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Theobroma cacao – The Food of the Gods, provides the raw material for the multi-billion dollar chocolate industry, and is also the main source of income for about 6 million smallholders around the world. Additionally, cocoa beans have a number of other non-food uses in the pharmaceutical and cosmetic industries. Specifically, the potential health benefits of cocoa have received increasing attention as it is rich in polyphenols, particularly flavonoids. At present, the demand for cocoa and cocoa-based products in Asia is growing particularly rapidly and chocolate manufacturers are increasing investment in this region. However, in many Asian countries, cocoa production is hampered due to many reasons including technological, political and socio-economic issues. This review provides an overview of the present status of global cocoa production and recent advances in biotechnological applications for cacao improvement, with special emphasis on genetics/genomics, in vitro embryogenesis and genetic transformation. In addition, in order to obtain an insight into the latest innovations in the commercial sector, a survey was conducted on granted patents relating to T. cacao biotechnology.
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Article type : Review
Cacao biotechnology: current status and future prospects
Anushka M. Wickramasuriya1 and Jim M. Dunwell2
1 Department of Plant Sciences, Faculty of Science, University of Colombo, Colombo-07, Sri Lanka
2 School of Agriculture, Policy and Development, University of Reading, UK
Correspondence:-
Anushka M. Wickramasuriya
Department of Plant Sciences, Faculty of Science, University of Colombo, Colombo-07, Sri Lanka
a.m.wickramasuriya@gmail.com
Other contact:-
Jim M. Dunwell
School of Agriculture, Policy and Development, University of Reading, UK
j.m.dunwell@reading.ac.uk
Running title: Cacao biotechnology
Keywords: Theobroma cacao, chocolate, somatic embryogenesis, genetics, genomics, breeding,
transformation
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Summary
Theobroma cacao The Food of the Gods, provides the raw material for the multi-billion dollar
chocolate industry, and is also the main source of income for about 6 million smallholders around the
world. Additionally, cocoa beans have a number of other non-food uses in the pharmaceutical and
cosmetic industries. Specifically, the potential health benefits of cocoa have received increasing
attention as it is rich in polyphenols, particularly flavonoids. At present, the demand for cocoa and
cocoa-based products in Asia is growing particularly rapidly and chocolate manufacturers are
increasing investment in this region. However, in many Asian countries, cocoa production is
hampered due to many reasons including technological, political and socio-economic issues. This
review provides an overview of the present status of global cocoa production and recent advances in
biotechnological applications for cacao improvement, with special emphasis on genetics/genomics, in
vitro embryogenesis and genetic transformation. In addition, in order to obtain an insight into the
latest innovations in the commercial sector, a survey was conducted on granted patents relating to T.
cacao biotechnology.
Introduction
The diploid tropical fruit crop species (2n = 2x = 20), Theobroma cacao (cacao) (Figure 1) is an
economically important agricultural commodity for millions of people worldwide. It is grown by
about 6 million farmers globally, and livelihoods of more than 40 million people depend on cocoa
(Beg et al., 2017; World Cocoa Foundation, 2012). The majority of world cocoa production
(approximately 80-90%) comes from smallholder farmers (World Cocoa Foundation, 2014). This crop
originated from the Amazonian basin (Motamayor et al., 2002; Wood and Lass, 1985), and today it is
cultivated in many regions of the humid tropics.
The cocoa beans are the primary source of raw material for the multi-billion dollar industry that
produces chocolate and associated confectionery products, with Switzerland being the country with
the highest consumption (Figure 2), though much of this is due to purchases by tourists to that
country. The economic significance of the chocolate industry has been recently reviewed (Squicciarini
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and Swinnen, 2016), with the global market for chocolate rising 13% from 2010 to reach US$101
billion in 2015.
This crop belongs to the Malvaceae family and more than 20 species are known within the
Theobroma genus (Wood and Lass, 1985). Among these, T. cacao is the only species that is cultivated
extensively (Wood and Lass, 1985). This species has three genetic groups based on morphological
and anatomical characteristics Criollo (T. cacao Spp. Criollo), Forastero (T. cacao Spp.
Sphaerocarpum) and Trinitario (Pridmore et al., 2000). Of these, the Criollo type is well known for its
superior flavour and provides the raw material from which fine flavour chocolates are produced; these
represent 5-10% of world chocolate production (Rusconi and Conti, 2010). However, increased
susceptibility to pest and diseases, low vigour and yield has made this variety less popular among
cacao growers. Today, most of the world’s chocolate production (approximately 80%) comes from the
Forastero type of cacao; this variety is favoured over the Criollo for its disease resistant and high
yielding nature and beans from this variety are relatively cheaper than those from the Criollo type
(Rusconi and Conti, 2010). The third genetic group, Trinitario is a hybrid produced from crosses
between Criollo and Forastero varieties. This variety was initially developed in Trinidad, and today it
is cultivated in many parts of South and Central America, Africa, Southeast Asia and Oceania for its
aroma, productivity and disease resistant character.
World cocoa production
The International Cocoa Organization (ICCO) estimated that more than 4.0 million metric tons of
cocoa beans were produced worldwide in 2015/16 (Pipitone, 2016). Of this total, it is also estimated
that Africa contributed approximately 74% (2.92 million tonnes) in the 2015/16 season. This is 5,000
tonnes less than the estimated production for 2014/15. Among the cocoa producing regions, Côte
d’Ivoire, Ghana and Cameroon contributed 1.57, 0.8 and 0.23 million tonnes, respectively, to global
production in 2015/16 (Pipitone, 2016). It is important to note that there is a discrepancy between the
cocoa bean production data published by the Food and Agriculture Organization of the United
Nations (FAO) and the production estimates by the ICCO. This is mainly due to the use of different
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sources to estimate the production data. Figures 3 and 4 in the present review are based on the data
published by the FAO. Figure 3 shows cocoa bean production (tonnes) from 1993 to 2013 in the
leading production regions in Africa, namely Côte d'Ivoire, Ghana, Nigeria and Cameroon. With the
exception of Cameroon, a slight decline in production from 2012 to 2013 is noted in the other African
countries considered.
In addition, the Americas region (16%, 0.64 million tonnes), and Asia and Oceania (10%, 0.4 million
tonnes) are ranked as the second and third largest producers of cocoa beans worldwide. At present,
Indonesia is the third largest producer after te d’Ivoire and Ghana, with an estimated production of
0.33 million tonnes in 2015/16 (Pipitone, 2016). However, production is still relatively low in many
Asian countries such as Malaysia, The Philippines, and Sri Lanka, which all have a tremendous
potential to grow cacao (Figure 4). In addition to the fact that cocoa bean production contributes
significantly to the economy of the growing regions, it also serves as a main source of income for
millions of smallholder farmers (Darkwah and Verter, 2014).
The demand for cocoa is increasing considerably (approximately 3% per year) (World Cocoa
Foundation, 2014). At present, global cocoa production is considered to be at risk, and it has been
reported that the world may experience a cocoa shortage by 2020 (Earth Security Group, 2015;
Jégourel, 2016). However, the surplus seen in the 2016/17 crop season (up to May 2017) in some
cacao growing areas such as Cote d’Ivoire and Ghana is favourable for the future cocoa sector; an
18% increase of the world production is expected for the current crop season as compared to that of
the previous season (International Cocoa Organization, 2017).
Uses of cocoa
This perennial shade grown tree crop provides biodiversity benefits. It is cultivated either as
monocultures or in association with other crops such as fruit crops (Guiltinan et al., 2008). Cocoa
beans are the key raw materials in the production of chocolate and other cocoa-based products.
However, the freshly harvested cocoa beans do not contain the determinants of chocolate aroma or
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flavour, and hence, post-harvesting processing of raw beans (fermentation, drying and roasting) is
essential for optimum flavour formation (De Vuyst and Weckx, 2016; Kadow et al., 2015; Kongor et
al., 2016b; Loureiro et al., 2017). The process of cocoa bean fermentation is trigged by action of
microorganisms (e.g. yeast, acetic acid bacteria and lactic acid bacteria) (de Melo Pereira et al., 2016;
Illeghems et al., 2015), and the flavour precursors such as organic acids, reducing sugars and free
amino acids are produced at the end of the process. In addition, the process of fermentation involves
significant reduction in polyphenols (epicatechin and catechin) and alkaloids (methylxanthines
caffeine, theobromine) found in raw cocoa beans that give rise to bitterness and unpleasant
astringency (Kadow et al., 2015; Lee et al., 2016).
Most production of cocoa takes place in the tropics, and the beans produced in this region used to be
principally processed elsewhere into cocoa powder and cocoa butter (Wood and Lass, 1985). Now,
although most of the cocoa grindings (38%) are carried out in the Europe and Russia region
(principally the Netherlands), the remainder is processed close to production areas in the Americas
(22%), Asia and Oceania (21%) and Africa (19%) (Pipitone, 2016). In addition to its use in
confectionery, cocoa products are also considered to have other functional properties (Konar et al.,
2016; Wilson and Hurst, 2015) and are used in a range of pharmaceutical and cosmetic products.
Cacao seeds are a rich source of polyphenolic antioxidants and consequently, it has been reported that
cocoa-based products contribute a greater proportion of the dietary intake of phenolic antioxidants
than do green tea, wine, soy beans and blueberries, which are known antioxidant rich food products
and beverages (Lee et al., 2003). The antioxidant properties of cocoa, particularly the high flavonoid
content are now of great interest due to its profound effects on human health. Specifically, the claim
that cocoa polyphenols could prevent cancer or delay/slow down the progression of cancer (chemo-
preventive agents) has received increased attention (Martin et al., 2013). Furthermore, flavonoids
extracted from cocoa have been shown to play a pivotal role in mediating innate and acquired
immunity (Ramiro-Puig and Castell, 2009), and also have been shown to have an effect on diet
induced obesity and insulin resistance (Dorenkott et al., 2014). Emerging data support the suggestion
that cocoa flavanols may serve as cardioprotective agents. These compounds have been reported to
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modulate mediators of inflammation (Keen et al., 2005). Cocoa flavanols and procyanidins (Bowser
et al., 2017; Liu et al., 2015) have been shown to have beneficial effects including decreased platelet
aggregation through increasing concentration of epicatechin and catechin in the plasma (Keen et al.,
2005; Murphy et al., 2003). Furthermore, cacao shell is a rich source of theobromine and vitamin D.
The pods contain a high level of potash that is used in soap production (Bart-Plange and Baryeh,
2003).
Cocoa bean quality
It is also important to maintain or enhance bean quality. Recently, several bean quality attributes, both
physical and chemical, that are required by the cocoa manufacturers/buyers, have been documented in
detail to encourage the cacao community towards the production of better quality cocoa
(CAOBISCO/ECA/FCC, 2015). These quality characteristics include flavour, purity or
wholesomeness (e.g. free from bacteria, infestation, allergens, mycotoxins, heavy metals and pesticide
residues), physical characteristics (e.g. consistency, yield of edible material bean, bean size and
uniformity, shell content, fat content and moisture content) and cocoa butter characteristics (e.g. free
fatty acid content). Some of these bean quality attributes, such as total fat content, acidity, total
phenols, organic acids, heavy metals, amino acids, caffeine, theobromine, pH, sugars, macro and
micro nutrient content, have been considered in the proposed Cocoa Quality Index (CQI) for
Forastero type beans (Araujo et al., 2014). Such an indexing system may represent a useful tool in
research programmes designed to improve bean quality for sustainable cocoa production. One
recommended source of information on cocoa quality is the Cocoa Atlas (Rohsius et al., 2010;
http://www.cocoa-atlas.org/). This DVD is funded by the German Cocoa and Chocolate Foundation
and produced by the Cocoa Research Group of the Biocenter Klein-Flottbeck, University of Hamburg,
Germany. It includes, in addition to global data, valuable information from 32 individual countries,
with information from each country divided into 12 sections with the following titles:- background
notes, cocoa growing areas, cocoa production, cocoa trade, foreign trade, aroma description, bean
weight/count, cut test, fat composition, free amino acids, further compounds, and pictures of samples.
Most recently, a Working Group on the Development of International Standards for the assessment of
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Cocoa Quality has been established by the Cocoa of Excellence (CoEx) programme and an initial
draft document on this theme is now available (Cocoa of Excellence, 2017).
The flavour profile of beans is a key quality measure in cocoa. For instance, the clone CCN 51, which
is planted extensively in Ecuador, exhibits many attractive agronomic traits like disease resistance,
high butter content and high productivity; however, it is less popular among fine flavour chocolate
manufacturers, especially due to the lack of fine flavour trait (Boza et al., 2014). In addition to the
cacao genotype, several other factors such as location where the trees are grown (i.e. soil condition),
the age of trees and post-harvest treatments (fermentation, drying and roasting) also affect cocoa bean
flavour (Kongor et al., 2016a). A comprehensive overview of factors affecting cocoa flavour
attributes has been published elsewhere (Afoakwa et al., 2008; Kongor et al., 2016a). Furthermore, a
sensory study conducted on raw cacao seeds and fruit pulp using a gas chromatography mass
spectrometry method has identified monoterpenes, methylketones, and secondary alcohols and their
respective esters as the main volatile aroma components in fine flavour clones such as SCA6 and
EET62 (Kadow et al., 2013). Analytical methods such as MS-fingerprinting (Qin et al., 2017; Tran et
al., 2015) and the near infrared spectroscopy (NIRS) method (Krähmer et al., 2015) have been
successfully employed in evaluation of cocoa biochemical quality parameters related to flavour
attributes and quality of fermentation, as efficient and routinely applicable approaches. These studies
have provided a foundation for understanding the molecular basis of fine aroma components in cocoa,
and thereby for the development of molecular markers linked to fine aroma quality in this species.
Good pre-harvest and post-harvest practices are key to maintaining many of the above mentioned
bean quality descriptors (CAOBISCO/ECA/FCC, 2015). For instance, selection of suitable planting
materials or the desired genetic background for cultivation is necessary to maintain the required
flavour, yield, bean size and colour, and cocoa butter content (CAOBISCO/ECA/FCC, 2015; Loureiro
et al., 2017). Furthermore, the quality of soil in which the cacao plants are grown is also a concern
today as there is some evidence for the presence of heavy metals, especially cadmium, in cocoa beans
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produced in some parts of the producing countries (Arévalo-Gardini et al., 2017;
CAOBISCO/ECA/FCC, 2015; Loureiro et al., 2017).
Cocoa bean quality is also influenced by post-harvest practices, especially the fermentation and
drying processes. For example, controlled drying of the fermented cocoa beans is a crucial step to
avoid development of off-flavours that may affect quality of beans. High-throughput molecular
analysis tools could be used for rapid and efficient identification of microbial population diversity
during cocoa fermentation and drying, and for development of microbial markers associated with the
process (Hamdouche et al., 2015). For instance, the powerful biotyping tool, matrix-assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) method, has recently been
used for molecular identification of micro-organisms involved in cocoa bean fermentation (Miguel et
al., 2017; Schwenninger et al., 2016). Recently, fermentation-like incubation systems or lab-scale
fermentation methods have proven to be applicable for the fermentation process of fresh cacao seeds
(Evina et al., 2016; Kadow et al., 2015). This system, which does not depend on microorganisms, may
provide a better alternative to the natural fermentation process that is usually difficult to control.
Furthermore, the experimental model described in Lee et al. (2016) for cocoa fermentation that
mimics on-farm cocoa fermentation process may speed-up fermentation studies at a laboratory level
in the future.
Another important factor that influences the quality of cocoa beans is the specific environmental
condition in which cacao plants are cultivated. The increasing atmospheric temperature and
evapotranspiration caused by global warming are likely to have a profound impact on global cacao
cultivation (Oyekale et al., 2009). Additionally, the climate in cacao growing regions has a
considerable impact on cocoa fermentation and drying processes. Läderach et al. (2013) have
projected that by 2050, the present cacao farming areas or cacao-favoured growing areas in Côte
d’Ivoire and Ghana may shift to areas with higher altitudes due to progressive increase in
temperatures. A more recent detailed study of this topic is that by Schroth et al. (2016). If the
predicted climate and weather variability continues, this may have an impact on the economic status
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of cocoa farmers and major cocoa producing countries; as result, global chocolate and confectionery
industry is likely to be affected due to a cocoa shortage. Breeding for climate-smart cacao varieties is
vitally important to long-term sustainability of cocoa production (World Cocoa Foundation, 2016).
This subject of climate-smart agriculture (CSA) is the basis of the on-going project “Mainstreaming
CSA practices in cocoa production in Ghana”, which aims to implement CSA practices with cacao
farmers (http://www.sustainablefoodlab.org/initiatives/climate-smart-agriculture/).
Cacao genetics and breeding
Cacao is a diploid fruit crop species with a relatively small genome, organized into ten chromosomes
(da Silva et al., 2017); i.e. the genome size is approximately double that of Arabidopsis thaliana, the
model dicot. Recently the published genome of the most cultivated type of cacao, T. cacao Matina 1-6
clone reports a genome size of 445 Mbp (Motamayor et al., 2013), which is considerably larger than
the previously published genome of a Criollo genotype (430 Mbp) (Argout et al., 2011). According to
the genome statistics reported by Argout et al. (2011), 28,798 protein coding genes from more than
682 gene families are present in the cacao genome. These include many genes related to disease
resistance, lipid biosynthesis (Zhang et al., 2015), flavonoid biosynthetic pathway and terpenoid
synthesis (Argout et al., 2011). The updated version of this Criollo sequence, with 99% of genes
anchored to the 10 chromosomes, was released in January 2017 and is accessible at the Cocoa
Genome Hub (http://cocoa-genome-hub.southgreen.fr/) (Argout et al., 2017). Availability of whole
genome sequences for several cacao varieties (Argout et al., 2011; Motamayor et al., 2013) has
allowed identification and characterization of novel genes of interest to breeders and also
development of molecular markers for marker assisted selection (MAS) (Lopes et al., 2011). The
release of cacao genome sequences has also provided the way for rapid identification, functional and
structural characterization of many gene families in cacao, through in silico computational studies and
expression analysis. For example, recently three legumain proteins, TcLEG3, TcLEG6 and TcLEG9,
which play diverse roles in programmed cell death, seed germination and seed development have been
identified and characterized through in silico analyses, three-dimensional modelling, and expression
analyses (Santana et al., 2016). Also, comprehensive genome-wide analysis of pathogenesis-related
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(PR) gene family in the two published T. cacao genomes has identified a set of candidate genes that
are likely to be involved in mediating defence responses against major pathogens such as
Phytophthora palmivora (Maora et al., 2017) and Colletotrichum theobromicola (Fister et al., 2016a).
Arrays constructed from subtractive libraries have also been used in an investigation of molecular
responses to cocoa black pod infection (Legavre et al., 2015). Such findings may contribute to a better
understanding of the genetics and genomics of T. cacao.
In terms of breeding targets, these can be divided into two main categories. The first is associated with
resistance to biotic stress, as unfortunately, outbreaks of diseases (Bailey and Meinhardt, 2016) in
major cacao growing areas have significantly affected production in South America and Africa. For
instance, Witches’ broom disease (WBD) (Almeida et al., 2017; Teixeira et al., 2015) caused by the
fungal pathogen Crinipellis perniciosa has reduced cacao yields in many cultivation areas in South
America including Ecuador and Brazil (Brown et al., 2005). In this context, a cacao osmotin-like
protein and various synthetic peptides (Falcao et al., 2016), and a phylloplanin (Freire et al., 2017)
have been shown to have be involved in the response to WBD. Also with reference to pathogens,
MALDI-TOF MS methods have been applied recently for the rapid identification of M. perniciosa,
Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C.
paradoxa, and C. fimbriata (dos Santos et al., 2017).
Another major disease problem in cacao is Cacao swollen shoot virus (CSSV) (Muller, 2016), which
is transmitted largely by mealy bugs (Wetten et al., 2016). Although efforts have been made to
eradicate the problem by removing infected trees, this has proved unsuccessful (Ameyaw et al., 2015)
and it is now hoped that a greater understanding of the genetic variation in both the virus (Abrokwah
et al., 2016; Chingandu et al., 2017a; Chingandu et al., 2017b) and its vector (Herrbach et al., 2016),
together with studies in more amenable model species (Friscina et al., 2017), will lead to progress in
understanding this important disease and related badnaviruses (Andres et al., 2017; Bhat et al., 2016).
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The second main breeding objective relates to physiological traits, as in addition to major pest and
disease outbreaks, cacao cultivation is also affected by several other factors, which include altered
short term climatic variation (e.g. El Niño), longer term global warming, high labour costs, depletion
of soil fertility, poor plant productivity, lack of breeding strategies to develop and distribute improved
varieties, and outdated farming practices (Zhang and Motilal, 2016). Specific breeding objectives
reported in one recent study include dwarfism or semi-dwarfism, which might enable smaller trees to
be planted at higher density, and photosynthetic efficiency, an important determinant of yield (Pereira
et al., 2017). As an alternative approach to breeding efforts to increase yields in the major production
areas, some cocoa producers are now considering new regions that might allow an extension in the
area under cultivation.
Importantly in terms of breeding strategies, cacao has a relatively longer juvenile period, namely 3-5
years. This makes selection of fruit-specific traits in breeding programs more time-consuming and
expensive, as the trees must be maintained for a longer period of at least three years to visually
observe such characters in pods. Moreover, this crop is primarily outbreeding (i.e. SCA 6 and EET 75
cacao clones), and therefore many populations are mostly heterozygous. This makes generation of
inbred lines from crosses more labour intensive, and doubled haploid lines (Dunwell, 2010) are not
easily generated. Moreover, the self-incompatibility that exists in some of the cultivated cacao clones
means that breeding populations are often highly heterogeneous with a wide range of yields (Royaert
et al., 2011). However, it should also be noted that genetic variability does exist in cacao populations,
and there are several self-compatible cacao clones, such CCN 51 and ICS 6 (Cervantes-Martinez et
al., 2006). Cacao trees also require a large area of land and high input of resources, including labour,
for their maintenance under field conditions. These characteristics have made this crop less attractive
as a model system, although like Arabidopsis, it has a relatively small genome.
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Because of the recalcitrant (do not survive drying) nature of its seeds the germplasm of this
allogamous tree crop must be conserved in field genebanks as a living collection (Motilal et al., 2013)
or by cryopreservation (Adu-Gyamfi and Wetten, 2012; Adu-Gyamfi et al., 2016) (see below). The
largest collections of cacao germplasm are those at the International Cocoa Genebank, Trinidad (ICG,
T) (https://sta.uwi.edu/cru/index.asp) (2400 accessions) and at the Centro Agronomico Tropical de
Investigación y Enseñanza (CATIE) in Costa Rica (https://www.catie.ac.cr/en/) (1146 accessions),
with another collection (c. 400 accessions), the International Cocoa Quarantine Centre, housed at the
University of Reading, UK (http://www.icgd.reading.ac.uk/icqc/). However, maintaining genetic
resources as living collections in situ or ex situ is practically difficult and is also an expensive process.
A significant number of mislabelled accessions has been reported in these field genebanks (Motilal
and Butler, 2003; Motilal et al., 2012). Thus, an efficient strategy to eliminate these mislabelled
and/or duplicated accessions in large cacao germplasm collections is required for efficient and
accurate management of genetic resources. In this context, DNA fingerprinting as a screening tool has
been extensively used in rapid and accurate identification of cacao accessions. Restriction fragment
length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPD), amplified fragment
length polymorphisms (AFLPs), microsatellites and single nucleotide polymorphisms (SNPs) are
some of the molecular markers commonly used in cacao molecular studies (Kuhn et al., 2012; Lanaud
et al., 1999; Laurent et al., 1994; Motilal and Butler, 2003; Santos et al., 2012a; Turnbull et al., 2004).
Livingstone et al. (2012) report an optimized 5’-nuclease (TaqMan)-based SNP assay for efficient
genotyping of cacao trees under field conditions. This simple, cost effective method would be a useful
technique for cacao breeders.
Development of high-density molecular-linkage maps and characterization of molecular markers
linked to major quantitative trait loci (QTL) have greatly accelerated breeding programmes in cacao
by facilitating examination of particular fruit-specific characters at a genotype level. The QTL
mapping studies of this species have been performed using different mapping populations i.e. F1 or
F2 mapping populations (Lanaud et al., 2009; Motamayor et al., 2013; Royaert et al., 2011; Schnell et
al., 2007) and association mapping or linkage disequilibrium mapping systems (Marcano et al., 2007;
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Stack et al., 2015). The first genomic map of cacao with a total of 193 loci covering 759 cM in 10
linkage groups was published by Lanaud et al. (1995). RFLP and RAPD markers were mainly used to
construct this genetic map, which was subsequently used to produce high-density molecular linkage
maps in several subsequent studies. For example, Risterucci et al. (2000) published a high resolution
molecular linkage map comprising 424 markers covering 885.4 cM over ten linkage groups; AFLP
and Simple Sequence Repeat (SSR) markers were employed to construct this high-density map, which
was considered as a good reference map for research activities in cacao (Clément et al., 2001).
Development and mapping of co-dominant microsatellite markers to the cacao genome has
accelerated genetic studies and breeding experiments. These PCR based co-dominant SSR markers
are highly polymorphic, and easily transferrable between/across populations and/or laboratories; they
can thus be used in MAS (Pugh et al., 2004). The high density linkage map described in this study has
465 markers with 268 SSR markers. A small number of such SSR markers have also been used in a
study of genetic diversity in historical cacao plantations in Brazil (Santos et al., 2015) and germplasm
assessments in Indonesia (Dinarti et al., 2015) and Cuba (Martínez et al., 2017). The collection of T.
cacao expressed sequence tags (ESTs) generated from a range of organs, genotypes and
environmental conditions is a valuable resource for discovery of important candidate genes and
molecular markers for cacao genetic improvements (Argout et al., 2008). For instance, Fouet et al.
(2011) discovered 174 EST-based SSRs markers by screening a cacao EST dataset and developed a
high density linkage map with 582 co-dominant markers including 384 SSR markers. Most recently,
da Silva et al. (2017) used a set of 20 EST-SSRs to examine the evolutionary relationship of species
within the Theobroma genus. Also, within the last few years, the use of SNP based co-dominant
markers in genetics has increased significantly, due to advances in high throughput sequencing
systems. A SNP-based linkage map for cacao was initially developed by Allegre et al. (2012). This
genetic linkage map contains a set of 1,262 markers spanning in a length of 734 cM, and of these
markers, 681 are EST based SNPs.
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Recently, a large number of SNPs has been detected by aligning RNA sequence (RNAseq) data of 16
cacao cultivars to the assembled Matina 1-6 transcriptome (Livingstone et al., 2015). In this study, a
saturated genetic linkage map with 2,589 SNPs was constructed. More importantly, this study led to
the development of an Illumina Infinium SNP array for cacao Cacao6kSNP array that consisted of
6,000 high quality SNPs. The newly developed array and the SNP data reported by Livingstone et al.
(2015) provide a valuable genomic resource for cacao breeding. The latest genetic linkage map of
cacao includes SNP data obtained from a large mapping population (459 trees) of a cross between
WBD resistant, TSH 1,188 and WBD tolerant (moderately resistant to WBD) CCN (Royaert et al.,
2016). It contains 3,526 SNP markers and has a length of 852.8 cM. In addition to genetic linkage
mapping studies, several recent studies have highlighted the importance of SNP based DNA
fingerprinting in assessing cacao bean authentication (Fang et al., 2014), cacao variety development
(Padi et al., 2017; Padi et al., 2015) and cacao genetic diversity (Cosme et al., 2016).
This rapid discovery of molecular markers also permits the efficient identification and study, in cacao,
of the genetic basis of QTL for many agronomic traits such as bean traits and the number of ovules
per ovary (Clement et al., 2003b), butter content and its hardness in cocoa beans (Araújo et al., 2009),
diseases resistance for Ceratocystis wilt (Santos et al., 2012b), resistance for Phytophthora species
(Akaza et al., 2016; Clement et al., 2003a; Efombagn et al., 2016; Flament et al., 2001; Lanaud et al.,
2004; Legavre et al., 2015; Motilal et al., 2016; Risterucci et al., 2003), resistance for WBD (Brown et
al., 2005; Faleiro et al., 2006; Motilal et al., 2016; Queiroz et al., 2003; Royaert et al., 2016), number
of filled seeds (Motilal et al., 2016), yield (Clement et al., 2003a; Crouzillat et al., 2000), and self-
compatibility/incompatibility (da Silva et al., 2016; Royaert et al., 2011). Furthermore, meta-analysis
on QTL related to disease resistance in cacao has been performed by Lanaud et al. (2009). Such
information would be of great use in MAS, which to date, is employed in many crop breeding
programs for development of improved cultivars including cacao. Recently, a semi-automated
genotyping platform for MAS known as amplicon sequencing (AmpSeq) has been successfully
applied for grapevine breeding program (Yang et al., 2016). This study showed the applicability of
this strategy in heterozygous crop breeding by generation of AmpSeq markers for several traits with
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high breeding value including disease resistance in grapevine. It is possible that such a high
throughput, cost-effective, flexible and rapid breeding strategy could be implemented with some
modifications to assist MAS in cacao in the future.
In addition to markers based on nuclear genomic DNA, several loci of chloroplast DNA (cpDNA)
such as matK, rbcL and trnH-psbA can be used as markers in DNA barcoding (Bieniek et al., 2015).
Recently, sequence variation of the trnH-psbA intergenic spacer has been analysed in 28 cacao
accession obtained from different farms in southern Mexico (Gutiérrez-López et al., 2016). It was
found that the indels located in this region could be considered as potential markers for development
of a DNA barcoding system in cacao. These markers are useful in identification of accessions in
situations when other marker systems can only discriminate between accessions on the basis of a very
small number of SSR markers (Gutiérrez-López et al., 2016).
Propagation methods and in vitro embryogenesis
Genetic improvement of cacao for improved traits has been hindered due to its narrow genetic base
and long life cycle (Li et al., 1998). It is estimated that approximately 30% of world cacao production
is lost due to pest and diseases, annually (Guiltinan et al., 2008). Therefore, an efficient propagation
method for cacao is essential to accelerate breeding programmes and to avoid production shortages in
the future. The cacao crop is grown with an approximate planting density of 1,100 trees per hectare
and it has been estimated that with a replanting rate of 10%, there is an annual requirement for one
billion units. This requirement is not being met at present, and some of the alternative propagation
options (Laliberte and End, 2015) are considered below.
Cacao is normally propagated by means of seeds. Additionally, to maintain a genetically stable
population, it is also propagated through a number of vegetative/asexual methods, of which a variety
of grafting methods (Miguelez-Sierra et al., 2017) are the most commonly practiced; these methods
have been reviewed in detail by Sena Gomes et al. (2015). However, these propagation systems are
not widely practiced in developing countries (Maximova et al., 2002). This could be due to the low
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rate of propagation and undesirable morphological features observed in some propagules, which often
lack normal dimorphic nature and display bush-like growth with a fibrous root system. Therefore,
maintenance of such material is a more labour intensive process and requires skilled workers (Traore
et al., 2003).
In cacao, in vitro embryogenesis or somatic embryogenesis (SE) is an alternative to traditional
propagation methods and allows rapid clonal propagation of true-to-type plants with normal
dimorphic architecture and tap-root formation. Importantly, this system has shown to be an effective
method in propagation of CSSV disease free plantlets (Quainoo et al., 2008). Moreover, a study
conducted by López and co-workers in 2010 found that de novo genetic mutations and epigenetic
modifications do not accumulate with aging of in vitro induced cacao calli (López et al., 2010b); this
conclusion is also relevant to a recent study of long-term SE (Quinga et al. 2017). Additionally, the
SE system has been utilized in cacao germplasm conservation through cryo-preservation (Adu-
Gyamfi and Wetten, 2012; Adu-Gyamfi et al., 2016) and in genetic transformation (da Silva et al.,
2008; Maximova et al., 2002). Induction of cacao somatic embryos has been observed from a range of
its tissues i.e. zygotic embryos (Pence et al., 1980), floral parts petals and staminodes (Alemanno et
al., 1996; Alemanno et al., 1997; Boutchouang et al., 2016; Li et al., 1998; Tan and Furtek, 2003), and
nucellar tissues (Figueira and Janick, 1993). The SE method developed by Li et al. (1998) was
applicable to many different cacao genotypes and later, Maximova et al. (2002) improved this system
to produce secondary somatic embryos from primary somatic embryos. Somatic embryo derived
plants have been successfully grown under field conditions (Maximova and Guiltinan, 2015;
Maximova et al., 2008). However, the efficiency of SE in this species is strongly influenced by
genotype, particularly in respect of the conversion rate of mature somatic embryos into complete
plants (Maximova et al., 2002). In addition, the type of explant used and its position i.e. flower bud
position (Boutchouang et al., 2016; Traore and Guiltinan, 2006), tissue culture media composition
(Traore and Guiltinan, 2006) and phenological parameters such as the periodicity of new leaf
development (Issali et al., 2008) are also have an impact on SE efficiency in cacao. Several
researchers have focused on optimizing in vitro culture media composition to improve somatic
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embryo differentiation in this species (Minyaka et al., 2008; Niemenak et al., 2012; Niemenak et al.,
2008; Traore and Guiltinan, 2006). Furthermore, establishment of a temporary immersion bioreactor
system for mass production of cacao somatic embryos was a breakthrough process in cacao
biotechnology research (Niemenak et al., 2008). Consequently, studies were designed to further
optimize somatic embryo induction in liquid suspension cultures (Niemenak et al., 2012). More
recently, an alternative method to induce cacao primary SE at a high efficiency has been achieved by
supplementing DKW medium with different ratios of kinetin to 2,4-dichlorophenoxyacetic acid (1.0 :
3.9 callus induction medium; 1.0 : 7.8 secondary callus growth medium) (Ajijah et al., 2016).
Furthermore, this methodology yielded a 65% plantlet conversion rate and a relatively low percentage
of somaclonal variation (López et al., 2010a). Other beneficial modifications to SE media have been
reported recently by Kouassi et al. (2017) and Modeste et al. (2017).
Further details and modified SE protocols developed by the various commercial chocolate companies
are also given in the various patent documents mentioned in the section below. Despite continuous
progress, the overall low efficiency and reproducibility of the methods developed as well as the
genotype dependent nature of the many steps involved in the SE process still present a significant
challenge for mass propagation of many elite cacao genotypes at the commercial scale required for
many parts of the cacao growing regions (da Silva et al., 2008). In an ambitious scheme, Nestlé’s
Cocoa Plan aims to produce and distribute at least 12 million plants of elite varieties that are disease
free, high yielding and high quality in terms of beans and taste by the year 2022 (Fair Labor
Association, 2012; Guillou et al., 2014). They aim to improve farmers’ income and living conditions,
avoid deforestation through sustainable production of cocoa, technology transfer, and distribution of
quality planting material for propagation in Côte d’Ivoire.
Understanding the molecular mechanism of SE would allow us to improve this process in
economically important crops, including cacao, and thereby provide an efficient system to speed-up
commercial plant production of many crops in the coming years. Recent advances in high throughput
sequencing systems and omics resources have facilitated generation of high resolution transcriptome
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data for plant embryogenesis, both in vivo and in vitro and thereby, to provide novel insights into the
molecular basis of embryogenesis (Wickramasuriya and Dunwell, 2015; Xu et al., 2012). In a relevant
recent example in cacao gene expression profiles of zygotic embryogenesis (ZE) and SE have been
generated using whole genome microarray (Maximova et al., 2014). This study reported that a large
number of genes including those encoding for transcription factors, genes related to flavonoid and
lipid biosynthesis were differentially expressed between the two embryo developmental processes.
Such results thus provide an insight into cacao SE at a molecular level, and the information provided
could be used to develop and characterize novel molecular markers for SE. In addition, proteome
profiles of cacao SE and their equivalent ZE at various developmental stages have been generated and
analysed through 2D PAGE and nano-LC-MC (Niemenak et al., 2015; Noah et al., 2013). Availability
of the genome sequence together with such recent proteomic and transcriptomic information for cacao
embryogenesis provides a good starting point for functional studies of many genes and their encoded
proteins essential for embryo development in this species.
In addition, several key regulators of plant embryogenesis have also been isolated and characterized
from cacao. Of these, the members of the Leafy cotyledon (LEC) gene family LEC1, LEC2 and
FUSCA3 (FUS3) serve as master regulators of embryo development, and they have been well
characterized in Arabidopsis (Lotan et al., 1998; Stone et al., 2001). Recently, a functional ortholog of
Arabidopsis LEC2 has been isolated and characterized in T. cacao (TcLEC2) by Zhang et al. (2014).
This gene was found to be expressed at a significant level in endosperm and cotyledons but not in
flower and leaf tissues (Zhang et al., 2014). Furthermore, a 20 fold higher level of TcLEC2 transcript
accumulation was observed in embryogenic calli than in non-embryogenic calli (Zhang et al., 2014).
Moreover, overexpression of TcLEC2 has led to increased expression of several seed specific genes in
leaves of cacao i.e. TcAGL15 (>129 fold), TcABI3 (>9 fold) and WRINKLED1 (WRI1) (>10 fold).
This also increased the embryogenic competency of cotyledon explants and regeneration capacity of
somatic embryos, supporting the fact that, as in many other plants, TcLEC2 is a key regulator of cacao
embryogenesis. Furthermore, a functional homologue of the LEC1-like gene has also been reported
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from the cacao genome (Alemanno et al., 2008); increased expression of this gene has been detected
in early stages of cacao zygotic and somatic embryogenesis.
Another well-studied regulator of embryogenesis is the AP2/ERF family member BABY BOOM
(BBM), which was first identified from Brassica napus microspore derived in vitro embryos
(Boutilier et al., 2002). Being significantly expressed in developing embryos and seeds, BBM is
considered as one of the key marker genes in embryogenesis (Boutilier et al., 2002; Ikeda et al., 2006;
Karami et al., 2009). A functional gene with a high degree of similarity to BBM in A. thaliana has
been isolated and characterized from cacao (TcBBM) (Florez et al., 2015). TcBBM expression has
been detected throughout embryogenesis in cacao; a higher level of expression has been detected in
SE than in ZE. In a manner similar to that in species including Arabidopsis (Boutilier et al., 2002) and
cereals (Lowe et al., 2016), overexpression of TcBBM in cacao has been found spontaneously to
induce somatic embryos in hormone-free media (Florez et al., 2015); thus, TcBBM could be used to
enhance the efficiency of SE in cacao. The most recent advance in this area is the report of an
inducible SE system by exploiting a dexamethasone activatable embryogenic transcription factor to
promote somatic embryo formation from juvenile leaves (Shires et al., 2017).
Similarly, kinases play an important role in plant embryogenesis. For example, somatic
embryogenesis receptor kinases (SERKs) are a subgroup of protein kinase genes that are expressed in
early stages of somatic and zygotic embryo development. These genes were initially isolated from in
vitro embryogenic cultures of carrot by Schmidt et al. (1997) and have subsequently SERKs been
identified and characterized in many species i.e. A. thaliana (Hecht et al., 2001), Solanum tuberosum
(Sharma et al., 2008), Cocos nucifera (Perez-Nunez et al., 2009), Zea mays (Baudino et al., 2001),
Momordica charantia (Talapatra et al., 2014) and cacao (de Oliveira Santos et al., 2005). This latter
gene (TcSERK) was highly expressed in embryogenic calli, and also in mature zygotic and somatic
embryos at a moderate level, suggesting that the functional copy of SERK found in cacao plays a key
role during the process of embryo development.
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Genetic transformation
Completion of whole genome sequencing for many economically important crops has significantly
contributed to their respective genetic improvement. In addition, identification and functional
characterization of novel genes and transfer of genes that regulate agronomically valuable traits such
as disease resistance have been achieved in many crops. The earliest attempt of cacao transformation
was recorded in 1994 by Sain et al. (1994) using the Agrobacterium-mediated gene transfer method.
Although transformed callus cells derived from leaf tissues were obtained, no plant regeneration was
recorded from those transformed cells. This was due to the lack of an efficient protocol to recover
plants from cacao leaf tissue derived calli at that time (Sain et al., 1994).
Subsequently, a more efficient method for stable genetic transformation and recovery of transformed
plants from transformed cacao cells was established by Maximova et al. (2003). This study employed
SE as a regeneration system together with Agrobacterium tumefaciens co-cultivation to obtain
transgenic plants. This system has been successfully used to produce transgenic cacao plants
overexpressing cacao class I Chitinase gene (TcChil1) (Maximova et al., 2006). These transgenic
plants showed enhanced fungal pathogen resistance against Colletotrichum gloeosporioides. Although
this transformation system was proven to be reproducible (Maximova et al., 2003; Maximova et al.,
2006), the reliable production of a large number of transgenic embryos remains a challenge.
Subsequently, several studies have been conducted in an attempt to improve the efficiency of the
transformation method described in Maximova et al. (2003) (Silva et al., 2009). Most recently, an
optimized method for transient transformation of cacao for several genotypes through Agrobacterium-
infiltration has been published by Fister et al. (2016b). As a tool this will allow more efficient in vivo
functional analysis of cacao genes, subcellular localization of proteins, and promoter analysis. It is
also possible that the use of rapidly flowering transgenic lines, as used in other perennial species
(Callahan et al., 2016), may be applicable to cacao. Most recently, an inducible SE system in cacao
has been developed by use of a transgenic plant expressing the LEC2 embryogenic transcription
factor (Shires et al., 2017).
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Although much effort has been devoted to improve cacao varieties through genetic transformation,
genetically modified (GM) cacao material has not been released commercially so far, and the studies
have been limited to laboratories and greenhouses. Though genetic transformation serves as a
valuable tool in crop improvements, the future of GM cacao is not clear as the consumer acceptance
of food from genetically modified organisms is still a controversial issue in some countries (Dunwell,
2014; Guiltinan et al., 2008).
It is now possible to effect gene-specific mutagenesis by genome editing, (e.g. CRISPR/Cas9) for
functional characterization of genes; this also serves as a promising approach for genetic manipulation
of disease resistance, and other genes in cacao. Whether or not such methods will be considered as a
form of GM is still uncertain, at least in Europe.
Resources available for cacao functional studies
Open source bioinformatic tools and web databases have greatly contributed to the rapid
developments in omics-based researches and thereby crop improvements. A list of tools/databases
freely accessible for cacao researchers and breeders is summarized in Table 1. The latest genome
sequence of cacao is available on the Cacao Genome Database (CGD,
http://www.cacaogenomedb.org/), which was developed in collaboration with MARS, USDA/ARS,
IBM, Clemson University Genomics Institute, PIPRA, HudsonAlpha Institute for Biotechnology,
National Center for Genome Resources, Indiana University and Washington State University Main
Lab Bioinformatics. In addition, tools such as BLAST, GBrowse can be accessed through this web
database.
Survey of patents relating to cacao
Often, very useful information about advances in scientific research can be obtained from a study of
patent databases, since information is often published here before appearing in more usual scientific
publications (Dunwell, 2012). In addition to academic institutes, private sector industries play a key
role in cacao research and development. Hence, a patent search analysis was performed using the
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Lens patent database (https://www.lens.org/lens/) to provide an overview of public and private sector
involvement, and application of biotechnology techniques in cacao research; structured search was
carried out on granted patents using the terms ‘Theobroma or ‘cacao’ or ‘cocoa’ in the abstract or
claims. A total of 2,732 granted patents were recorded prior to 13th July 2017. Thus, because it is not
feasible to summarize all these granted patents in this review only a selected summary is provided
here (Tables 2). In brief, granted patents related to plant breeding and biotechnology applications
were filtered based on the International Patent Classification (IPC) codes; a total of 10 granted patents
with following IPC codes were identified, and listed in Table 2: A01G 17/00, A01H 4/00, A01H 5/08,
C12N 5/00, C12N 5/02, C12N 5/04, C12N 15/82, C12N 15/84 and C12N 15/87 (definition of these
IPC codes can be obtained through the World Intellectual Property Organization (WIPO) page
http://www.wipo.int/classifications/ipc/en/). We identified several granted patents relating to the
production of cacao somatic embryos through optimized tissue culture techniques. Not surprisingly,
several studies are funded either fully or partially by the leading chocolate manufacturers. Notable
publications of particular relevance to this review include those describing methods for SE with
granted patents from Hershey Foods (US 5312801) in 1994, Penn State Research Foundation
(US6150587) in 2000, Nestle S.A. (US 8921087) in 2014, and most recently a granted patent from
Mars Inc. (AU 2014/353082 B2) in April 2017. This patent on the production of cacao plants claims
micropropagation via direct SE and is available at
https://www.lens.org/images/patent/AU/2014353082/B2/20170420/AU_2014_353082_B2.pdf. This
method uses explants such as staminodes and petal base tissues for induction of primary embryos in a
medium supplemented with 6-benzylaminopurine (BAP). Subsequently, the epicotyl segments
removed from primary embryos are placed in an induction medium containing BAP to induce direct
secondary embryos, followed by further embryo development in a medium containing gibberellic
acid, if needed. All the cultures are maintained in the light (photoperiod: 16:8 (light: dark) at a
temperature of 23-29oC for a sufficient period of time to obtain embryos. Although several attempts
have been made in the past to develop a direct SE system for cacao micropropagation, there has been
only limited success (Pence et al., 1980). Therefore, the detailed information provided in this patent
related to a direct SE method may provide additional valuable information for cacao researchers
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working on this subject. However, there is no patent information yet on the use of novel breeding
techniques such as Zinc Finger Nuclease (ZFN) or CRISPR/Cas9 technology, cisgenesis and RNA-
dependent DNA methylation (RdDM) in cacao plants.
Conclusion
There is a great demand for high quality cocoa beans. Thus, to ensure long term sustainability of
cocoa production, future research should focus on the development of improved cacao varieties that
can both tolerate changing climates, but also meet the stringent quality criteria demanded by the
chocolate industry. Implementation of modern molecular tools in cacao biotechnology research will
undoubtedly be an integral part of this process.
Conflict of Interest
The authors declare no conflict of interest.
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Figure legends
Figure 1. Cacao tree with multiple pods.
Figure 2. Annual consumption of chocolate per year (modified from Statista.org).
Figure 3. Cocoa bean production in Africa Côte d'Ivoire, Ghana, Nigeria and Cameroon from
1993 to 2013. Primary axis left: Côte d'Ivoire and Ghana; Secondary axis right: Nigeria and
Cameroon.
Source: FAOSTAT (http://faostat3.fao.org/browse/Q/*/E, retrieved on 7th February 2017).
Figure 4. Cocoa bean production in Asia Indonesia, Malaysia, Philippines and Sri Lanka from
1993 to 2013. Primary axis left: Indonesia; Secondary axis right: Malaysia, Philippines, Sri Lanka.
Source: FAOSTAT (http://faostat3.fao.org/browse/Q/*/E, retrieved on 7th February 2017).
Accepted Article
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Tables
Table 1. A list of web based resources available for cacao.
Resource
URL
CacaoNet Global Network for Cacao Genetic
Resources
https://sites.google.com/a/cgxchange.org/cac
aonet/home/partners-of-cacaonet
International Cocoa Germplasm Database (ICGD)
http://www.icgd.reading.ac.uk/index.php
Cacao Genome Database
http://www.cacaogenomedb.org/
Cocoa Genome Hub
http://cocoa-genome-hub.southgreen.fr/
CocoaGenDB
http://cocoagendb.cirad.fr/
CEMID Cocoa EST Marker Information
Database
http://riju.byethost31.com/cocoa/?ckattempt
=1
Ensembl Plants
http://plants.ensembl.org/Theobroma_cacao/
Info/Index
Dicots PLAZA 3.0
http://bioinformatics.psb.ugent.be/plaza/versi
ons/plaza_v3_dicots/organism/view/Theobro
ma+cacao
Phytozome v11.0
https://phytozome.jgi.doe.gov/pz/portal.html
#!info?alias=Org_Tcacao
GenBank NCBI
http://www.ncbi.nlm.nih.gov/genome/?term=
cocoa
Witches’ Broom Disease Transcriptome Atlas
http://bioinfo08.ibi.unicamp.br/wbdatlas/
Accepted Article
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Table 2. A list of granted patents, in chronological order, on T. cacao biotechnology, prior to July 2017.
Publication Number
Publication Year
Title
US 4301619 A
1981
Plant tissue produced by non-agricultural proliferation of
Cacao embryos
US 4291498 A
1981
Method for production of mature asexual Cacao
embryos, and product thereof
US 4306022 A
1981
Cocoa bean cell culture
US 4545147 A
1985
Asexual embryogenesis of callus from Theobroma
Cacao L.
US 5312801 A
1994
Somatic embryogenesis and plant regeneration of Cacao
US 6150587 A
2000
Method and tissue culture media for inducing somatic
embryogenesis, Agrobacterium-mediated transformation
and efficient regeneration of Cacao plants
US 8921087 B2
2014
Cocoa somatic embryogenesis
US 8969655 B1
2015
Modulation of flavonoid content in Cacao plants
US 9428759 B2
2016
Methods for increasing the production of phenolic
compounds from Theobroma Cacao
AU 2014/353082 B2
2017
Production of plants using somatic embryogenesis
Accepted Article
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Figure 1. Cacao tree with multiple pods.
Figure 2. Annual consumption of chocolate per year (modified from Statista.org).
Accepted Article
This article is protected by copyright. All rights reserved.
Figure 3. Cocoa bean production in Africa Côte d'Ivoire, Ghana, Nigeria and Cameroon from
1993 to 2013. Primary axis left: Côte d'Ivoire and Ghana; Secondary axis right: Nigeria and
Cameroon.
Source: FAOSTAT (http://faostat3.fao.org/browse/Q/*/E, retrieved on 7th February 2017).
Figure 4. Cocoa bean production in Asia Indonesia, Malaysia, Philippines and Sri Lanka from
1993 to 2013. Primary axis left: Indonesia; Secondary axis right: Malaysia, Philippines, Sri Lanka.
Source: FAOSTAT (http://faostat3.fao.org/browse/Q/*/E, retrieved on 7th February 2017).
0
100,000
200,000
300,000
400,000
500,000
600,000
0
200,000
400,000
600,000
800,000
1,000,000
1,200,000
1,400,000
1,600,000
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Production (tonnes)
Production (tonnes)
Year
Côte d'Ivoire
Ghana
Nigeria
Cameroon
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
9,000
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
900,000
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Production (tonnes)
Production (tonnes)
Year
Indonesia
Malaysia
Philippines
Sri Lanka
... Hybrid cacao varieties are planted in most producer countries in the world, including those in West Africa, where around 70% of the world cocoa beans are produced [9]. Besides the potential exploration of heterosis, hybrids offer additional advantages when compared to clones. ...
... With the dramatic reduction of sequencing and genotyping costs in recent years, cacao has experienced significant advances in genomic resources and knowledge [9]. Two cacao genomes have been sequenced and assembled [22,23], thousands of SNPs were discovered and gathered in user-friendly, high-precision genotyping platforms [24][25][26], and a large number of germplasm accessions genotyped. ...
... A considerable number of studies have been published showing a variety of applications of molecular markers, both microsatellites and SNPs in cacao breeding and germplasm characterization (reviewed in [9]). Clone fingerprinting for certification of identity in germplasm collections and progeny trials has been the most operationally useful application. ...
Article
Full-text available
Cacao is a globally important crop with a long history of domestication and selective breeding. Despite the increased use of elite clones by cacao farmers, worldwide plantations are established mainly using hybrid progeny material derived from heterozygous parents, therefore displaying high tree-to-tree variability. The deliberate development of hybrids from advanced inbred lines produced by successive generations of self-pollination has not yet been fully considered in cacao breeding. This is largely due to the self-incompatibility of the species, the long generation cycles (3-5 years) and the extensive trial areas needed to accomplish the endeavor. We propose a simple and accessible approach to develop inbred lines based on accelerating the buildup of homozygosity based on regular selfing assisted by genome-wide SNP genotyping. In this study we genotyped 90 clones from the Brazilian CEPEC´s germplasm collection and 49 inbred offspring of six S1 or S2 cacao families derived from self-pollinating clones CCN-51, PS-13.19, TSH-1188 and SIAL-169. A set of 3,380 SNPs distributed across the cacao genome were interrogated on the EMBRAPA multi-species 65k Infinium chip. The 90 cacao clones showed considerable variation in genome-wide SNP homozygosity (mean 0.727± 0.182) and 19 of them with homozygosity ≥90%. By assessing the increase in homozygosity across two generations of self-pollinations, SNP data revealed the wide variability in homozygosity within and between S1 and S2 families. Even in small families (
... Its production is concentrated in Africa (63.2%), followed by Asia (17.4%) and Latin America (14.1%), where Ecuador and Brazil are the major producing countries (Wickramasuriya & Dunwell, 2018). ...
Article
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Cacao (Theobroma cacao L.) is an important commercial crop and agricultural commodity worldwide; for some Latin American countries, it is an essential part of export products. In Colombia, this crop has promising conditions to extend and strengthen this agriculture sector. However, their productivity is low under current agricultural practices, mainly due to insufficient modernization and inadequate or no management of their nutritional schemes. This publication reviewed the different findings currently in the scientific literature regarding the factors that determine the nutritional status of cacao plants, such as the function and distribution of minerals, nutritional efficiency, soil properties, establishment systems, organic and organic and inorganic sources examined. Additionally, it highlighted the importance of using and expanding diagnostic tools to determine nutritional needs and the design of effective programs according to the particular conditions of each region and the genotypes planted. This conceptual journey highlights the existing theoretical and experimental gap in the identification of the factors that determine the nutritional status of the plantations and their effect on the implementation of the fertilization programs used today. Information together provides elements to adequately address this agronomic practice and the economic impact on farmers and the cacao production chain.
... El cacao (Theobroma cacao L.), es una especie frutal de la familia Malvaceae, originaria de las selvas húmedas tropicales de la región colombo-brasileña en américa del sur (Salazar et al., 2018). Esta planta es uno de los principales cultivos perennes del mundo, su producción está concentrada en África (63,2%), seguido de Asia (17,4%) y América Latina (14,1%), siendo Ecuador y Brasil los principales países productores de cacao (Wickramasuriya & Dunwell, 2018). El gran aprecio por este fruto deriva principalmente por ser materia prima clave para la elaboración del chocolate, además de su uso en la industria cosmética y farmacológica (ICCO, 2017;Van Vliet & Giller, 2017). ...
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Entre las diferentes acciones encaminadas a mejorar la tecnificación del cultivo de cacao (Theobroma cacao L.) se encuentra la optimización de sus condiciones de crecimiento. Identificar sus requerimientos nutricionales, es una de las estrategias que permite mejorar el material vegetal. Entre los nutrientes esenciales, se seleccionó el potasio como uno de los elementos fundamentales para la regulación de procesos hídricos, enzimáticos, iónicos y de osmorregulación. Así, se buscó profundizar en la optimización de los niveles de potasio para diferentes genotipos de cacao. En este estudio se evaluó el efecto de diferentes concentraciones y fuentes de K+ sobre características morfo-fisiológicas de cinco genotipos de T. cacao. Principalmente se encontraron diferencias en altura, longitud de raíz y biomasa, entre los clones CCN-51, FSA-13 y FEAR-5, siendo los dos primeros los que registraron valores superiores. Igualmente, se encontró una redistribución de la biomasa de la raíz y el vástago en las plantas crecidas con la menor concentración de potasio (0,16g/planta). Particularmente el índice raíz: vástago para el genotipo CCN-51 fue superior. Respecto a los parámetros fisiológicos se encontraron valores altos en conductancia estomática y fluorescencia de la clorofila para todos los genotipos independientemente de los tratamientos evaluados. Además, las variaciones genéticas entre los genotipos muestran respuestas diferenciales para las condiciones nutricionales evaluadas. Finalmente, los resultados sugieren que las plantas de cacao lograron un desarrollo adecuado al ser suplementadas con 0,16 g de K+ /planta en esta etapa de crecimiento, y ambas fuentes, KNO3 y KCl, son adecuadas para el suministro de este nutriente.
... T. Cacao is a small evergreen tree native to South America (Baliga et al., 2014). The seeds of T. cacao are often used in the food industry especially for chocolate making (Wickramasuriya and Dunwell, 2018). T. cacao has anti-HIV activity. ...
... The list of the main species found in the forest restoration initiatives studied is presented in Table 2. Information on the main species found in forest restoration models ( Table 2) was obtained from specialized sources [31][32][33]. Plants such as acai berry, banana, coffee, cocoa, coconut, ginger, and mangosteen, for example, provide important agricultural products with benefits for human health [34][35][36][37][38][39][40]. ...
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Mobilizing funds is a major challenge to achieve scalable Forest Landscape Restoration projects. While pure ecological restoration may not be a feasible investment from the private perspective, combining native species with non-timber forest products (NTFP) species may be a solution for reaching large scale and financially sustainable forest restoration. This study addresses potential species combinations for 12 restoration models, three models being based in pure ecological restoration and nine models being based on agroforests with NTFP, their economic costs, and benefits in tropical forests in Brazil, Peru, Cambodia, and Indonesia. A total of 12 semi-structured interviews were conducted to capture the models’ productivity and prices. As for the prices that the producers did not know, specialized stores were consulted in the cities of the collection. The starting investment to restore 01 hectare (ha−1) of tropical forest ranged between US $104 and $7736, with an average of $1963 ha−1 and a standard deviation of $2196 ha−1, considering the 12 cases evaluated in 2018 and 2019. From nine restoration models that had economic purposes, financial indicators showed a median net present value (NPV) of $1548 ha−1, and a median internal rate of return of 22%, considering a discount rate of 10%. The NPV varied between $−685 ha−1 and $55,531 ha−1. Costs of pure ecological restoration were on average 42% lower than agroforestry systems, but did not produce direct income from NTFP, therefore yielding negative NPV. The study demonstrated the economic feasibility of seven of nine models that had economic objective, showing that there are promising business cases for private investment in tropical forest restoration.
... In the past decades, the advent of genotyping and the reduction in its cost by next-generation sequencing (NGS)-based genotyping methods have made whole-genome sequencing (WGS) and resequencing feasible for numerous crops including durian [5][6][7]. This progress made molecular breeding more feasible in several crops in recent years [8][9][10][11][12]. ...
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Durian (Durio zibethinus L.) is a crop of economic and health importance globally. Efforts are being made to revamp China’s only successful commercial-scale durian plantations in Hainan; however, their genetic base is unknown. Therefore, the present study was undertaken to assess the genetic base and population structure of 32 genotypes in durian plantation sites in Hainan, China, and develop simple sequence repeat (SSR) markers by whole genome sequencing through restriction site-associated DNA sequencing technology to facilitate germplasm conservation and breeding. The results from identity by state (IBS), phylogenetic tree, population structure, and principal component analysis grouped the 32 genotypes into two clusters/sub-populations. Based on IBS, genotypes in Cluster I are largely duplicated genotypes; however, results from the model-based population structure demonstrated that most of the genotypes in Sub-population II shared a common genetic background with those in Sub-population I/Cluster I. The results revealed that the core durian collection in the plantation sites in Hainan include D24, D101, MSW, JH, D163, HFH, and NLX-5. In addition, we developed a total of 79,178 SSR markers with varied lengths and amplicon sizes. The genetic diversity and population structure reported in this study will be useful for durian conservation and utilization. In addition, the discovered and developed SSR markers will lay the foundation for molecular breeding via marker-assisted selection, quantitative trait loci mapping, and candidate gene discovery and validation.
... Moreover, it is widely known that cacao is beneficial to human health. However, cacao production is hampered in Asian countries due to biotic/abiotic stresses [32]. R2R3-MYB genes play important roles in plant development and responses to biotic and abiotic stresses, which means that these genes might have the potential to be exploited for cacao production improvement. ...
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The MYB gene family is involved in the regulation of plant growth, development and stress responses. In this paper, to identify Theobroma cacao R2R3-MYB (TcMYB) genes involved in environmental stress and phytohormones, we conducted a genome-wide analysis of the R2R3-MYB gene family in Theobroma cacao (cacao). A total of 116 TcMYB genes were identified, and they were divided into 23 subgroups according to the phylogenetic analysis. Meanwhile, the conserved motifs, gene structures and cis-acting elements of promoters were analyzed. Moreover, these TcMYB genes were distributed on 10 chromosomes. We conducted a synteny analysis to understand the evolution of the cacao R2R3-MYB gene family. A total of 37 gene pairs of TcMYB genes were identified through tandem or segmental duplication events. Additionally, we also predicted the subcellular localization and physicochemical properties. All the studies showed that TcMYB genes have multiple functions, including responding to environmental stresses. The results provide an understanding of R2R3-MYB in Theobroma cacao and lay the foundation for a further functional analysis of TcMYB genes in the growth of cacao.
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Composite materials based on renewable and biodegradable natural fibres derived from agricultural waste and wood industry residues are being used in an increasing number of applications. These products are an environmentally friendly and cost-effective alternative to traditional petroleum-based materials, as they significantly reduce the use of fossil fuels and greenhouse gas emissions. Furthermore, these materials have excellent mechanical qualities and require less energy to manufacture. Wood-based industries and agriculture, on the other hand, produce significant amounts of organic waste and residues that are still underutilised as low-value energy resources, and organic waste is commonly disposed of using traditional waste management techniques like landfilling, anaerobic digestion, or composting. Natural fiber-reinforced polymer composites (NFPCs) made from organic agriculture and wood industry waste and residues are an environmentally friendly, sustainable, and cost-effective option. Green tribology is generally a new field that discovers applications in different tribo frameworks. It has the potential to give answers for the issues of vitality and ecological contamination from a universal point of view. The demand for eco-friendly material for tribological applications in automotive sector is rising since it reduces the impact of asbestos based components in the environment. The availability, sustainability, simplicity of assembling and value addition of agro waste fibers has enticed scientists to consider its possibility of an alternate to synthetic reinforcement. The new research also extended to what degrees they fulfil the necessary particulars for the amelioration of wear properties of green composites for tribological applications. This review endeavours to summarise and emphasize the principles and basics of Green sustainable tribology. A comprehensive overview of various crop residues, its availability and the characterization are also presented. Furthermore, an attempt to devote a gathering of ongoing advancements of wear performance of agro waste areca husk fiber (AHF) reinforced composites. These aspects may prompt a solid establishment for further advancements of agro waste fiber in the region of green tribology enlightens the importance of using recyclable and biodegradable polymers for environmental safety.
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Les chocolatiers bean-to-bar redéfinissent le chocolat en mettant l’accent sur le processus de transformation et la biodiversité cacaoyère. Basée sur une ethnographie multisituée à Montréal et à Piura (nord péruvien), cette recherche retrace le trajet de la fève de cacao le long d’une filière de chocolat bean-to-bar pour analyser l’articulation entre les valeurs des économies morales des chocolatiers et des producteurs de cacao fin. Grâce à une incursion dans l’histoire du chocolat, l’économie morale des chocolatiers est définie comme un positionnement contre l’industrie du chocolat conventionnel et ses standards. Alors que les valeurs des chocolatiers gravitent autour d’une éthique gustative engendrant des préoccupations pour la biodiversité cacaoyère et les droits des cacaoculteurs, celles des producteurs visent plutôt à renforcer leurs rôles familiaux et communautaires et à mettre en place un projet de développement local collectif. Cette étude suggère qu’une analyse des économies morales peut rendre compte de l’articulation entre des économies capitalistes et précapitalistes et des phénomènes qui y sont créés, dans le cas présent, la production d’une localité cacaoyère. Bean-to-bar chocolate makers redefine chocolate by drawing attention to the transformation process and cacao biodiversity. Based on a multi-sited ethnography in Montreal and Piura (northern Peru), this research follows the cacao bean through a bean-to-bar chocolate filière to study how the values of chocolate makers and fine cacao producers' moral economies are embedded. Through an incursion into the history of chocolate, the moral economy of chocolate makers is defined as a posture against the conventional chocolate industry and its standards. While chocolate makers' values gravitate towards a taste ethic that generates preoccupations for cacao biodiversity and cacao farmers' rights, producers' values aim to reinforce their family and community roles and to implement a collective project of local development. This research puts forward moral economy as a conceptual tool for studying the embeddedness between capitalist and pre-capitalist economies and the phenomena that emerge from this nexus, in this case, the production of a cacao locality.
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Cocoa tree (Theobroma cacao L.) is cultivated mainly in tropical regions and produces beans that are used for chocolate manufacture. Worldwide, cocoa bean production is threatened by biotic stresses, mainly fungus, oomycetes, virus and other pests. The understanding of the determinism of the plant-pathogen interactions as well as the different and integrated ways to manage the cocoa diseases at field level began the focus of several research groups. Here, we did an overview of the several cocoa diseases, of the traditional breeding methods as well as the molecular assisted ones recently developed, of the molecular and omics resources currently available, and of the new biotechnology approaches—including genome edition and nanotechnologies—that are used at basic and applied research levels. We also described the main germplasm and collections worldwide as well as the use of the cocoa diversity as main source of disease resistance.
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Background Theobroma cacao L., native to the Amazonian basin of South America, is an economically important fruit tree crop for tropical countries as a source of chocolate. The first draft genome of the species, from a Criollo cultivar, was published in 2011. Although a useful resource, some improvements are possible, including identifying misassemblies, reducing the number of scaffolds and gaps, and anchoring un-anchored sequences to the 10 chromosomes. Methods We used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined four Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions and reduced the number of scaffolds. We then used genotyping by sequencing (GBS) methods to increase the proportion of the assembly anchored to chromosomes. Results The scaffold number decreased from 4,792 in assembly V1 to 554 in V2 while the scaffold N50 size has increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. Conclusion Theobroma cacao Criollo genome version 2 will be a valuable resource for the investigation of complex traits at the genomic level and for future comparative genomics and genetics studies in cacao tree. New functional tools and annotations are available on the Cocoa Genome Hub (http://cocoa-genome-hub.southgreen.fr). Electronic supplementary material The online version of this article (10.1186/s12864-017-4120-9) contains supplementary material, which is available to authorized users.
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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.
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In Theobroma cacao L., declined embryogenic potential was observed in regenerated somatic embryos from long-term secondary somatic embryogenesis (SE). In order to explore the relationship between DNA methylation and the long-term secondary SE, the embryogenic potential and global DNA methylation levels in young (12 months-old), aged (36 months-old) and extra somatic embryogenesis (39 months-old) subjected to different 5-Azacytidine (5-azaC) treatments were comparatively assessed. Global DNA methylation levels increased in aged somatic embryos with long-term in vitro culture, but 5-azaC-supplemented treatments resulted in unaltered levels. In addition, DNA methylation pattern during SE was not affected by 5-azaC. DNA methylation increased during SE expression. Interestingly, the extra SE induction showed that aged somatic embryos can recovery the embryogenic potential in treatment supplemented with 5-azaC at specific concentration. The outcome of this study suggested that the long-term SE in cacao induced the decline on embryogenic potential, which can be reversible trough 5-azaC supplementation. Besides, increased DNA methylation levels might be a response to the stress conditions that plant cells were exposed to during SE.
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Selecting parents and evaluating progenies is a very important step in breeding programs and involves approaches such as understanding the initial stages of growth and characterizing the variability among genotypes for different parameters, such as physiological, growth, biomass partitioning and nutrient translocation to the aerial part. In these cases, facilitating tools can be used to understand the involved gene dynamics, such as diallel crosses and genetic and phenotypic correlations. Our main hypothesis is that the contrasting phenotypes of these parental genotypes of cocoa used are due to genetic factors, and progenies derived from crosses of these parental genotypes are useful for breeding programs related to plant architecture, physiological parameters and translocation of mineral nutrients. We aimed to evaluate the combining abilities in progenies of cacao (Theobroma cacao L) originating from contrasting parents for canopy vigor. Emphasis was given to the evaluation of morphological and physiological parameters and the phenotypic and genotypic correlations to understand the dynamics of the action of the genes involved, as well as in expression profile from genes of gibberellins biosynthesis pathway in the parents. Fifteen F1 progenies were obtained from crosses of six clones (IMC 67, P4B, PUCALA, SCA 6, SCA 24 and SJ 02) that were evaluated in a randomized complete block design with four replicates of 12 plants per progeny, in a balanced half table diallel scheme. It is possible to identify and select plants and progenies of low, medium and high height, as there is expressive genetic variability for the evaluated parameters, some of these on higher additive effects, others on larger nonadditive effects and others under a balance of these effects. Most physiological parameters evaluated show that for selection of plants with the desired performance, no complex breeding methods would be necessary due to the high and medium heritability observed. Strong genetic components were observed from many of the correlations, which indicate the possibility to formulate selection indices for multi-traits, such as dwarfism or semidwarfism, tolerance to increase of leaf sodium concentrations and maintenance of the photosynthetic apparatus integrity under these conditions. Additionally, plants with higher carbon fixation, better water use, higher carboxylation efficiency and greater magnesium accumulation in leaves can be selected.
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Peru is one of the leading exporters of organic cacao beans in the world. However, the accumulation of heavy metals in cacao beans represents a problem for cocoa bean export and chocolate quality. The aim of this study was to investigate the distribution and accumulation of heavy metals in cacao leaves and cocoa beans in three major cacao growing regions of Peru. The study was conducted in cacao plantations of 10 to 15 years old in three regions of Peru: North (Regions of Tumbes, Piura, Cajamarca, and Amazonas); Center (Regions of Huánuco and San Martin) and South (Junin and Cuzco). Samples of leaf and cacao beans were collected from 70 cacao plantations, and the nature of cacao clone or genotype sampled was recorded. The concentrations of heavy metals such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in leaves and beans were determined using atomic absorption spectrophotometer. Overall, concentrations of heavy metals were below the critical limits; however, the presence of high levels of Cd in cacao grown in Amazonas, Piura, and Tumbes regions is of primary concern. Plantations of cacao with different cacao clones show differences in Cd accumulation both in leaves and cocoa beans. Therefore, it is promising to screen low Cd accumulator cacao genotypes for safe production of cacao on lightly to moderately Cd contaminated soils. Also, synergism between Zn and Cd present both in plant and soil suggests that Zn has a direct effect on Cd accumulation in cacao.
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swollen shoot virus (CSSV) [Badnavirus, Caulimoviridae] causes swollen shoot disease of cacao (Theobroma cacao) L. in West Africa. Beginning in ~2002, tests once effective for detection of CSSV failed to detect virus in ~50-70% of symptomatic cacao plants in Ghana and Cote d’Ivoire, suggesting the possible emergence of an uncharacterized CSSV variant(s). Asymptomatic and symptomatic cacao and non-cacao plant samples from Cote d’Ivoire were subjected to polymerase chain reaction (PCR) amplification using eight different sequence-specific and/ or degenerate primer pairs with an expected size amplicon of 375-1100 base pairs. The frequency of PCR-amplification was variable, depending on sample-primer combination. Virus was not detectable in all symptomatic samples, despite characteristic CSSV-like symptoms, and several asymptomatic samples were CSSV-positive. Phylogenetic analysis using Maximum Likelihood of DNA sequences determined from the amplicons resulting from each primer pair resolved two to three groups, two that were closely related to previously reported CSSV isolates, and a third previously undescribed group. Based on the badnavirus species threshold at ≥ 80% pairwise nucleotide (nt) identity for the taxonomicallyinformative RT-RNase H region of the genome, analysis of a partial fragment corresponding to this locus resolved four CSSV groups, at 66-99% nt identity, among the PCR-amplifiable field isolates. Also, sequence analysis of as many as seven additional regions of the CSSV genome revealed extensive within-genome variability. These findings provide robust evidence for extensive genomic variation among multiple, divergent CSSV variants associated with swollen shoot disease symptoms in cacao in West Africa. Keywords: Badnavirus; Caulimoviridae; dsDNA virus; Emergent plant virus; Mealybug-transmitted virus; Pararetrovirus
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Phytophthora palmivora causes pod rot, a serious disease on cocoa widespread throughout the producing regions. In order to ascertain the genetic determination of cocoa resistance to P. palmivora, a study was carried out on two progenies derived from crosses between a heterozygous, moderately resistant Forastero clone, T60/887, and two closely related and highly susceptible Forastero clones, one completely homozygous, IFC2, and one partially heterozygous, IFC5. The cumulative size of both progenies was 112 individuals. Plants were subjected to natural and artificial inoculation of P. palmivora in Côte d'Ivoire. The genetic maps of T60/887 and of IFC5 were constructed using amplified fragment length polymorphism (AFLP) markers and microsatellites. The map of T60/887 comprised 198 markers assembled in 11 linkage groups and representing a total length of 793 cM. The map of IFC5 comprised 55 AFLP markers that were assembled into six linkage groups for a total length of 244 cM. Ratio of rotten over total number of fruit under natural infection was measured for each tree over two harvests. Artificial inoculations were performed on leaves and pods. These tests were weakly correlated with the pod rot rate in the field. Five quantitative trait loci (QTLs) of resistance were detected for T60/887 but none were common between the three traits measured. Stability and reliability of the experimental procedures are discussed and revealed the difficult use of these artificial tests on adult trees for a good prediction of field resistance.Key words: Theobroma cacao, Phytophthora palmivora, cocoa black pod disease, genetic map, quantitative trait locus (QTL).
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Phylloplanins are potential antimicrobial compounds that can be found on the surface of some plants. These compounds were initially identified in Nicotiana tabacum as proteins secreted by short glandular trichomes (STG), which play an important role in controlling infection caused by Peronospora tabacina, especially by acting on the phylloplane, the first site of plant-pathogen interaction. A protein similar to phylloplanins, containing an Ole e 1 domain, was identified in the Theobroma cacao genome database. In this study, we analyzed the Open Read Frame (ORF) and the promoter of T. Cacao phylloplanin (Tcphyll) using bioinformatics tools, the expression pattern of its transcript by RT-qPCR and the promoter activity driving the β-glucuronidase (GUS) expression in transgenic tobacco. The results indicate that Tcphyll encodes a protein of 158 amino acid residues, with signal peptide and potential glycosylation and phosphorylation sites, which are grouped with phylloplanins instead of allergenic pollen grain proteins. Lower levels of Tcphyll transcripts were found in flowers, and higher levels were found in shoot apical meristem, 2–3 cm segment with leaf primordia and, rich in glandular trichomes. Accumulation of transcripts in meristems increased on the first day after inoculation with Moniliophthora perniciosa, and it was reduced after the second day. Promoter analysis identified 40 cis-acting regulatory elements; the GUS expression, under the control of 731 base pairs of the Tcphyll promoter, was observed in tall and short glandular trichomes. The results indicate that TcPHYLL is the first characterized phylloplanin from a perennial tree.
Article
Background Cocoa the food of the Gods, has served the mankind from several decades. The consumption of the cocoa based products is increasing day by day. In fact, the high polyphenol content of cocoa, coupled with its widespread presence in many food items, render this food of particular interest from the nutritional and health viewpoints. The income of around 40 to 50 million people depend on cocoa farming, contributing an annual cocoa production worldwide at 4.2 million tons valued at $11.8 billion and growing at a rate of 3% per year from past decade. Scope and approach In this review the global status, market scenario and processing of the cocoa into different products has been outlined which may be helpful for the concerned communities. As per the reports the market for cocoa is growing at the rate of 18–20% in India and at 3% globally. The SWOT analysis has been carried out to identify the global strength, opportunities and threats. Key findings and conclusion Global demand for cocoa is increasing, as the world continues its insatiable appetite for chocolate products. Silent stakeholders are likely to be future targets of NGO campaigns and consumer scrutiny. Managed well through MSIs critics could enable the silent stakeholders to be encouraged into collaboration. Demand for cocoa is predicted to rise by 30% by 2020 but without empowering and investing in small-scale farmers, the industry will struggle to provide sufficient supply. Cocoa prices are volatile and influenced by many factors – from extreme weather, pests and disease to speculation and political instability in producing countries. Even as cocoa prices rise, farmers have not been capturing their fair share. Fair-trade can be part of a solution, helping to ensure decent incomes for farmers and a long-term supply of quality product to companies.
Article
The phylloplane is the first contact surface between Theobroma cacao and the fungus Moniliophthora perniciosa, which causes witches' broom disease (WBD). We evaluated the index of short glandular trichomes (SGTs) in the cacao phylloplane and the effect of irrigation on the disease index of cacao genotypes with/without resistance to WBD, and identified proteins present in the phylloplane. The resistant and susceptible genotype CCN51 and Catongo presented a mean index of 1600 and 700 SGTs•cm-2, respectively. The disease index in plants under drip irrigation was reduced by approximately 30% compared with plants under sprinkler irrigation prior to inoculation. Leaf water wash (LWW) of the cacao inhibited the germination of spores by up to 98%. Proteins from the LWW of CCN51 were analyzed by 2D SDS-PAGE followed by ms/ms. The gel showed 71 spots and identified a total of 42 proteins; 28 from the plant and 14 from bacteria. Proteins related to defense, to synthesis of defense metabolites and involved in nucleic acid metabolism were identified. The results support the hypothesis that the proteins and water-soluble compounds secreted to the cacao phylloplane participate in the defense against pathogens. They also suggest that SGTs can contribute to the resistance of cacao.