LEAF CHARACTERIZATION AND PROXIMATE COMPOSITION OF

THE SEEDS OF THEOBROMA CACAO ACCESSIONS FROM BOKI

LOCAL GOVERNMENT AREA, CROSS-RIVER STATE, NIGERIA

BY

EGWUATU, CHINECHEREM CYNTHIA

2018/242206

DEPARTMENT OF PLANT SCIENCE AND BIOTECHNOLOGY,

UNIVERSITY OF NIGERIA, NSUKKA.

SUPERVISOR: MR. D. C. URAMA

OCTOBER, 2023.

i
 TITLE PAGE

LEAF CHARACTERISATION AND PROXIMATE COMPOSITION OF THE SEEDS OF

Theobroma cacao ACCESSIONS IN BOKI LOCAL GOVERNMENT AREA, CROSS-
RIVER STATE, NIGERIA

i
 CERTIFICATION

This is to certify that EGWUATU, CHINECHEREM CYNTHIA with registration number

2018/242206, an undergraduate student of the department of Plant Science and
Biotechnology has satisfactorily completed the requirements for the research work for the
course PSB 492. This project has been approved by the Department of Plant Science and
Biotechnology for the award of Bachelor's of Science (B.Sc) Degree. The work embodied in
this project has not been submitted in part or full for any other diploma or degree of this or
any other university.

Approved by:

_____________________ _____________________
MR. D. C. URAMA. PROF. C. C. ONYEKE
(SUPERVISOR) (HEAD OF DEPARTMENT)
Date: ________________ Date: _________________

_________________________
EXTERNAL SUPERVISOR

_______________________
Date

ii
 DEDICATION

This work is dedicated to Almighty God, and to my family.

iii
 ACKNOWLEDGEMENT
I am grateful to Almighty God who has made this work a success. To my supervisor, Mr. D.
C. URAMA for his unending support and encouragement during the cause of this journey,
Mr. Eugene; words can't express my gratitude, thank you so much sir. Also, to Mr. Felix
Nwafor for helping and assisting me during the laboratory analysis, thank you sir.
To my amiable parents Mr and Mrs Leonard Egwuatu, you are the best. Thank you for your
unwavering support during this journey. To my brother Chinonso thank you and may God
enrich you, to my brother Chibuike, thank you. My gratitude goes to my friends and Catholic
Students Choir UNN as well. My gratitude as well goes to the Head of Department, Prof. C.
C. Onyeke for his fatherly love towards every student of this great department.

iv
 TABLE OF CONTENTS
Title Page - - -- - - - - - - i
Approval Page - -- - - - - - - ii
Dedication - - -- - - - - - - iii
Acknowledgment - -- - - - - - - iv
Table of Contents - -- - - - - - - v
List of Tables - - -- - - - - - - viii
List of Plates - - -- - - - - - - ix
Abstract - - -- - - - - - - x
CHAPTER ONE
1.0 Introduction - - - - - - - - - 1
1.1 Background of the Study - - - - - - - - 1
1.2 Justification of the Study - - - - - - - - 2
1.3 Aims and Objective of the Study - - - - - - - 3
CHAPTER TWO
2.0 Literature Review - - - - - - - - - 4
2.1 Review on Cocoa - - - - - - - - - 4
2.1.1 Scientific Classification of Cocoa - - - - - - 4
2.1.2 Morphology and Botanical Description of Cocoa - - - - 4
2.1.3 Ecology and Distribution - - - - - - - 7
2.1.4 Cocoa Composition - - - - - - - - 7
2.1.5 Production and Processing of Cocoa - - - - - - 8
2.1.6 Importance of Cocoa Tree - - - - - - - 10
2.2 Review on Leaf Composition and Seed Proximate Composition- - - 11

CHAPTER THREE
3.0 Materials and Methods - - - - - - - - 15
3.1 Sample Collection- - - - - - - - - 15
3.2 Sample Preparation - - - - - - - - 17
3.3 Planting of the Seeds - - - - - - - - 18
3.4 Leaf Characterization - - - - - - - - 18
3.4.1 Leaf Number - - - - - - - - - 18
3.4.2 Leaf Length - - - - - - - - - 18
3.4.3 Leaf width - - - - - - - - - 18
3.4.4 Number of Veins - - - - - - - - 18
3.4.5 Petiole Length - - - - - - - - - 18
3.5 Proximate Composition Analysis - - - - - - - 20
3.5.1 Determination of Total Ash Content - - - - - - 20
3.5.2 Determination of Crude Protein- - - - - - - 20
3.5.3 Determination of Crude Fat Content using Hexane - - - - 21
3.5.4 Determination of Crude Fiber - - - - - - - 21
3.5.5 Determination of Moisture Content - - - - - - 22
3.6 Statistical Analysis - - - - - - - - 22

v
CHAPTER FOUR
4.0 Results - - - - - - - - - - 23
4.1Results of the Proximate Composition Analysis - - - - - 23
4.2 Results for Leaf Characterization - - - - - - - 26
4.3 Relationship between Cocoa Leaf Morphological Traits - - - - 29
4.4 Relationships between Cocoa Seeds Proximate Characteristics - - - 31
4.5 Relationships between Cocoa leaf Morphological Traits and Seed Proximate - 33
Characteristics

CHAPTER FIVE

5.0 Discussion and Conclusion - - - - - - - 35

5.1 Discussion - - - - - - - - - - 35
5.1.1 Proximate Composition of Cocoa Seeds - - - - - 35
5.1.2 Leaf Morphological Characteristics - - - - - - 38
5.2 Conclusion - - - - - - - - - 40
5.3 Recommendation - - - - - - - - - 40
REFERENCES

vi
 LIST OF TABLES

Table 1: Showing Accessions, Communities and their Perceived Tolerance Level from 16
Boki LGA
Table 2: Proximate Composition in Seeds of Different Cocoa Accessions from Boki 25
LGA
Table 3: Leaf Morphological Characteristics of Different Cocoa Accessions from Boki 28
LGA
Table 4: Relationships between Cocoa Leaf Morphological Traits - - - 30
Table 5: Relationships between Cocoa Seeds Proximate Characteristics - - 32
Table 6: Relationships between Cocoa Leaf Morphological Traits and Seed - 34
Proximate Characteristics

vii
 LIST OF PLATES

Plate 1: Cocoa processing and production - - - - - - 10

Plate 1: Image of dried cocoa seeds - - - - - - - 17
Plate 2: Image of young Cocoa seedlings - - - - - - 19
Plate 3: Image of Grounded Cocoa Seeds - - - - - - 19

viii
 ABSTRACT

Cocoa (Theobroma cacao L.) belonging to the family Malvaceae is one of the important
beverage crops after tea and coffee. Twelve cocoa accessions were obtained from Boki Local
Government Area, and two from Cocoa Research Institute Cross-River State. The seeds were
split into two; one part was ground for lab analysis while the other was planted for leaf
morphological evaluations. The study presents a comprehensive assessment of cocoa
accession from Boki Local Government Area, focusing on both seed proximate composition
and leaf characterisation (leaf morphological traits). The cocoa accessions were analyzed for
their proximate composition, including crude fat, moisture content, total ash, crude protein,
crude fiber, total carbohydrate, and caloric value. Additionally, leaf morphological
characteristics such as leaf length, leaf width, number of veins and petiole length were
measured for each accession. The results revealed notable variations in the proximate
composition among the accessions, highlighting significant diversity in their nutritional
profiles. Specifically, crude fat content ranged from 23.50% to 35.00%, while moisture
content varied from 6.25% to 8.82%. Total ash content showed values ranging from 2.91% to
3.57% and crude protein content exhibited a range of 6.96% to 8.18%. Crude fiber content
varied from 11.12% to 12.67% and total carbohydrate content ranged from 33.95% to
46.00%. Caloric values ranged from 426.48% Kcal/g to 481.76 Kcal/g. Regarding leaf
morphological traits, differences were observed in leaf length, leaf width, number of veins,
and petiole length across the accessions. The leaf length varied from 5.67 cm to 8.00 cm, leaf
width ranged from 11.77 cm to 18.57 cm, the number of leaves ranged from 5.67 to 8, the
number of veins showed variations from 4.43 to 7.13 and petiole length ranged from 1.40 cm
to 4.50 cm. Furthermore, correlation analysis conducted to investigate relationships between
the different morphological traits and proximate characteristics revealed varying degrees of
correlation, indicating potential associations between certain leaf characteristics and seed
composition. It was also discovered that among the accessions evaluated KAN-BOK-TL was
the best as it contains all the evaluated traits in a suitable moderation. This study provides
valuable insights into the diversity of cocoa accessions from Boki Local Government Area,
shedding light on potential avenues for selective breeding and understanding of the
relationships between morphological traits and seed composition in cocoa plants, which are
crucial for the effective agricultural management and genetic improvement programs.

ix
 CHAPTER ONE

INTRODUCTION

1.1 Background of the study

Cocoa (Theobroma cacao L.) belonging to the family Malvaceae is one of the important

beverage crops after tea and coffee. Theobroma means “Food of God '' (Arunkumar and

Jegadeeswari, 2019). Cacao (Theobroma cacao L.) is a neotropical species native to

Amazonian lowland rainforests and is now grown in more than 50 countries for the

livelihoods of 40 to 50 million people worldwide. The main producers of cacao are Cote

d’Ivoire (32.2%), Ghana (19.3%), Indonesia (16.4%), Brazil (6.2%), Cameroon (6.1%) and

Nigeria (5.6%), and both marketing and consumption involves many countries around the

world (Li et al., 2019). Since the introduction of the crop into Nigeria in about 1874

(Oyedele, 2007; Oke and Omotayo, 2012), it has grown to be a major export crop (Afoakwa

et al., 2011).

Cocoa plant varieties are classified into three types: Criollo, Forastero and Trinitario(Rossi et

al., 2021). Cacao (or cocoa) beans/seeds are technically not beans or legumes, but rather the

seeds of the fruit of the Theobroma cacao tree. The pod shaped fruit is botanically classified

as baccate-like (berry-like) and each pod produces approximately 35-50 seeds surrounded by

a sweet pulp (Cuatrecasas, 1964). Cacao pods appear as tiny cucumber-like projections

emerging from small flowers and grow rapidly in the earliest stage. When pods are 15 to 17

weeks old, the ovules begin to continuously solidify from unsolidified gel-like material until

the seeds visually appear to be solid. As the pods develop over the last 8 weeks, the seeds

enlarge and deepen in colour until they are violet (Lehrian and Keeney, 1980).

The quality of the cocoa beans/seeds depends on many factors such as the genotype, the

agronomic management and the climatic conditions (Brunetteo et al., 2005). The main

1
component of cocoa beans is lipid fraction, approximately 50%, mainly constituted by neutral

lipids, with a predominant fraction of triglyceride molecules. Protein fraction constitutes 10–

15% of the dry weight of cocoa seeds, and it is composed of 52% and 43% of albumin and

globulin fractions, respectively (Antonella et al., 2013). The seed is also used in the

cultivation of the cocoa tree.

Leaf characterization that deals with the physical arrangement of the leaves on the branch of

the tree. The leaves are oblong, acuminate, and glabas with prominent central ribs. When

new, depending on the clone or cultivar, they have a colour ranging from green (more or less

rosy) to violet, depending on the amount of anthocyanin present. When old, the leaves lose

their pigmentation, becoming pale green, and finally, dark green and stiff (Rodrigues et al.,

2018).

The Petiole with 2 joined pulvini, one at the base and the other at the point of insertion of

the leaf. Stipules are 2 and are deciduous. Lamina elliptical-oblong or ovate-oblong, simple,

10-45 cm long; generally smooth, sometimes hairy, rounded and obtuse at the base, pointed

apex (Orwa et al., 2009).

1.2 Justification of Study

There has been researches on Theobroma cacao accessions by many researchers on different

areas but none has been able to give a detailed account on the leaf characterization and

proximate composition of the seed especially with regard to the accessions found in Boki

Local Government Area of Cross-River State, Nigeria.

This study is very significant because it bridges the gap left by other researchers in areas of

detailed information on leaf characterization and proximate composition of seed accessions in

Boki Local Government Area of Cross-River State, Nigeria.

2
1.3 Aims and Objective of the study

The aim of this study is to determine the leaf characterization and proximate composition of

the seeds of Theobroma cacao accessions in Boki Local Government Area of Cross-River

State, Nigeria.

The objectives include the following:

1. Morphological characterization of the leaves across different accessions.

2. The proximate composition of minerals found in the seeds of the different Theobroma

cacao accessions.

3
 CHAPTER TWO

LITERATURE REVIEW

2.1 Review on Cocoa

2.1.1 Scientific Classification of Cocoa

Domain: Eukaryota

Kingdom: Plantae

Subkingdom: Tracheobionta (Vascular plants)

Superdivision: Spermatophyta (Seed plants)

Division: Magnoliophyta (Flowering plants)

Phylum: Spermatophyta

Subphylum: Angiospermae

Class: Magnoliopsida (Dicotyledons)

Subclass: Dilleniidae

Order: Malvales

Family: Malvaceae/Sterculiaceae

Genus: Theobroma

Species: Theobroma cacao L.

(Umaharan, 2018).

2.1.2 Morphology and Botanical Description of Cocoa

The cacao plant (Theobroma cacao L.) belongs to the Malvaceae family and Malvales order

and is one of the 22 species of the Theobroma genus (Arguello et al., 1999; De Almeida and

Valle 2007). Cacao (Theobroma cacao L.) is a perennial crop of significant economic

importance in producing countries of West Africa, South America and Southeast Asia. The

4
traditional classification of T. cacao assumes 3 horticultural races or main types: Criollo,

Forastero and Trinitario. The conventional classification of cacao into Criollo and Forastero

is based on distinct morphological, historical and commercial traits. Trinitario is an

intermediate type between Criollo and Forastero, a hybrid group with traits that include the

total variation of the species (Motamayor et al., 2002).

Theobroma cacao L. is a small but economically important tree. It is an evergreen, 4–8 m tall,

of the Sterculiaceae family, native to the tropical region of the Americas (Rusconi and Conti,

2010). Cacao (or cocoa) beans/seeds are technically not beans or legumes, but rather the

seeds of the fruit of the Theobroma cacao tree. The pod shaped fruit is botanically classified

as baccate-like (berry-like) and each pod produces approximately 35-50 seeds surrounded by

a sweet pulp (Cuatrecasas, 1964). Cacao pods appear as tiny cucumber-like projections

emerging from small flowers and grow rapidly in the earliest stage. When pods are 15 to 17

weeks old, the ovules begin to continuously solidify from unsolidified gel-like material until

the seeds visually appear to be solid. As the pods develop over the last 8 weeks, the seeds

enlarge and deepen in colour until they are violet (Lehrian and Keeney, 1980).

The quality of the cocoa beans/seeds depends on many factors such as the genotype, the

agronomic management and the climatic conditions (Brunetteo et al., 2005). The main

component of cocoa beans is lipid fraction, approximately 50%, mainly constituted by neutral

lipids, with a predominant fraction of triglyceride molecules. Protein fraction constitutes 10–

15% of the dry weight of cocoa seeds, and it is composed of 52% and 43% of albumin and

globulin fractions, respectively (Antonella et al., 2013). Each seed contains a significant

amount of fat (40–50% as cocoa butter) (Rusconi and Conti, 2010). The seed is also used in

the cultivation of the cocoa tree.

5
Leaf characterization deals with the physical arrangement of the leaves on the branch of the

tree. The leaves are oblong, acuminate, and glabas with prominent central rib. When new,

depending on the clone or cultivar, they have a colour ranging from green (more or less rosy)

to violet, depending on the amount of anthocyanin present. When old, the leaves lose their

pigmentation, becoming pale green, and finally, dark green and stiff (Rodrigues et al., 2018).

The Petiole with 2 joined pulvini, one at the base and the other at the point of insertion of the

leaf. Stipules are 2 and are deciduous. Lamina elliptical-oblong or ovate-oblong, simple, 10-

45 cm long; generally smooth, sometimes hairy, rounded and obtuse at the base, pointed apex

(Orwa et al., 2009). Flowers are small and yellowish-white or pink in colour, borne on the

trunk and branches.

Classifications based on where cacao is grown have divided the cacao genotypes into

varieties, cultivars, types, or populations (Chessman, 1944; Thomas et al., 2012).

Morphological descriptors are useful because they can help select the best accessions for

breeding programs (Engels et al.,1980). Studies on morphological diversity have been carried

out on flowers, fruits, and leaves of accessions from cacao germplasm (Engels, 1986; Ayestas

et al., 2013), which revealed the existence of two morphological groups: Criollo/Trinitario

and Forastero, with variation between the two groups due to several genetic mixtures among

the genotypes (Motilal et al., 2010). The classification reported by Engels (1986) was

confirmed later by N'Goran (1994), using seed and pod characteristics. However, recent

studies from Motamayor et al. (2008) questioned the existence of only these two genetic

groups, and instead they proposed 10 genetic clusters. Flower trials used earlier by Enríquez

and Soria (1967), and more recently by Lachenaud et al. (1999), allowed for the detection of

great variability among cacao cultivars. All of these results indicate that morphological

markers could be used to structure the diversity of different populations and avoid misleading

classifications (Efombagn et al., 2009).

6
2.1.3 Ecology and Distribution

Cacao tree is wildly grown in tropical and subtropical areas around the world including in

Africa, such as Côte d'Ivoire, Ghana, Nigeria, and Cameroon, in American countries, like

Brazil, Ecuador, Columbia, and Mexico, in Caribbean and Southwestern Pacific countries,

such as the Dominican Republic and Papua New Guinea, and in Southeast Asian countries,

like Indonesia, Malaysia, and Vietnam (FAOSTAT, 2015). Nigeria cocoa production has

steadily grown from 165,000 tons in 1999-2000 to 250,000 tons in 2013-2014 mainly as a

result of high grower prices and to a limited extent also to the government support as outlined

in the 2011 Cocoa Transformation Action Plan (Wessel and Quist-Wessel, 2015).The total

harvested area amounts to 640,000 ha and the average yield is about 400 kg per ha.

Traditionally, cacao is cultivated under the shade of a selectively thinned forest (Lobão et al.,

2007).

2.14 Cocoa Composition

The term “cocoa component” is intended to refer to a fraction derived from shell-free cocoa

nib and includes chocolate liquor, partially- or fully-defatted cocoa solids, cocoa extracts,

cocoa butter and cocoa nib. In particular, the bioactive constituents of cocoa components

exhibit pharmacologic effects in reducing inflammatory processes (Sola et al., 2012). This is

based on their ability to downregulate pro-inflammatory cytokines and their downstream

biochemical pathways (Corti et al., 2009). In addition, according to Lee et al.(2003) phenolic

and flavonoid contents and total antioxidant capacities of cocoa are higher than that of other

phytochemical-rich foods.

Cocoa is unusually rich in polyphenols, but accurate characterisation, let alone quantification

of the poly-phenol content has only been developed relatively recently (ZumbeÂ, 1998). In

addition to polyphenols, cocoa contains methylxanthine compounds, predominantly

7
theobromine and caffeine, but in lower amounts than those of theobromine. The cocoa bean is

an extremely rich source of many essential minerals, including magnesium, copper,

potassium and iron. Moreover, cocoa and cocoa products are rich in iron, but they are

relatively low in potassium (Katz et al., 2011).

2.1.5 Production and Processing of Cocoa

The first harvest takes place after approximately three years (hybrid/improved variety) or 4 –

5 years (traditional variety coming from the nursery) after planting. The cocoa tree can

produce twice a year for more than 30 years. Harvest the pods at regular intervals of 10 - 15

days (do not go over three weeks). Harvest the pods at optimum maturity (when the fruit

turns three quarters yellow, vermilion, orange or red, depending on the pod colour of the

variety. Harvesting is done by cutting the stalk with a machete, a pruning pole, pruning shears

or a sickle. Avoid damaging the flower cushions which will produce the flowers and fruits of

the subsequent harvests.

Next, remove the pods from the plantation and transport them to the pod breaking site. The

second process is the pod breaking stage. The pods are broken no more than 5 days after

harvesting. Separate the healthy pods from the damaged pods to differentiate between the

grades. Open the pods with sticks that have no sharp edges so as to extract the seeds without

damaging them. When breaking the pods, any defective beans, the rachis and cortex debris

must be removed (Adabe and Emilienne, 2014).

Fermentation is essential for rapid reduction of the beans ability to germinate and to develop

the flavour and aroma precursors of the chocolate. The freshly extracted beans are placed on

banana leaves (or cocoyam leaves) inside baskets, wooden boxes, clean fermentation tanks or

on the ground in the fermentation area and under shelter. The beans are then covered with

leaves and left to ferment for 4 – 7 days. Every other day (2nd and 4th day), stir them around

8
and check the humidity. The beans then change colour (from white/mauve to brown). The

inside is light brown or reddish. Well-fermented beans have a shiny appearance, without

mold and their cotyledons break easily. They release a chocolate aroma. After fermentation,

remove the remains of the pulp by washing the beans or mixing them with sawdust and dry

banana leaves.

The beans are then dried naturally or artificially. Natural or solar drying is the simplest and

most popular method and takes 8 – 15 days. In small operations, the beans are often spread

out on bamboo or straw mats placed in the sunlight, on sheets of black plastic, etc. Stir them

around frequently for 5 days. Sort them to remove defective or flat beans. Once dry, their

average weight is one gram with a moisture content of approximately 7 %. Place them in a

dry, sheltered and well-aerated spot to protect them from damp (rain, humid nocturnal air)

and avoid the risk of mold developing. Natural drying can be optimised by using improved

driers that give better results. Depending on the model, the beans can easily be sheltered from

rain or protected by different structures. To reduce drying time, it is possible to use artificial

driers, with hot air for between 15 and 48 hours. The heat is produced by a wood- or gas-fired

furnace. Make sure that the beans don't come into contact with the smoke or with the heating

plate. There must be a system for ventilation and controlling the parameters, particularly the

temperature, as the taste qualities of the cocoa beans change above 55 °C. Storage involves

keeping the cocoa completely dry to avoid mould, insect damage and the formation of free

fatty acids. The dried cocoa beans are placed in jute bags on a pallet to avoid contact with the

ground and walls. The storage location must be dry, clean, well-aerated and protected from

rodents and humidity to ensure the quality of the product. In the case of insect attacks,

fumigate the storage area (Adabe and Emilienne, 2014). Once this phase of processing is

done the beans are then used in the manufacture of cocoa butter, chocolate, cocoa liquor,

chocolate cake e.t.c.

9
Plate 1: Cocoa processing and production

Source: Adabe and Emilienne, 2014.

2.1.6 Importance of Cocoa Tree

The benefits of planting cacao alongside other trees, such as fruit trees, include shade for the

cacao tree, increased biodiversity on the farm, weed inhibition (reducing the need for

chemical herbicides), and additional food and money for the farmer’s family (Prajapati,

2021). Cacao-growing cooperatives are shifting to a more systematic approach to organic

farming, investing in pieces of training and systems to ensure high harvests while protecting

the environment.

10
Cacao-growing cooperatives are transitioning to a more deliberate approach to organic

farming, investing resources in training and programs to ensure strong yields while

preserving the local environment. Chemicals have been used sparingly on cacao fields in the

past because growers couldn’t afford them. Unfermented cocoa beans can be used to extract

cocoa bean fat, which can then be used to make soap. Cacao butter is made from seeds and is

used in skin lotions, cosmetics, and suppository bases (Prajapati, 2021).

For local people, the cocoa tree produces a wide range of products, including fibre for cloth,

thread, and paper, wood for construction, creating utensils, and other items; and home

coverings, among other things. Potassium oxide can be recovered from pod husk ash in the

form of potassium hydroxide, a helpful alkaline in the saponification process (Prajapati,

2021).

2.2 Review on Leaf Characterization and Seed Proximate Composition

Raji (2108) evaluated the proximate composition of cocoa beans obtained from IPELE Farm

Owo, in Ondo State, Nigeria. The cocoa beans were sundried after fermentation for 5 days

and dishelmed. The bean and shell were separately milled into powder. The chemical

composition of both seed and shell flour/powder were investigated according to AOAC

(1980). The proximate composition of the beans of the cocoa are: Moisture (12%), Ash (6%),

Lipid (17.2%), Protein (2.98%), Fibre (34.8%), and carbohydrate (34.98%).

Bertazzo et al. (2012) worked on the composition of cocoa beans and discovered that the

protein fraction constitutes 10-15% of the dry weight of cocoa seeds.

Adeigbe et al. (2021) as well evaluated Variability and character association of bean

biochemical traits of cocoa (Theobroma cacao) genotypes in four Nigerian field banks:

Providing a platform for nutrition-based selection. He worked with fermented and well dried

11
beans from seventy-seven cocoa genotypes belonging to four field banks in Nigeria, where he

evaluated for seventeen proximate and nutritive traits and he discovered their variability and

genotype by trait associations. The cocoa genotypes showed significant differences (p ≤ 0.05)

in moisture, protein, fat, crude fibre, ash. The values below show the mean nutritive traits

(proximate) of the 13 cocoa clones in the local clone plots. Out of the thirteen genotypes, was

recorded; protein content (12.46%), fibre content ranged from 7.42% to 8.73% with a mean

of 8.14%, Fat (23.82%-21.51%), mean ash content was 4.14%-4.80%, moisture content

(9.71%-10.70%).

Ronaldo and Ronan (2011) evaluated four wild species of four wild species, one semi-

cultivated and one cultivated species of Theobroma L., indigenous to Brazil and introduced in

the Bahian cocoa-growing region leaf traits indicated significant differences (P\0.0001)

between each of the six taxa analysed. Petiole length, leaf area and leaf weight were those

traits with higher variation among leaf data. PeL ranged between 1.04cm (T. obovatum) and

2.31cm (T. cacao) and T. bicolor presented the higher means for leaf area (358.95cm 2), and

T. grandiflorum for leaf weight (7.10 g). T. cacao leaf traits were significantly different from

wild species, however there were no differences to T. grandiflorum. The smaller means for

most leaf traits is exhibited by T. obovatum, with the exception of the petiole diameter, which

did not differ from species with large leaves. Similar results were reported by Engels et al.

(1980) for some Theobroma species with raw data.

García et al. (2022) evaluated 113 cacao accessions from the Huarangopampa germplasm

bank in Peru. The morphological traits of the leaf of cocoa studied included agronomic

descriptors such as: leaf length (cm), leaf width (cm) and petiole length (cm). Group 1 didn't

show the above parameters. Group 2 consists of a set of 30 cacao accessions, the highest

values were found for characteristics such as leaf width (11.32 ± 2.09), petiole length (2.46 ±

0.65).

12
Suárez et al. (2018) evaluated on non-destructive estimation of the leaf weight and leaf area

in cacao (Theobroma cacao L.), regression models to predict leaf area and leaf weight in

cacao (Theobrama cacao L.) were fitted using leaves from cultivated plants under nursery

conditions and from plants of commercial plantations, both located in the Amazon

Investigations Center CIMAZ at Macagual, Caquetá, Colombia. A total of 2895 leaves were

collected in such a way to cover a wide range of leaf sizes. Width, length, weight, and leaf

area were measured for each leaf. The total number of leaves was randomly divided into

training and validation sets. The training set was used for model fitting and selection, the

other for measuring model prediction ability. Leaf area and leaf weight were modelled using

different linear regression models based on length and width of leaf. Polynomial regressions

involving both length and width of leaves provided very good models to estimate the

expected area (R2 = 0.98) and weight (R2 = 0.91) of leaves.

Widyasary and Susandarini (2020) evaluated morphological variability and taxonomic

affinity of cocoa (Theobroma cacao L.) clones from Central Sulawesi, Indonesia; leaf

morphology was studied. Variations in leaf characters had been reported on six species of

cocoa in Brazil, in which three quantitative characters showed the highest variation, i.e.

petiole length, leaf area, and leaf weight. These results were consistent with the study on

morphological diversity on cocoa in Cameroon, in which differences between cocoa clones

originating from southern and northern Cameroon were in quantitative characters on fruits.

Similarly, reported that fruit qualitative traits and bean qualitative traits were important and

accounted for the agro-morphological variability observed in 184 accessions of cocoa from

the farmer’s fields and filed genebank collections in Nigeria.

William et al. (2015) worked on morphological characterization of elite cacao trees

(Theobroma cacao L.) in Tumaco, Nariño, Colombia; also evaluated in the study was leaf

13
morphology. The first group (G1) comprised 37.25% of the genotypes; there wasn’t any leaf

characterisation. The second group (G2) comprised 19.6% of the genotypes; this group had

the lowest leaf length/width ratio index (2.60 vs 2.76cm). The third group (G3) was made up

of 23.52% of the genotype; leaf length (35.95 vs 38.71cm) and leaf width (13.18 vs 14.29cm)

present. The fourth group (G4) had 6.86% of the genotypes; absence of leaf characterization.

The fifth group (G5) had 12.75% of the genotype; they had the highest leaf length/width ratio

(2.76 vs 3.05cm).

14
15
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample Collection

Ripe pods of Theobroma cacao accessions were harvested from farmers fields located in

Boki Local Government Area of Cross-River State, Nigeria. The accessions included: BA-

BOK-SC1, BA-BOK-TL1, ABON-BOK-TL, ABON-BOK-SC, AEB-BOK-SCA, AEB-

BOK-TLA, KAN-BOK-TL, KAN-BOK-TL, ORI-BOK-TL, ORI-BOK-SC, WU-BOK-TL,

WU-BOK-SC, CRIN-SGa, CRIN-F3HY. They were properly identified and authenticated by

Mr. Felix Nwafor, a plant taxonomist in the Department Plant Science and Biotechnology,

University of Nigeria, Nsukka.

The table below represented the communities from where the accessions were gotten from

and their perceived tolerance level.

16
Table 1: Showing Accessions, Communities and their Tolerance Level from Boki LGA

Accessions Communities Perceived Tolerance Level

BA-BOK-SC1 Bashua SC1 (Susceptible)

BA-BOK-TL1 Bashua TL1 (Tolerant)

ABON-BOK-TL Abontakom TL (Tolerant)

ABON-BOK-SC Abontakom SC (Susceptible)

AEB-BOK-SCA Abo Ebum SCA (Susceptible)

AEB-BOK-TLA Abo Ebum TLA (Tolerant)

KAN-BOK-TL Kanyang TL (Tolerant)

KAN-BOK-TL Kanyang TL (Tolerant)

ORI-BOK-TL Oriemkpang TL (Tolerant)

ORI-BOK-SC Oriemkpang SC (Susceptible)

WU-BOK-TL Wula TL (Tolerant)

WU-BOK-SC Wula SC (Susceptible)

CRIN-SGa Cocoa Research Institute TL (Tolerant)

Seed Garden Accession

CRIN-F3HY Cocoa Research Institute SC (Susceptible)

F3 Hybrid

17
3.2 Sample Preparation

The pods were cut open using a knife to extract the seeds. The seeds were divided into two

for planting and laboratory analysis. The seeds for lab analysis were dried at room

temperature for 7 days and then in a dehydrator (dehydrator ST-02, China) at 50 0 C for 48

hours. The dried seeds were then ground using a blender (KENWOOD 6000 BT, China)and

stored in an air-tight amber bottle until use.

3.3 Planting of the Seeds

Nurseries were raised in the botanical garden of the Department of Plant Science and

Biotechnology, University of Nigeria, Nsukka. Polythene bags were filled with topsoil mixed

with poultry manure. Two seeds were sown in each polybag laid out in a Complete

Randomised Design (CRD) in the screen house. The bags were watered every two days and

weed removed by hand picking.

Plate 2: Image of dried cocoa seeds.

18
3.4 Leaf Characterization

The parameters used in leaf characterization include: leaf number, leaf length, leaf width,

number of veins and petiole length.

3.4.1 Leaf Number

The numbers of leaves per stalk were counted numerically by direct observation.

3.4.2 Leaf Length

This was measured using a metre rule in centimetres. The leaf length was measured from the

point the petiole ended.

3.4.3 Leaf Width

The leaf width was measured using a metre rule and the area measured was the broadest part

of the leaf.

3.4.4 Number of Veins

The number of veins per leaf was counted numerically by direct observation.

3.4.5 Petiole Length

This was measured using a metre rule.

19
Plate 3: Image of young Cocoa seedlings.

Plate 4: Image of Grounded Cocoa Seeds.

20
3.5 PROXIMATE COMPOSITION ANALYSIS

Using adapted techniques from the Association of Official Analytical Chemists (AOAC,

2005), approximate composition (ash content, crude protein content, crude fat content, crude

fibre and moisture content) was assessed.

3.5.1 Determination of Total Ash

A silica dish that had already been weighed received two grams (2 g) of the powdered

material. A weighing balance was used to determine the weight of the silica dish after it had

been ashed (heated) at 600oC for 3 hours in a muffle furnace. The ash's weight was

determined by the difference in weight. Below is the formula used to determine the % ash

content:

weig h t of as h
Ash = x 100
weig h t of sample

(AOAC, 2005).

3.5.2 Determination of Crude Protein

To ascertain the crude protein concentration, the micro-Kjeldahl technique (1979) was

applied. A 50 ml Kjeldahl flask was filled with one gram of the dry, ground material. With a

pinch of catalyst (a combination of selenium oxide, K 2SO4, and CuSO4), 20 millilitres of

concentrated H2SO4 were carefully added. A fume hood was used to constantly heat the

mixture until a clear, greenish-clear solution emerged. The mixture was cooked for an

additional 2 minutes after the digest had cleared, and then it was allowed to cool. To prevent

caking, 10 ml of distilled water was added to the sample, which resulted in 50 ml of distilled

water.A receiver flask containing 20 ml of boric acid indicator solution was positioned

beneath the dispenser of the distillation apparatus, and 10 ml of the digested sample was

delivered into the Kjeldahl apparatus. After adding 10 ml of a 40% NaOH solution through a

funnel stop stopper, the distillation process started. After being collected through the

21
condenser tip, the distillate (35 ml) was titrated with 0.01 M of HCL until a pink tint was

seen. The following formula was used to determine the protein concentration:

titre value x 1.1 x 0.01 x 100 x 50

Nitrogen =
1000 x 1 g x 10 ml
Protein = % Nitrogen x 6.25 (where 6.25 is a constant) (AOAC, 2005).

3.5.3 Determination of Crude Fat Content using Hexane

A clean beaker was heated up for 30 minutes and allowed to cool. The seed is then heated in

a desiccator and let it all turn blue. 1g of the cocoa sample is measured using a weighing

balance then poured into a conical flask, 20 ml of hexane is poured into the conical flask that

contains the cocoa sample and heated for 3 minutes. The flask is removed from the

desiccators and weighed and extracted filtered into a beaker. Place the extract in a dry oven

for 3 minutes. The heated extract is placed in a desiccator to cool. The final weight is

weighed and the (%) fat calculated using the formula below:

C−B 100
Fat = x
A 1
Where B = Weight of empty flask

A = weight of sample

C = weight of flask + oil after drying (AOAC, 2005).

3.5.4 Determination of Crude Fibre

The ground sample was heated to boiling for 30 minutes in 150 ml of 0.218 M pre-heated

H2SO4 in a 400 ml beaker, cooled, and then filtered. With hot water, the residue was cleaned

three times. This was then heated to boiling by adding 128 moles of previously heated KOH.

An antifoaming agent was applied in a few drops. Following a 30-minute boil, it was filtered.

The residue was thoroughly cleaned three times with hot water and a further three times with

22
acetone before being dried in a crucible at 103°C for an hour and weighed (W2). After being

ashed at 500oC, it was once more weighed (W3).

Using the following formula, the percentage of fibre was calculated.

W 2−W 3
% Fibre = x 100
W1
Where W1 = weight of the ground sample used

W2 = Weight of residue after drying at 103oC

W3 = Weight of ash (AOAC, 2005).

3.5.5 Determination of moisture content:

Five grams (5 g) of the ground sample were dried to a constant weight at 600 oC in a hot air

circulating oven to determine the sample's moisture content. The change in weight following

drying was used to calculate the moisture content, as indicated below:

W 2−W 3 100
X
W1 1

Where W1=initial weight of empty crucible

W2 = Weight of crucible + sample before drying

W3 = Final weight of crucible + sample after drying (AOAC, 2005).

3.6 Statistical Analysis

In this course of analysis, one way analysis of variance (ANOVA) was used to statistically

analyse the data used obtained in the study. The level of statistical significance was set at

p < 0.05% (95% confidence interval). The software used was SPSS version 25.

23
 CHAPTER FOUR

RESULTS

4.1 Results of the Proximate Composition Analysis

The cocoa powder was analysed for proximate composition and the results are displayed in

Table 2 below. The cocoa powder shows the presence of crude fat, moisture content, total

ash, crude protein, crude fibre, total carbohydrates and caloric value. The result showed a

significant difference (p < 0.05) in crude fat, moisture content, total ash, crude fibre, total

carbohydrate and caloric value but there was a slight significant difference in the crude

protein.

CRIN-SGa had the highest amount of crude fat with the mean value of 35.00±1.00%, while

CRIN-F3HY had the least amount of crude fat with the mean value of 23.50 ± 1.50%. The

highest value recorded with CRIN-SGa was not significantly different from BA-BOK-SC1

and BA-BOK-TL1. On the other hand, CRIN-F 3HY with the lowest crude fat was not

significantly lower (p > 0.05). When compared to ABON-BOK-TL, ABON-BOK-SC, AEB-

BOK-SCA, AEB-BOK-TLA, KAN-BOK-TL,KAN-BOK-TL, ORI-BOK-TL, ORI-BOK-SC,

WU-BOK-TL, WU-BOK-SC.

ABON -BOK-TL had the highest amount of moisture content with the mean value of 8.82 ±

0.02%, while AEB-BOK-TLA had the lowest amount of moisture content with the mean

value of 6.25 ± 0.15%. Whereas AEB -BOK-TL was significantly different from all the other

accessions except KAN-BOK-TL and CRIN-F3HY with an average of 8.15 ± 1.10%, 7.75 ±

0.25% and 8.01 ± 0.02% respectively.

24
ORI-BOK-TL had the lowest amount of total ash with the mean value of 2.91 ± 0.02% while

CRIN-F3HY had the highest amount of total ash with the mean value of 3.57 ± 0.10%.

CRIN-F3HY with the mean value of 3.75 ± 0.10% was significantly different from every

other accession. While the lowest value in ORI-BOK-TL was not significantly different (p >

0.05) from BA-BOK-SC1, ABON-BOK-TL, AEB-BOK-SCA and AEB-BOK-TLA.

ABON-BOK-TL had the lowest amount of crude protein with the mean value of 6.69 ±

0.05% while ORI-BOK-TL had the highest amount of crude protein with the mean value of

8.18±0.04%. BA-BOK-TL1, ABON-BOK-SC, KAN-BOK-TL, ORI-BOK-SC, WU-BOK-

TL, WU-BOK-SC, CRIN-SGa and CRIN-F3HY were statistically similar from the rest.

CRIN-F3HY had the lowest amount of crude fibre with mean value of 11.12 ± 0.14% while

AEB-BOK-TLA had the highest amount of crude fibre with the mean value of 12.67 ±

0.04%. AEB-BOK-TLA with the mean value of 12.67 ± 0.04% was significantly different

from all other accessions except AEB-BOK-SCA, ABON-BOK-TL and CRIN-SCa.

CRIN-SGa with the mean value of 33.95 ± 1.13% had the lowest total amount of

carbohydrates while ORI-BOK-SC with the value of 45.77 ± 0.72% was the highest. WU-

BOK-TL, BA-BOK-TL1 and CRIN-SCa were significantly different from the total

carbohydrate content in ORI-BOK-SC.

CRIN-SGa had the highest amount of caloric value with the mean value of 481.76 ± 4.04

Kcal/g while the accession with the lowest amount of caloric value was KAN-BOK-TL with

the mean value of 428.86±1.78 Kcal/g. CRIN-SCa with the mean value 481.76 ± 4.04 Kcal/g

was significantly different from every other accession except BA-BOK-SC1, BA-BOK-TC1,

AEB-BOK-SCA, ORI-BOK-TL and WU-BOK-TL.

25
Table 2 : Proximate Composition in Seeds of Different Cocoa Accessions from Boki
LGA
Crude fat Moisture Total ash Crude Crude Total Caloric
(%) content (%) protein fibre (%) Carboh value
(%) (%) ydrate (Kcal/
(%) g)
BA-BOK-SC1 30.00 ± 6.83 ± 2.99 ± 7.33 ± 12.10 ± 40.77 462.36
0.00abcd 0.02cde 0.01 gh
0.04d 0.06bcde ± ±
0.12abc 0.32abcd
BA-BOK-TL1 33.50 ± 7.30 ± 3.07 ± 7.65 ± 12.18 ± 36.30 477.30
4.50ab 0.46bcde 0.03efg 0.07bc 0.01bcd ± ±
4.13cd 24.26ab
ABON-BOK-TL 24.50 ± 8.82 ± 3.13 ± 6.96 ± 12.06 ± 44.54 426.48
0.50de 0.02a 0.01 fgh
0.05e 0.04ab ± ± 2.44e
0.57ab
ABON-BOK-SC 26.00 ± 6.70 ± 3.17 ± 7.68 ± 11.72 ± 44.74 443.66
2.00cde 0.35de 0.04 cde
0.00b 0.37ef ± 1.25a ±
13.02cde
AEB-BOK-SCA 29.00 ± 6.88 ± 3.01 ± 8.03 ± 12.50 ± 40.60 455.48
2.00bcde 0.03bcde
0.02 fgh
0.03a 0.01ab ± ±
1.96abc 10.28abcd
e

AEB-BOK-TLA 27.00 ± 6.25 ± 2.96 ± 7.50 ± 12.67 ± 43.64 447.52

0.00cde 0.15e 0.05 gh
0.06c 0.04a ± ±
0.09ab 0.64bcde
KAN-BOK-TL 24.50 ± 8.15 ± 3.19 ± 7.73 ± 12.07 ± 44.37 428.86
0.50de 1.10ab 0.01 cde
0.06b 0.02cde ± ± 1.78e
1.63ab
KAN-BOK-TL 25.00 ± 6.45 ± 3.34 ± 8.10 ± 12.19 ± 44.93 437.08
1.00de 0.15e 0.02b 0.08a 0.03bcd ± 0.93a ± 5.56de
ORI-BOK-TL 27.50 ± 6.89 ± 2.91 ± 8.18 ± 11.79 ± 42.75 451.18
0.50cde 0.04bcde
0.02h 0.04a 0.00def ± ±
0.40ab 2.78abcde
ORI-BOK-SC 24.50 ± 7.05 ± 3.14 ± 7.65 ± 11.90 ± 45.77 434.18
0.50de 0.14bcde
0.07 de
0.01bc 0.03cde ± 0.72a ± 1.58de
WU-BOK-TL 31.50 ± 7.02 ± 3.24 ± 7.79 ± 11.46 ± 39.01 470.66
2.50abc 0.27bcde
0.01 bcd
0.08b 0.21fg ± ±
2.48bcd 12.30abc
WU-BOK-SC 26.50 ± 6.80 ± 3.12 ± 7.70 ± 11.46 ± 44.44 447.04
1.50cde 0.45cde 0.01 def
0.02b 0.11fg ± ±
0.95ab 9.64bcde
CRIN-SGa 35.00 ± 7.75 ± 3.29 ± 7.74 ± 12.27 ± 33.95 481.76
1.00a 0.25abcd
0.02bc 0.11b 0.03abc ± 1.13d ± 4.04a

26
CRIN-F3HY 23.50 ± 8.01 ± 3.57 ± 7.81 ± 11.12 ± 46.00 426.72
1.50e 0.02abc 0.10a 0.02b 0.14g ± 1.22a ± 8.52e

27
4.2 Results for Leaf Characterization

The leaf characterization was carried out basically by observation using sight and the use of

metre rule in centimetres. The parameters recorded include: Number of leaves, Leaf length,

Leaf width, Number of veins and Petiole length. The result showed significant differences in

the Leaf width, leaf length and number of veins. Moreso, there was no significant difference

across the accessions in the number of leaves.

In the recorded petiole length, ABON-BOK-TL, ABON-BOK-SC, AEB-BOK-TLA, KAN-

BOK-TL, KAN-BOK-TL, ORI-BOK-SC, WU-BOK-TL, WU-BOK-SC, CRIN-SGc, CRIN-

F3HY showed no significant difference although they were significantly different from the

other accessions.

Table 3 showed that AEB-BOK-SCA had the highest number of leaves with 8.00 ± 0.00

while ABON-BOK-TL and BA-BOK-TL1 had the fewest number with 5.67 ± 0.33. It was

observed that all were significantly similar.

The leaf length showed a difference range from a high 18.57 ± 3.43 cm to a low 11.77 ± 0.07

cm in WU-BOK-SC and ORI-BOK-TL respectively. ORI-BOK-TL with the mean value of

11.77 ± 0.07 cm was significantly different from WU-BOK-SC (p < 0.01) with the mean

value of 18.57 ± 3.43 cm.

The leaf width recorded showed that ABON-BOK-SC had the highest leaf width value with

the mean value of 7.13 ± 0.47 cm while ORI-BOK-TL had the lowest leaf width value with

the mean value of 4.43 ± 0.07 cm. It was observed that ABON-BOK-SC with the mean value

7.13 ± 0.47 cm was significantly similar with all other accessions except BA-BOK-SC1,

AEB-BOK-SCA, KAN-BOK-TL, KAN-BOK-TL, ORI-BOK-TL, CRIN-SGa and CRIN-

F3HY

The number of veins recorded showed ORI-BOK-TL and WU-BOK-TL with the highest

mean value of 12.67 ± 0.67 cm while the fewest was recorded in BA-BOK-SC1 with a mean

28
value of 9.00 ± 0.00 cm. BA-BOK-SC1 with the mean value of 9.00 ± 0.00 was significantly

different from ORI-BOK-TL, WU-BOK-TL and WU-BOK-SC.

BA-BOK-SC1 and ORI-BOK-TL recorded a ranged difference in mean values from a high

4.50 ± 0.00 cm and to a low 1.40 ± 0.10 cm in the petiole length. BA-BOK-SC1 with the

mean value of 4.50 ± 0.00 was significantly different from ORI-BOK-TL with mean value of

1.40 ± 0.10 while BA-BOK-TL1 and AEB-BOK-SCA were significantly similar with mean

values of 2.33 ± 0.17 respectively. While the rest of the accessions were all significantly

similar.

29
 Table 3: Leaf Morphological Characteristics of Different Cocoa Accessions from Boki
LGA
NO. OF LEAF LEAF PETIOLE
NO. OF
LEAVES LENGTH WIDTH LENGTH
VEINS
(cm) (cm) (cm)

BA-BOK-SC1 6.00 ± 0.00a 12.00 ± 0.00cd 4.50 ± 0.00d 9.00 ± 0.00e 4.50 ± 0.00a

BA-BOK-TL1 5.67 ± 0.33a 13.03 ± 0.77cd 5.53 ± 0.27bcd 9.67 ± 0.33de 2.33 ± 0.17cd

ABON-BOK-TL 5.67 ± 0.33a 14.60 ± 1.72abcd 6.03 ± 0.69abcd 10.67 ± 0.33bcd 2.83 ± 0.17bc

ABON-BOK-SC 6.33 ± 0.33a 16.33 ± 0.67abc 7.13 ± 0.47a 10.00 ± 0.58cde 3.00 ± 0.00bc

AEB-BOK-SCA 8.00 ± 0.00a 16.27 ± 2.13abc 4.87 ± 0.63d 11.00 ± 0.00bc 2.33 ± 0.17cd

AEB-BOK-TLA 6.67 ± 0.33a 17.67 ± 0.17ab 6.67 ± 0.17abc 11.67 ± 0.67ab 3.00 ± 0.00bc

KAN-BOK-TL 7.00 ± 1.53a 15.17 ± 1.42abcd 5.10 ± 0.21cd 10.00 ± 0.00cde 3.00 ± 0.29bc

KAN-BOK-TL 7.67 ± 0.67a 14.30 ± 0.60abcd 4.83 ± 0.33d 10.00 ± 0.00cde 2.67 ± 0.17bc

ORI-BOK-TL 6.33 ± 0.33a 11.77 ± 0.07d 4.43 ± 0.07d 12.67 ± 0.67a 1.40 ± 0.10d

ORI-BOK-SC 7.67 ± 0.33a 15.67 ± 1.30abcd 5.83 ± 0.44abcd 11.00 ± 0.00bc 2.67 ± 0.17bc

WU-BOK-TL 7.33 ± 0.67a 15.33 ± 0.17abcd 5.67 ± 0.33abcd 12.67 ± 0.67a 3.33 ± 0.17bc

WU-BOK-SC 7.33 ± 0.67a 18.57 ± 3.43a 7.00 ± 1.00ab 12.67 ± 0.33a 3.17 ± 0.83bc

CRIN-SGa 7.67 ± 0.33a 15.33 ± 1.33abcd 5.30 ± 0.60cd 11.67 ± 0.33ab 3.33 ± 0.33bc

CRIN-F3HY 7.67 ± 1.86a 13.33 ± 0.83bcd 4.67 ± 0.74d 9.67 ± 0.33de 3.17 ± 0.17bc

30
4.3 Relationship between Cocoa Leaf Morphological Traits
The result as presented on Table 4 showed that significant and positive relationship exist

between leaf length and leaf width (r = 0.819, p<0.01), leaf length and number of veins (r =

0.302, p<0.05), and leaf length and petiole length (r = 0.317, p<0.05). Moreso, number of

leaves showed a weak relationship with leaf length (r = 0.283, ns), leaf width (r = 0.116, ns),

number of veins (r =0.192, ns) and petiole length (r = 0.097, ns). Leaf width also showed

weak relationship with number of veins (r = 0.219, ns) and petiole length (0.260, ns).

31
Table 4: Relationships between Cocoa Leaf Morphological Traits
Petiole
No. of leaves Leaf length Leaf width No. Of veins
length
No. Of leaves 1 .283 .116 .192 .097
Leaf length 1 .819** .302* .317*
Leaf width 1 .219 .260
No. Of veins 1 -.266
Petiole length 1

32
4.4 Relationships between Cocoa Seeds Proximate Characteristics
The result presented in Table 5 revealed crude fat had a significant and negative relationship

with total carbohydrate (r = -0.979, p<0.01) while it showed a significant and positive

relationship with caloric value (r = 0.985, p<0.01). Moreso, significant and negative

relationships were seen in moisture content and crude protein (r = -0.393, p<0.05).

Moreover, crude fat showed a weak and negative relationship with moisture content (r = -

0.154, ns) and total ash (r =-0.200, ns) as well as a weak and positive relationship with crude

protein (r = 0.063, ns) and crude fibre (r = 0.171, ns). Similarly, moisture content showed a

negative and weak relationship with crude fibre (r = -0.155, ns), total carbohydrate (r = -0.17,

ns) and caloric value (r = -0.305, ns) as well as a weak and positive relationship with total ash

(r = 0.338, ns). Total ash and crude fibre (r = -0.466, p<0.05) had a negative and significant

value. Total ash also showed a weak and positive relationship with crude protein (r = 0.121,

ns) and total carbohydrate (r = 0.129, ns) with a weak and negative relationship with caloric

value (r = -0.242, ns).Crude protein showed a weak and negative relationship with crude

fibre(r = -0.096, ns) and total carbohydrate (r = -0.055, ns) while it showed a weak and

positive relationship with caloric value (r = 0.127, ns). Lastly, crude fibre revealed a weak

and negative correlation with total carbohydrate (r = -0.220, ns) with a weak and positive

correlation with caloric value (r = 0.121, ns).

33
 Table 5: Relationships between Cocoa Seeds Proximate Characteristics
Moisture Total Crude Crude Total Caloric
Crude
content ash protein fibre Carbohydrate value
fat (%)
(%) (%) (%) (%) (%) (Kcal/ g)
Crude fat (%) 1 -.154 -.200 .063 .171 -.979** .985**
Moisture content (%) 1 .338 -.393* -.155 -.017 -.305
Total ash (%) 1 .121 -.466* .129 -.242
Crude protein (%) 1 -.096 -.055 .127
Crude fibre (%) 1 -.220 .121
Total Carbohydrate
1 -.931**
(%)
Caloric value (Kcal/ g) 1

34
4.5 Relationships between Cocoa leaf Morphological Traits and Seed Proximate
Characteristics
Table 6 showed a significant and positive relationship in leaf length and carbohydrate (r =

0.374, p<0.05), number of veins and total ash (r = 0.494, p< 0.01) and number of veins and

crude proteins (r = 0.527, 0.01) while a significant and negative relationship was seen in

petiole length and total ash (r = -0.546, p<0.01). In the other parameters, there were no

significant relationships while it showed weak positive and negative relationships.

35
 Table 6: Relationships between Cocoa Leaf Morphological Traits and Seed Proximate

Characteristics

Caloric
Moisture Crude Crude Total
Crude Total value
content protein fibre Carbohydrate
fat (%) ash (%) (Kcal/
(%) (%) (%) (%)
g)
No. Of leaves -.070 .107 .268 .221 -.285 .050 -.069

Leaf length -.355 -.189 -.095 .338 -.046 .374* -.307

Leaf width -.175 -.278 -.273 .140 .163 .214 -.130

No. Of veins -.006 .116 .494** .527** -.041 -.076 -.037

Petiole length -.019 -.277 -.546** -.171 .124 .098 .034

36
 CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1 Discussion

5.1.1 Proximate Composition of Cocoa Seeds

The chemical composition of cocoa beans has been studied extensively by Adeigbe et al.

(2021). Proximate composition of food simply includes moisture, ash, lipid, protein and

carbohydrate contents. The results observed in this study supports the theory of the presence

of these compounds in Theobroma cacao accessions found in Boki LGA.

The crude fat content from this study ranged from 23.5% to 35% which corroborates with

Adeigbe et al. (2021) whose value for fat was 21.51% to 23.82% but does not correlate with

Raji (2018) whose value was 17.2% mean value. However, according to Mustiga et al. (2019)

environmental conditions and genetic composition of the accession could have played a

crucial role in the Fatty acid variation observed (Mustiga et al., 2019). CRIN-SGa was the

accession with the highest amount of fat content and this was anticipated because it has

undergone certain improvements from the research institute. Dietary guidelines from the

World Health Organization and the Dietary Reference Intakes recommend a total fat intake is

between 20 and 35% of total calories (Liu et al., 2017). Fats help our bodies absorb important

vitamins—called fat-soluble vitamins—including vitamins A, D and E (National Academies

Press (US), 1989). Therefore, this cocoa accession can be consumed for daily fibre

requirement.

The moisture content is an indication of the shelf life of the product; the lower the moisture

content the longer the shelf life (Adeigbe et al., 2021) . Moisture content in this study varied

from approximately 6.2% to 8.8%. This result was in deviation from previous reports of

Adigbe et al. (2021) whose value was 9.71%-10.70% and Raji 2018 whose value was 12%.

The cause in variation between studies was likely as a result of the porosity of the husk

37
enclosing the cotyledons. AEB-BOK-TLA is the accession recommended for this study with

regards to its lower moisture content for storage purposes. Cocoa beans must be dried to

about 55% to 7% moisture content level to be safe for storage (Kotey et al., 2022).

Early studies of Voigt et al. in the 1990s confirm that cocoa seed storage proteins play an

important role in flavour development since essential precursors of the cocoa-specific aroma

components are formed from their degradation during the fermentation process. In this study,

the protein value varies approximately from 6.92%to 8.18%. This deviates from the study of

Bertazzo et al. (2012) whose value was 10-15%, Raji 2018 (2.98%) and Adeigbe et al. (2021)

(12.46%). It has been speculated that the chemical composition of cocoa beans, especially the

proteins, polysaccharides and polyphenols are determined by the genotypes. This infers that

the expression of nutritional components may not be due to environmental factors alone; it

may well be under genetic control (Adeigbe et al., 2021). At least 10% of your daily calories,

but not more than 35%, should be from protein, according to the Institute of Medicine (Jaret,

2008). The cocoa accessed does not possess the required amount needed in a day, so it should

be augmented when consumed.

Crude fibre daily dietary requirement for according to Smiley (2023), the Daily Value for

fibre is 28 grams per day for adults on a 2,000-calorie diet. This number may also depend on

the age or sex of the individual (Smiley, 2023) . Crude fibre content ranges from

approximately 11.1% to 12.5% in this study. This deviates from Raji (2018) whose fibre

value was 34.8% and Adeigbe et al. (2021) whose value was 7.42% to 8.73%. These

variations occurred not just as a result of environmental factors alone; it may as well be under

genetic control (Adeigbe et al., 2021). In this study the recommended accession for this trait

was AEB-BOK-TLA as it has the highest amount of crude fibre. However, the evaluated

cocoa accessions did not contain the amount of fibre needed in a daily proportion but should

be supplemented with other food sources.

38
Carbohydrates play an important role in the human body. They act as an energy source, help

control blood glucose and insulin metabolism, participate in cholesterol and triglyceride

metabolism, and help with fermentation (Holesh et al., 2023). Total carbohydrate content in

this study showed a variation from around 33.95% to 46%. This correlates with Raji (2018)

whose value was 34.98%. The Institute of Medicine set an acceptable macronutrient

distribution range (AMDR) for carbohydrates of 45–65% of total calories (Slavin and

Carlson, 2014). The accession in this study with the highest amount of carbohydrate was

CRIN-SGa. Products made from seeds of this accession could be consumed to obtain the

required carbohydrate needed in a day.

Calories are a measure of energy. "Small" calories (cal) estimate the amount of energy

required to raise the temperature of exactly one gram of water by one degree Celsius at one

atmospheric pressure, and “big” calories, also known as kilogram calories (Cal), are more

commonly known and refer to the calories in food (Osilla et al., 2022). Caloric value per

gram ranges from approximately 426.5 to 481.8 Kcal/g. Montagna et al. (2019) carried out a

comparative study on cocoa dark chocolate and light chocolate and discovered that cocoa has

355 Kcal/g. This does not correlate with results available in this study. This variation could

have occurred as a result of environmental factors and can also be linked with genetic

differences in accessions (Adeigbe et al., 2021). The estimate used for Nigeria is slightly

lower at 2251 calories per day and is a weighted average of the minimum caloric intake for

women (2117), men (2900) and children (783-2958, depending on age) (Thomas and Turk,

2023). In this study the accession with the highest amount of caloric value was CRIN-SGa

and it has to be augmented with other energy sources when consumed to meet up with the

daily required intake.

Ashing is an important step in proximate or specific mineral analysis. Ash refers to the

inorganic (mineral) residue remaining after the combustion or complete acid-facilitated

39
oxidation of organic matter in food (Harris and Marshall, 2017). In this study total ash varies

from approximately 2.9% to 3.6%. This deviates from the study of Raji (2018) whose ash

value was 6% and Adeigbe et al. (2021) whose value was 4.14% to 4.80%. The variation in

result could be as a result of climate change, poor farm management, and diseases which are

the major limiting factors to bountiful cocoa production around the world (Etaware, 2022).

5.1.2 Leaf Morphological Characteristics

The leaf is an important organ for plant photosynthesis, respiration and transpiration. Its size

and shape will affect photosynthetic efficiency and plant growth closely related to plant

growth potential, nutrient supply, yield, quality and resistance (Nicotra et al., 2011). In this

study, parameters evaluated included the number of leaves, leaf width, petiole length, leaf

length and number of veins. Meanwhile, petiole length can be a determining factor in the

efficiency of photosynthate transport by affecting canopy architecture, and thus the rate of

canopy light interception and photosynthetic efficiency, ultimately improving the yield (Ross

et al. 2000; Zhu et al. 2010). The petiole length ranges from approximately 1.4 to 4.5 cm and

the study correlates with Ronaldo and Ronan, 2011 (2.31 cm) with regards to petiole length

as well as Gracia et al. (2022) (2.46 cm). BA-BOK-SC1 was observed to have longer petioles

which selection of this accession could advance the improvement of increased photosynthetic

activities in cocoa. Leaf length ranges from approximately 11.77 to 17.67cm. This does not

correlate with William et al. (2015) in his study (35.95 vs 38.71cm) with regards to leaf

length of his third accession. Variation in the leaf size and shape has been shown to be

correlated with climatic factors. In addition, other environmental factors, such as light

intensity and nutrient availability, can influence leaf size and shape (Li et al., 2015). The

number of leaves during flowering serves as an important parameter for bunch development

in bananas since these leaves are related to the plant photosynthetic rate and reflect the

40
potential yield (Nomura et al., 2017). Since leaves are essential for photosynthesis and

produce the bulk of biomass, the number of leaves will also influence yield (Trimble, 2022).

The number of leaves varies from 5.67 to 8 across different accessions. In this study, leaf

length ranges from approximately 11.77 to 17.67 cm, this does not correlate with William et

al. (2015) whose value was 35.95 vs 38.71 cm. Selection of accession with increased number

of leaves (AEB-BOK-SCA) will be recommended for improved productivity in cocoa.

Water supply and demand in leaves are primarily determined by stomatal density (SD, water

demand) and minor leaf vein density (VLA, water supply) (Zhang et al., 2022). In this study

the number of veins was also evaluated and it ranges from 4.5 to 7.13 cm. The accessions

with higher leaf veins were ORI-BOKI-TL and WU-BOK-TL and could be selected for

breeding cocoa with high water conductance translating to increased yield.

Factors that may affect leaf width include: soil nutrient availability, temperature, light, and

water availability (Wang et al., 2022). Also leaf width was also evaluated and it varies from

about 4.43 to 7.13 cm, William et al. (2015) correlates with the study with regards to leaf

width of his first and fifth accession respectively (2.76 cm).

41
5.2 Conclusion

The study evaluated the leaf characterization and proximate composition of seed of

Theobroma cacao which provides valuable insights into their nutritional and physiological

characteristics. Leaf morphology, including traits like leaf weight, leaf length, petiole length,

number of veins e.t.c, plays a crucial role in photosynthesis and overall plant health.

Understanding these traits can aid in the assessment of plant vigour and adaptation to various

environmental conditions. Furthermore, the proximate composition analysis offers essential

information about cocoa seeds, protein, fat, moisture content, total ash, total carbohydrate.

In the relationship between the cocoa leaf characteristics and seed proximate composition; it

was observed that the longer the leaf length the higher the total carbohydrate content of the

seed of which ORI-BOK-SC could be recommended for further evaluation. Also, the petiole

length was related with total ash content of the seed, therefore, the shorter the petiole length

the smaller the total ash content of the seed. Therefore, for an increase in ash content, KAN-

BOK-TL could be recommended for selection for further breeding. Lastly the number of

veins as well played a role in the total ash content and also in the crude protein content of the

seeds. The higher the number of veins, the higher the ash content of the seed and accession

KAN-BOK-TL could be further improved for ash and protein content via selection based on

increased number of veins.

5.3 Recommendations

Having carried out thorough analysis on the 14 cocoa accessions studied in this work KAN-

BOK-TL was the best accession as it possessed all the evaluated traits in the study in

moderate amounts. Further research in this area and using this accession holds promise for

continued advancements in cocoa cultivation and its global significance in the agricultural

industry.

42
43
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