacao that plays a crucial role in converting stearic acid (18:0, saturated fatty acid) into oleic acid (18:1, unsaturated fatty acid). The expression of TcSAD1 was highly correlated with the change of fatty acid composition during cacao seed development. The activity of TcSAD1 rescued all the Arabidopsis ssi2 (a fatty acid desaturase) related mutant phenotypes, further supporting its in vivo functions. The discovery of the critical function of TcSAD1 offers a new strategy for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs, to help pyramid genes for quality traits such as cocoa butter content. Moreover, because of the significance of fatty acid biosynthesis and lipid accumulation during cacao seed development, to further explore the regulatory mechanism, I functionally characterized of a master regulator, TcLEC2 gene, which controls both zygotic and somatic embryo development of cacao. Transient overexpression of TcLEC2 induced the expression of a variety of seed specific genes in cacao leaves. Furthermore, functions of TcLEC2 were explored during somatic embryogenesis, which is an in vitro propagation system for cacao. My results suggested that the activity of TcLEC2 determines the embryogenic capacity of the cacao tissue explants and correlated with embryogenic capacity of cultured cells. Transgenic embryos overexpressing TcLEC2 produced a significantly higher number of embryos compared to non-transgenic embryos; however, most of these transgenic somatic embryos exhibited abnormal phenotypes, and the development normally ceased at globular stage. This discovery may have future applications in increasing the efficiency of cacao mass propagation programs. Notably, in addition to major storage compounds in cacao seeds, fatty acids also function as signals involved in defense responses. I found that the endogenous level of 18:1 was modulated by exogenous glycerol application. Glycerol application on cacao leaves increased the level of glycerol-3-phosphate and lowered the level of 18:1 through an acylation reaction, which further triggered the defense responses. 100mM glycerol was sufficient to induce the accumulation of ROS, activate the expression of a variety of pathogen-related genes, and confer enhanced resistance against fungal pathogen Phytophthora capsici. My results demonstrated the potential of foliar glycerol application to become an environmentally safe means to induce the plant defense responses and fight important plant diseases in the field. Together, my Ph.D. dissertation makes major contributions to three important research areas in cacao: (1) identification of the key gene regulating fatty acid composition in cocoa butter, (2) improvement of large-scale propagation system (somatic embryogenesis) of cacao, (3) enhancement of cacao foliar disease resistance. This thesis not only provides useful knowledge of the regulatory mechanisms of important quality traits at the molecular and genetic levels, but also demonstrates the potential of taking advantage of cacao genomic resources to accelerate cacao basic research and breeding programs. <pdf>
Shi, Z. (2010). Functional analysis of non expressor of PR1 (NPR1) and its paralog NPR3 in theobroma cacao and arabidopsis thaliana (Order No. 3442953). Available From ProQuest Dissertations & Theses A&I. (853752677). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/853752677?accountid=13158
Arabidopsis NON EXPRESSOR OF PR1 (NPR1) is a key transcription regulator of the salicylic acid (SA) mediated defense signaling pathway. The NPR gene family consists of NPR1 and five other NPR1-like genes in Arabidopsis. This research focuses on the functional analysis of an NPR1 ortholog from Theobroma cacao L. and characterization of one of the NPR1 paralogs, NPR3, in both Arabidopsis and cacao. To identify the function of NPR3 in Arabidopsis, I first examined the gene expression pattern of NPR3 and found it to be strongly expressed in developing flower tissues. Interestingly, an npr3 knockout mutant displayed enhanced resistance to Pseudomonas syringae tomato pv. DC3000 (P.s.t.) infection of immature flowers. Gene expression analysis also revealed increased basal and induced levels of PRI transcripts in npr3 developing flowers. To investigate the possible mechanism of NPR3-dependent negative regulation of defense response, I tested the physical interactions of NPR3 with both TGA2 and NPR1 in vivo, which suggests that NPR3 represses NPR1-dependent transcription by inhibiting the nuclear localization of NPR1 through direct binding to TGA2 and NPR1. To characterize the NPR1 ortholog from cacao, I isolated TcNPR1 gene from genotype of Scavina6, and demonstrated that it expresses constitutively in all the tested tissues. To functionally analyze this gene, a bacterial growth assay was carried out with npr1-2 transgenic lines overexpressing TcNPR1, and a reduced level of bacterial growth demonstrated that TcNPR1 can partially complement Arabidopsis the npr1-2 mutation. In addition, TcNPR1 was shown to translocate into nuclei upon SA treatment in a manner identical to Arabidopsis native NPR1. To further explore the NPR gene family in cacao, I identified a total of four NPR-like genes from the cacao genome, and phylogenetic analysis indicated that the duplications of three clades in this gene family occurred before the divergence of Arabidopsis and cacao. To identify the functional ortholog of Arabidopsis NPR3, I isolated a putative TcNPR3 gene and demonstrated that its expression level was higher in un-open flowers and older leaves, a pattern similar to Arabidopsis NPR3. A complementation test of TcNPR3 expressed in the Arabidopsis npr3-3 null mutant showed that TcNPR3 can functionally substitute for the Arabidopsis NPR3 gene, demonstrating that TcNPR3 is the functional ortholog of AtNPR3. To obtain the genome-wide transcriptional responses of SA treatment in cacao, I used microarray analysis to measure gene expression in two cacao genotypes (ICS1 and Scavina6), three leaf developmental stages (A, C and E) and two treatments (water and SA). After validating the microarray results with RT-PCR, I identified differentially expressed genes from all twenty-four pair-wise comparisons. Interestingly, chloroplast and mitochondrial genes are enriched in SA-induced Scavina6 but those genes are underrepresented in ICS1, suggesting that the oxidative burst and hypersensitive response during defense response may vary between the two genotypes. In all, this research will not only offer us the knowledge of defense response mechanism and signal transduction regulation in Arabidopsis and cacao, but also provide molecular tools for selecting cultivars with enhanced disease resistance for cacao breeders and farmers. <pdf>
Liu, Y. (2010). Molecular analysis of genes involved in the synthesis of proanthocyanidins in theobroma cacao (Order No. 3420238). Available From ProQuest Dissertations & Theses A&I. (750368282). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/750368282?accountid=13158
The flavonoids catechin and epicatechin, and their polymerized oligomers, the proanthocyanidins (PAs, also called condensed tannins), accumulate to levels of up to 15% of the total weight of dry seeds of Theobroma cacao L. These compounds have been associated with several health benefits in humans including antioxidant activity, improvement of cardiovascular health and reduction of cholesterol levels. They also play important roles in pest and disease defense throughout the plant. This research focuses on molecularly dissecting the proanthocyanidin biosynthetic pathway of Theobroma cacao. To this end, I first isolated candidate genes from T.cacao (Tc) encoding key structural enzymes of this pathway which include, anthocyanidin reductase (ANR), leucoanthocyanidin dioxygenase (LDOX, also called anthocyanidin synthase, ANS) and leucoanthocyanidin reductase (LAR). I performed gene expression profiling of candidate TcANR, TcANS and TcLAR in various tissues through different developmental stages and also evaluated PA accumulation levels in those tissues. My results suggested that all PA candidate genes are co-regulated and positively correlated with PA synthesis. To functionally analyze the candidate genes, I used the model plants Arabidopsis and tobacco as expression platforms. Results from Arabidopsis mutant complementation tests and transgenic tobacco plants constitutively overexpressing cacao genes demonstrate that the candidate structural genes isolated from cacao are true ANS, ANR and LAR genes and all actively involved in PA synthesis in cacao. To further explore the transcriptional regulation of the PA synthesis pathway, I then isolated and characterized an R2R3 type MYB transcription factor TcMYBPA from cacao. I examined the spatial and temporal gene expression patterns of TcMYBPA in cacao and found it to be developmentally expressed in a manner consistent with its involvement in PAs as well as anthocyanin synthesis. Complementation test of TcMYBPA in Arabidopsis tt2 mutant suggested that TcMYBPA could functionally substitute Arabidopsis TT2 gene. Interestingly, except PA accumulation in seeds, I also observed an obvious increase of anthocyanidin accumulation in hypocotyls of transgenic Arabidopsis plants. This is consistent with gene expression analysis which showed that the entire PA pathway could be induced by overexpression of TcMYBPA gene, including DFR, LDOX (ANS) and BAN (ANR). Therefore I concluded that the isolated TcMYBPA gene encodes an R2R3 type MYB transcription factor and is involved in the regulation of both anthocyanin and PA synthesis in cacao. This research will not only offer us the knowledge of secondary metabolites production in cacao, but also provides molecular tools for breeding of cacao varieties with improved disease resistance and enhanced flavonoid profiles for nutritional and pharmaceutical applications. <pdf>
Miller, C. (2009). An integrated in vitro and greenhouse orthotropic clonal propagation system for Theobroma cacao L (United States -- Pennsylvania: The Pennsylvania State University), pp. 158. <pdf>
Xia, H. (2009). Structure and function of endosperm starch from maize mutants deficient in one or more starch-branching enzyme isoform activities (United States -- Pennsylvania: The Pennsylvania State University), pp. 261. <pdf>
Marelli, J. (2008). Solanum lycopersicum as a model system to study pathogenicity mechanisms of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao (United States -- Pennsylvania: The Pennsylvania State University), pp. 178. <pdf>
Swanson, J.-D. (2005). Flower development in Theobroma cacao L.: An assessment of morphological and molecular conservation of floral development between Arabidopsis thaliana and Theobroma cacao (United States -- Pennsylvania: The Pennsylvania State University), pp. 201. <pdf>
Antunez de Mayolo, G. (2003). Genetic engineering of Theobroma cacao and molecular studies on cacao defense responses (United States -- Pennsylvania: The Pennsylvania State University), pp. 148. <pdf>
Cakirer, M.S. (2003). Color as an indicator of flavanol content in the fresh seeds of Theobroma cacao L. (The Pennsylvania State University). <pdf>
Tomscha, J.L. (2001). Phosphatase secretion mutants in Arabidopsis thaliana (United States -- Pennsylvania: The Pennsylvania State University), pp. 103. <pdf>
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