INTRODUCTION

Background of the Study

Theobroma cacao belongs to the Malvaceae family and subfamily Sterculioidea. It is

considered one of the most valuable cultivated crops in the Philippines, which could provide

a possible source of income for large and small-scale farmers. There is a rising global

demand for the crop, which has tripled since 1970 (Cabugsa et al., 2019; Solpot, 2020;

Zuidema et al., 2005). Caraga is among the top producing regions of cacao in the country.

Currently, cultivated cocoa trees have been categorized into three main groups. The

first majority group is Criollo, which has a nicely flavored bean but is susceptible to diseases.

The second group is Forastero, which has a high yield and is highly tolerant to diseases,

although the bean aroma is weak. The third group is Trinitario, which is the hybrid group of

both criollo and forastero, thus it has inherited the high-quality traits of both groups (Patel &

Watson, 2018).

Production constraints due to diseases and climate change phenomena such as El Niño

and La Niña are among the major challenges facing the cocoa industry. Cocoa plants are

sensitive to drought stress and this negatively affects its natural processes eventually

disrupting its metabolism and photosynthesis (Zakariyya, Setiyawan, & Susilo, 2017).

Studies that could address this problem continue to develop and one possible solution to this

dilemma is to plant cacao trees that are potentially tolerant to drought. Thus, the mechanism

of plant adaptation under drought stress must be well understood first.

Leaf stomata play an essential role in the survival of plants under drought conditions.

Stomatal closure commonly happens as an adaptive response of plants to the onset of drought

condition in order to regulate the rate of transpiration or uptake of carbon dioxide by leaves

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(Bosabalidis & Kofidis, 2002; Agurla et al., 2018). The ability of crops to maintain water-

potential values and turgor under water limiting conditions is an important physiological

adaptation to drought. This maintenance of turgor and stable water potential values can be

attained through stomatal control. Thus, the capacity of cacao to regulate stomatal closure as

a way to sustain a high relative water content would be a good indicator and basis for future

efforts towards breeding drought-tolerant cacao (Medina & Laliberte, 2017)

According to the Philippine Atmospheric, Geophysical and Astronomical Services

Administration (PAGASA) (2021), the climate of the country can be divided into two major

seasons: (1) the rainy season, from June to November; and (2) the dry season, from

December to May. The dry season may be subdivided further into (a) the cool dry season,

from December to February; and (b) the hot dry season, from March to May. Plants respond

to the stress brought about by the dry season through conserving water in their bodies, which

involves controlling their stomata and regulating their relative water content (Yamasaki &

Dillenburg, 1999). It is also important to take note that the adaptation of plants to specific

regions they inhabit depends on varying environmental conditions caused by both abiotic and

biotic factors and that species of plants respond to growth stressors differently.

The goal of this study is to investigate the stomatal and leaf water status changes in

the locally propagated Theobroma cacao plant (BR 25) variety at VPO cacao farm, located

in Rosario Agusan del Sur, during months of March to May 2021, in response to the probable

drought stress because of the dry season.

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Objectives of the Study

This study aims to evaluate the stomatal and leaf water status characters of the leaves of

Theobroma cacao BR 25 variety during the hot dry season. The specific objectives are as

follows:

1. determine the rainfall distribution, temperature, and relative humidity at the time of

observation from the month of March to May;

2. examine the stomatal traits of the cocoa plants during the months of March to May;

3. determine the thickness and relative water content of the cocoa plant leaves during the

months of March to May;

and

4. Provide Statistical data Analysis.

Significance of the Study

This study is found to be timely and relevant. The results of this study are significant

to the following:

Local Residents: The dissemination of information from the results of this study will be of

great help for the residents to be informed about propagating cocoa tree varieties that are

potentially drought resistant.

Local Government Unit (LGU): This study can help LGU to formulate or strengthen laws

that are relevant to the cacao industry.

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Department of Agriculture (DA): This study will help provide information that can be

helpful in the propagation and production of a more preferrable cacao variety by the cacao

farmers.

Future Researchers: This study will provide baseline information about the mechanism on

the potential drought response of locally propagated Theobroma cacao species and may

greatly aid them as they conduct related studies in the future.

Scope and Limitations of the Study

This study will focus only on the collection of leaves from BR25 variety of

Theobroma cacao species from VPO cacao plantation (located in Rosario Agusan del Sur),

examination of stomatal traits and leaf water status. Only rainfall distribution, temperatures,

and relative humidity will be the environmental conditions that will be obtained and

considered. Sampling will be done once a month and the study will be conducted on a span of

three months from March 2021 to May 2021 in ASSCAT Laboratory Bunawan Agusan del

Sur.

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Definition of Terms

Hot dry season – the span of time and the desired condition the study will be conducted.

Relative water content – physiological character that will be measured to determine the

water status of the samples in the study.

Stomata – morphological feature to be observed, counted, and measured on the surface of the

samples.

Theobroma cacao – species to be examined in the study.

BR25 – variety of cacao plant.

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 Conceptual Framework

Independent Vvariables Dependent Variables

 Relative water content

 Theobroma cacao leaf
 BR 25
 PBC 123

Figure 1. Schematic Diagram of the Study

Figure 1 shows the schematic diagram of the study depicting both the independent

and dependent variables.

REVIEW OF RELATED LITERATURE

Drought

It is uncommon for African and Asian regions to experience inadequate precipitation

for an extended period of time. This phenomenon is called drought. This is primarily

characterized by a lack of rain or snow that are below the normal levels. As a result, soil

moisture is reduced, the stream flow diminished, crops are damaged, and there is water

supply shortage (National Geographic, 2020). Even water abundant tropical countries can be

affected by drought and suffer severe damage. In 2019, several provinces in the Philippines

experienced drought in the months of March to May. In fact, Zamboanga City was declared

under a state of calamity due to El Niño (Flores, 2019).

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 The devastating effects of drought to agricultural production are prominent as factors

like temperature and precipitation control crop growth and yield (Ray, Fares, & Risch, 2018).

Similarly, the cultivation and production of Theobroma cacao, an economically important

fruit that are primarily grown in Mindanao, is also heavily impacted by this climate

instability.

Theobroma cacao

Theobroma literally means “food of the gods” and is commonly known as cacao or

cocoa plant, is a small evergreen tree that grows up to five to eight meters tall in tropical

rainforest that is its natural habitat. It is native in Latin America but has been introduced to

African and Asian countries as a crop plant (Plants of the World Online, n.d.). It is known

worldwide for its beans that are used in making one of the world’s most delicious and popular

product, the chocolate (Nunes et al., 2020).

The cacao plant is characterized by having green, alternate, entire, and unlobed leaves

that measure 10–40 cm (4–16 in) in length and 5–20 cm (2–8 in) in width (Bureau of Plant

Industry, n.d.). The leaf surfaces are hairless, and the leaf base is rounded while the leaf

texture is described as shiny and leathery. It produces throughout the year clustered, small,

and yellowish flowers with pink calyx that grow directly from the trunk and older branches

which are pollinated by midges or tiny flies. The fruit is called a pod. It is ovoid in shape and

can reach up to 15–30 cm (6–12 in) long and 8–10 cm (3–4 in) wide. It can contain 20-60

seeds embedded in a white pulp; the beans used in the production of chocolate and cocoa

butter (Bureau of Plant Industry, n.d.; Plants of the World Online, n.d.).

The extensive historical medicinal uses of cacao were already documented in the

ancient Aztecs manuscripts, while it was introduced to Europe as both primary remedy and

a means to deliver other medicines in the middle of the 1500s (Bennet, 2003; Dillinger et al.,

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2000). Cocoa is rich in phytochemicals mainly polyphenols that are antioxidants and further

exhibit potential health benefits for chronic diseases such as inflammation, cardiovascular

diseases, diabetes, cancer, and neurodegenerative disorders (Baharum et al., 2014; Kim, Lee,

& Lee, 2011).

There are many cacao varieties that are cultivated worldwide but only nine (9) select

clones were registered and approved by the National Seed Industry Council to be cultivated

here in the Philippines and these are BR 25, K 1, K 2, UIT 1, ICS 40, UF 18, S 5, and K 4

(Bureau of Agricultural Research, 2012). Both BR 25 and UF 18 are hybrids of Criollo and

Forastero varieties that are also commonly grown in the Puentespina Farms in Malagos and

which were once nominated as among the world’s best cacao beans during the International

Cocoa Awards in 2017 (Rappler, 2017).

Drought Response of Cacao

As in other plants, drought stress is a common limiting factor that negatively affects

growth and pod production of cacao even though its production usually occurs in tropical

areas having high rainfall (Bae et al., 2008). According to Kalefetoğlu and Ekmekçi (2005),

the drought stress triggers varying responses in plants in the morphological, physiological,

biochemical, and molecular aspects allowing specific plants to adapt to the challenging

condition. Drought could be classified into two categories: water deficit or desiccation. Water

deficit condition involves moderate loss of water leading to stomatal closure and gas

exchange limitations, while desiccation is characterized by excessive loss of water leading to

the disruption of important metabolic processes in the plant (Kalefetoğlu & Ekmekçi, 2005).

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 Cacao has four main types of physiological response namely osmotic adjustment,

varying stomatal conductance, a range of root responses, and genetic responses (Medina &

Laliberte, 2017). Some cacao cultivars have the potential to be drought tolerant because of

their capacity for osmotic adjustment and their ability to allocate carbohydrates in the roots so

that they could escape drought by successfully penetrating deeper in the soil, however, both

of these traits are not solely an adequate basis in selecting drought tolerant species. On the

other hand, further and extensive studies are yet to be conducted to validate and understand

the mechanism of the genetic response of the cacao plant in drought conditions (Medina &

Laliberte, 2017).

Stomatal Adjustments

Leaf stomata plays an essential role in the survival of plants under drought

conditions. Stomatal closure commonly happens as an adaptive response of plants to the

onset of drought condition in order to regulate the rate of transpiration or uptake of carbon

dioxide by leaves (Bosabalidis & Kofidis, 2002; Agurla et al., 2018). The ability of crops to

maintain water-potential values and turgor under water limiting conditions is an important

physiological adaptation to drought. This maintenance of turgor and stable water potential

values can be attained through stomatal control. Thus, the capacity of cacao to regulate

stomatal closure as a way to sustain a high relative water content would be a good indicator

9
and basis for future efforts towards breeding drought-tolerant cacao (Medina & Laliberte,

2017).

The study of Zakariyya et al. (2017) showed that clones of cacao differ in their

stomatal traits in terms of density, width, length, and aperture. Varieties that have smaller

number of stomata, narrowest stomal width, and lowest stomatal length aperture were

considered as more resistant to drought. Anita-Sari and Abdoellah (n.d.) also confirmed the

positive correlation of increasing length of light intensity exposure to stomatal density and

opening width of stomata. It was also found that factors like humidity had negative

correlation with air temperature and light intensity, however, light intensity had positive

correlation with air temperature.

Water Status

The volume of imports for cocoa in the Philippines remains to be increasing as seen

over the past 10 years implying that the demand remains to be palpable which calls for

serious attention from the government and the stakeholders to develop a competitive and

sustainable cacao industry (Lirag, 2021). It is therefore important to acquire cacao cultivars

that exhibit desirable characteristics adapted to specific ecological conditions of a region

demonstrating vital traits that are key to the success of farming programs that will be

implemented (Hunter, 1990).

As previously mentioned, another important drought resistant mechanism is the

ability of plants to maintain leaf turgor at low water potential values due to osmotic

adjustment (de Almeida et al., 2002). The status of water in the plant body can be reflected

10
through the relative water content and water-saturation deficit determining its resistance to

drought. De Almeida, Tezara, and Herrera (2016).

MATERIALS AND METHODS

Gathering of Materials

Materials needed for the study such as laboratory materials will be borrowed from the

ASSCAT laboratory. Other essential equipment and consumables will be provided and

purchased by the proponent.

Sample Collection

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 A letter will be sent to the owner of the Cacao Plantation located at Rosario, Agusan

del Sur, asking permission to conduct the said study. Cocoa plants of the same variety and

age, preferably between 5-10 years old, will be marked as the plants of interest that will be

examined in the study. Mature leaf samples from the identified trees will be collected every

month for three months, leaves collected must be from the mid-section, in order to minimize

age effects. quickly put into airtight bags, and will be taken to ASSCAT laboratory for

further analysis.

Determination of Environmental Conditions

At the schedule of sample collection and observation, environmental conditions in the

study area (Brngy. Sta. Cruz, Rosario Agusan del Sur) will be noted during the month of

March to May 2021. Monthly average rainfall, minimum and maximum temperatures and

relative humidity will be identified by looking into the PAGASA climate and weather

forecast via their official website.

.Relative Water Content

To determine the relative water content (RWC), it must be calculated following the

formula (Afzal, Duiker, & Watson, 2017)

RWC = 100 x (actual leaf weight - dry weight / turgid leaf weight - dry weight)

where actual leaf weight is the weight of the sample placed on the balance, turgid leaf weight

is the weight of the leaf sample at full turgor, and dry weight is the weight of the leaf sample

dried in an oven.

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 In order to determine the leaf at its full turgor, the leaf samples' petioles will be

submerged in distilled water right away, and the whole leaves, as well as the container

holding the water, will be sealed in dark polyethylene bags. These bags will be stored in the

dark at 7 ͦ C for 8 h in a refrigerator to ensure that leaves reached full turgidity (Burquez,

1987).

Individual leaves collected will be immediately weighed in an analytical balance to

obtain the fresh mass. To obtain the turgid mass, leaves will be allowed to rehydrate through

soaking its petiole in a container with distilled water under dim light and naturally fluctuating

temperature conditions in the laboratory (Yamasaki & Dillenburg, 1999). During this

imbibition period, the samples will be taken out of water every 10 minutes and the surface

will be dried lightly with tissue paper and weighed until the weight of the leaf remains

constant over time. The samples will be oven dried at 60-80°C for 24h and weighed after

being cooled down to determine dry weight. All weighing will be done to the nearest mg.

Statistical Analysis

With the help of a statistician, the data collected will be compared utilizing mean,

standard deviation, and t-tests. The significant differences in the stomatal and traits of the

leaves investigated will be statistically analyzed using the JASP website. Statistical inference

will be carried out at a significance level of P = 0.05.

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Photograph

Documentation will be done during the time of study for further evidences. Then, those

photos will be used for further identification and verification. It is also useful for the

preparation of instructional materials.

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