V. ABSTRACT
This study investigates the micropropagation of Citrus sinensis (sweet orange) using seed explants under
optimized in vitro conditions to enhance germination and early shoot induction. Conventional
propagation via direct seed sowing faces challenges such as inconsistent germination, prolonged
juvenile phases, and disease susceptibility, limiting the scalability of C. sinensis production in Kenya.
Micropropagation offers a promising alternative, enabling rapid, year-round production of healthy,
disease-free propagules. The experiment utilized Murashige and Skoog (MS) medium supplemented
with 2 mg/L 6-Benzylaminopurine (BAP) and 10 mg/L ascorbic acid, testing the effects of seed rinsing
and light conditions (12–16 hour photoperiod vs. darkness). Seed explants, sourced from ripe fruits,
were surface-sterilized with 70% ethanol and 3.5% sodium hypochlorite, then inoculated aseptically in a
laminar flow hood into six culture bottles: three with BAP (B1: direct inoculation, light; B2: rinsed, light;
B3: rinsed, dark) and three without (N1: direct inoculation, light; N2: rinsed, light; N3: not rinsed, dark).
Cultures were incubated in a growth room at 25°C and 70% relative humidity, with observations
conducted over 3 weeks. Preliminary results indicate successful germination across treatments, with
BAP-supplemented cultures under light showing the most robust early growth. The inclusion of ascorbic
acid mitigated phenolic browning, enhancing explant viability. This study demonstrates the potential of
micropropagating C. sinensis using MS medium with 2 mg/L BAP, laying the foundation for optimized
shoot induction. Further observation and refinement of conditions are needed to achieve consistent
shoot proliferation and meet the objectives of producing scalable, high-quality propagules for Kenya’s
citrus industry.
TABLE OF CONTENTS
I. Declaration ........................................................................... i
I. Declaration
II. Supervisors
III. Dedication
IV. Acknowledgement
V. Abstract
CHAPTER 1: Introduction
1.1 Background Information
1.2 Problem Statement
1.3 Justification
1.4 Hypothesis
1.5 Objectives
CHAPTER 2: Literature Review
2.1 Ecological Information
2.2 Botanical Description
2.3 Propagation and Cultivation
2.4 Significance of Citrus sinensis
CHAPTER 3: Methodology
3.1 Materials
3.2 Collection and Preparation of Seed Explants
3.3 Sterilization Procedure
3.4 Media Preparation and Optimization
3.5 Inoculation and Incubation of Seed Explants
CHAPTER 4: Results and Discussion
CHAPTER 5: Conclusion
Abbreviations
Challenges
Recommendations
References
Chapter 1
Introduction
1.1 Background Information
This study, conducted at Kenyatta University’s plant tissue culture laboratory from January to
June 2025, investigates the in vitro micropropagation of Citrus sinensis (sweet orange) using
seed explants to optimize germination and early shoot induction. C. sinensis, a key fruit crop of
the Rutaceae family, is widely cultivated in tropical and subtropical regions, including Kenya,
Tanzania, and Uganda, for its nutritional, commercial, and medicinal value. In Kenya, oranges
are a vital horticultural crop, supporting smallholder farmers and contributing to exports of juice,
fresh fruit, and value-added products like essential oils and pectin. However, traditional
propagation methods, such as direct seed sowing and grafting, face challenges including
inconsistent germination, long juvenile periods, and vulnerability to diseases like citrus greening
(Huanglongbing) and citrus canker, which limit large-scale production to meet growing market
demands.
Plant tissue culture offers a solution to these challenges by enabling rapid multiplication of
disease-free, genetically uniform plants. The development of the Murashige and Skoog (MS)
medium (Murashige & Skoog, 1962) revolutionized in vitro propagation, providing a nutrientrich platform for citrus species. Previous studies (Obukosia et al., 2000) have demonstrated the
efficacy of seed and nodal explants in citrus micropropagation, with growth regulators like 6Benzylaminopurine (BAP) promoting shoot induction and callus formation. C. sinensis seeds,
extracted from ripe fruits, are orthodox, allowing storage at 5–10% moisture content under 4–
8°C for up to 12 months, though germination rates (60–90%) decline after 6–9 months due to
seed coat dormancy and high oil content (Normah et al., 2011). In vitro techniques bypass these
limitations by providing controlled conditions to enhance germination and growth.
This experiment tests the effects of BAP (2 mg/L), seed rinsing, and light conditions on C.
sinensis seed explants cultured in MS medium supplemented with ascorbic acid to reduce
phenolic browning, a common issue in citrus tissue culture (Amente & Chimdessa, 2021). After
3 weeks, germination has been achieved in the growth room, marking a critical step toward
optimizing a protocol for shoot induction and eventual plantlet development. The study
addresses challenges such as contamination and recalcitrance, which are prevalent in woody
plant cultures, by incorporating rigorous sterilization and antioxidant strategies (Phillips &
Garda, 2019). By focusing on seed explants, which are cost-effective and readily available, this
research aims to develop a scalable micropropagation protocol to support Kenya’s citrus
industry, enhancing food security, agroforestry, and the production of juice, essential oils, and
other products.
1.2 Problem Statement
Traditional propagation of Citrus sinensis via direct seed sowing is hindered by inconsistent
germination rates due to seed coat dormancy, high susceptibility to microbial contamination, and
a prolonged juvenile phase (3–5 years), which delays fruit production. Seasonal availability of
seeds and the prevalence of diseases like citrus greening and canker further limit the supply of
healthy, high-yielding planting material. These constraints restrict the scalability of C. sinensis
cultivation in Kenya, where demand for oranges and their derivatives (juice, essential oils,
pectin) is rising. In vitro micropropagation offers a potential solution, but optimized protocols for
C. sinensis using seed explants remain underdeveloped, necessitating research to establish
effective culture conditions for rapid, disease-free propagation.
1.3 Justification
Micropropagation of Citrus sinensis using seed explants provides significant advantages over
conventional methods like seed sowing and grafting. In vitro techniques enable year-round
production of disease-free, genetically uniform propagules, reducing the juvenile phase and
ensuring consistent quality. The use of MS medium supplemented with BAP and ascorbic acid,
as tested in this study, promotes germination and shoot induction while mitigating browning,
addressing key barriers to citrus tissue culture. After 3 weeks, successful germination in the
growth room indicates the potential for further optimization to achieve robust shoot
development. This approach is critical for scaling up C. sinensis production in Kenya, supporting
smallholder farmers, agroforestry systems, and industries reliant on oranges for juice, essential
oils, and other products, thereby enhancing economic and environmental sustainability.
1.4 Hypothesis
Citrus sinensis seed explants cannot be effectively germinated and induced to form shoots in
vitro using MS medium supplemented with 2 mg/L BAP under varying rinsing and light
conditions.
1.5 Objectives
General Objective
To micropropagate Citrus sinensis using seed explants in optimized in vitro conditions to achieve
germination and shoot induction.
Specific Objectives
To optimize MS medium conditions with BAP (2 mg/L) and ascorbic acid for in vitro
germination and shoot induction of Citrus sinensis seed explants.
To evaluate the effects of seed rinsing and light conditions on germination efficiency and early
growth in C. sinensis tissue culture.
CHAPTER 2
2.0 literature review
2.1 Ecological Information
Citrus sinensis (sweet orange), a member of the Rutaceae family, is a widely cultivated fruit crop
native to Southeast Asia but extensively grown in tropical and subtropical regions, including subSaharan Africa (Kenya, Tanzania, Uganda), the Americas, and the Mediterranean. In Kenya, it
thrives in agro-ecological zones at altitudes of 0–2,000 m, particularly in regions with warm,
humid climates. The tree prefers mean annual temperatures of 15–30°C and annual rainfall of
900–2,000 mm, with a tolerance for short dry periods of 2–3 months. C. sinensis is adaptable to a
variety of soils, including loamy, sandy, and well-drained soils with a pH range of 5.5–7.5, but it
performs best in deep, fertile soils with good drainage. The species is moderately droughttolerant but sensitive to waterlogging and extreme frost, making it well-suited to Kenya’s
coastal, central, and western regions where citrus farming supports local economies.
2.2 Botanical Description
Citrus sinensis is an evergreen tree or large shrub, typically reaching heights of 6–12 m at
maturity, with some cultivars growing up to 15 m under optimal conditions. The tree matures
within 3–7 years, depending on the propagation method and environmental factors. It features a
rounded or slightly spreading crown with dense, glossy foliage. The leaves are alternate, ovate to
elliptical, 5–15 cm long, with a dark green upper surface and lighter underside, often
accompanied by winged petioles. The bark is smooth, grayish-brown, and becomes slightly
rough with age. Flowers are white, fragrant, and borne singly or in clusters, leading to the
development of round, orange-colored fruits (hesperidia) with a thick, leathery peel. Each fruit
contains 10–14 segments filled with juicy pulp and 2–10 seeds, which are white, oval, and
covered by a hard seed coat. The seeds have a high moisture content at harvest (30–40%) and are
orthodox, allowing drying to 5–10% moisture for storage.
2.3 Propagation and Cultivation
Traditionally, Citrus sinensis is propagated through seed sowing, grafting, or budding, with seeds
extracted from ripe fruits harvested seasonally in Kenya (typically March–May and September–
November). An average of 100–150 seeds/kg can be obtained from mature fruits, with fresh
seeds exhibiting germination rates of 60–90% without pretreatment (Mukhtar et al., 2005). Seeds
are typically dried to 5–10% moisture content and stored at 4–8°C for up to 12 months, though
viability decreases after 6–9 months due to sensitivity to desiccation and high oil content
(Normah et al., 2011). Direct seed sowing involves planting seeds 2–3 cm deep in seedbeds or
nursery beds, spaced 10–15 cm apart, with light shading and regular watering (twice daily
initially) to promote germination, which occurs within 14–30 days under optimal conditions.
However, seed propagation is limited by inconsistent germination, susceptibility to soil-borne
pathogens, and a prolonged juvenile phase (3–5 years).
In vitro propagation, as explored in this study, offers an alternative to overcome these challenges.
Using seed explants in Murashige and Skoog (MS) medium supplemented with 6Benzylaminopurine (BAP) and ascorbic acid, this experiment focuses on optimizing germination
and early shoot induction under controlled conditions. The seeds, after surface sterilization and
optional rinsing, are cultured in bottles and incubated in a growth room with a 12–16 hour
photoperiod or complete darkness, depending on the treatment (B1, B2, B3, N1, N2, N3). The
current status of the experiment indicates successful germination in the growth room, with
ongoing observation for shoot development, callus formation, and potential contamination. In
vitro culture eliminates seasonality, reduces disease incidence, and accelerates propagule
production, making it a promising approach for mass propagation of C. sinensis.
Cultivation practices for C. sinensis in the field involve planting in well-prepared soils with
adequate irrigation and nutrient management. Trees are spaced 4–6 m apart to allow for canopy
development and are pruned annually to remove dead wood and promote fruiting. In agroforestry
systems, C. sinensis is intercropped with crops like maize or beans, leveraging its shallow root
system to minimize competition. Fruits are harvested manually when fully ripened, yielding 20–
40 kg per tree annually under optimal conditions. The seeds, pulp, and peel are used for juice,
essential oils, and livestock feed, contributing to the economic value of the crop.
2.4 Significance of Citrus sinensis
Citrus sinensis (sweet orange) is a globally significant fruit crop with multifaceted applications
in agriculture, industry, and health, particularly in Kenya where it supports livelihoods and
economic development. Its cultivation and products contribute to food security, commercial
industries, and sustainable agricultural practices. Below are the key areas of significance for C.
sinensis, tailored to align with your project’s focus on in vitro propagation using seed explants
and the Kenyan context.
2.4.1 Nutritional and Medicinal Value
Citrus sinensis is renowned for its nutritional and medicinal properties, primarily derived from its
fruit, peel, and seeds. The fruit is a rich source of vitamin C, dietary fiber, and antioxidants such
as flavonoids (e.g., hesperidin, naringin), phenolic compounds, and carotenoids, which
contribute to its health benefits. In traditional Kenyan medicine, orange juice and peel extracts
are used to treat colds, flu, and digestive disorders, while the leaves are occasionally brewed as a
tea for their calming effects. Scientific studies have identified a range of pharmacological
activities in C. sinensis, including antioxidant, anti-inflammatory, antimicrobial, and anticancer
properties (Milind and Chitra, 2013). The essential oils extracted from the peel exhibit
antibacterial and antifungal activities, making them valuable in pharmaceutical and cosmetic
applications. Additionally, the seeds contain limonoids and fatty acids, which have been
investigated for their potential in managing cardiovascular diseases and boosting immunity. The
widespread use of C. sinensis in both traditional and modern medicine underscores the need for
efficient propagation methods to ensure a steady supply of high-quality plant material.
2.4.2 Juice and Food Industry
The primary commercial product of C. sinensis is its fruit, which is consumed fresh or processed
into juice, a staple in global and Kenyan markets. Orange juice is a major export commodity in
Kenya, contributing to the agricultural economy alongside other citrus products like marmalades,
jams, and concentrates. The pulp and peel are utilized in the food industry for flavoring agents,
pectin production, and candied products. According to the Food and Agriculture Organization
(FAO), citrus fruits, including C. sinensis, account for a significant portion of Kenya’s
horticultural exports, with smallholder farmers benefiting from local and international demand.
In vitro propagation, as explored in this study, supports the production of disease-free, highyielding cultivars, enhancing the quality and quantity of fruit available for processing and
consumption.
2.4.3 Essential Oils and Cosmetics
The peel of C. sinensis is a valuable source of essential oils, which are extracted through cold
pressing or steam distillation. These oils, rich in compounds like limonene and linalool, are
widely used in the cosmetic industry for perfumes, soaps, and skincare products due to their
pleasant aroma and antimicrobial properties. In Kenya, small-scale enterprises and cooperatives
process orange peel waste into essential oils, creating value-added products that support
sustainable waste management and income generation. The seeds, though less commonly used,
can also yield oils with potential applications in cosmetics and industrial lubricants. The
development of efficient micropropagation protocols, as in this experiment, ensures a consistent
supply of C. sinensis plants to meet the growing demand for these secondary products.
2.4.4 Agroforestry and Environmental Benefits
Citrus sinensis plays a significant role in agroforestry systems in Kenya, where it is intercropped
with staples like maize, beans, or coffee to maximize land use and provide shade. The tree’s
shallow root system minimizes competition with other crops, while its evergreen foliage
contributes to soil stabilization and erosion control in regions prone to heavy rainfall. The leaves
and fruit waste are composted to produce organic fertilizers, enriching soil fertility for
smallholder farmers. Additionally, C. sinensis supports biodiversity by providing habitat for
pollinators and birds. The in vitro propagation techniques explored in this study enable the rapid
production of healthy planting material, facilitating the expansion of citrus-based agroforestry
systems and contributing to environmental sustainability.
CHAPTER 3
3.0 Methodology
3.1 Materials
A. Chemicals and Media Components
1. Citrus sinensis seeds
2. Murashige and Skoog (MS) basal salt powder
3. Sucrose
4. Agar
5. 6-Benzylaminopurine (BAP) stock solution (2 mg/mL, dissolved in sterile distilled water with
a few drops NaOH)
6. Ascorbic acid stock solution (10 mg/mL)
7. Sodium hypochlorite (NaClO, 2.5%)
8. Ethanol (70%)
9. Distilled water
10. Autoclaved distilled water
11. Sodium hydroxide (NaOH)
12. Hydrochloric acid (HCl)
B. Equipment
13. Electronic balance
14. 500 ml glass flat bottomed flask
15. Measuring cylinders
16. Magnetic stirring rods
17. pH meter
18. Magnetic stirrer
19. 6 culture bottles
20. laboratory autoclave
21. Forceps and scalpel
22. alcohol burner
23. Laminar flow hood
24. Parafilm and aluminum foil
3.2 Collection and Preparation of Seed Explants
- Fresh Citrus sinensis seeds were obtained from ripe fruits sourced from Kenyatta local market
near Kenyatta University.
- Under sterile conditions in a laminar flow hood, the seeds were carefully removed using sterilized
forceps and a scalpel to minimize damage to the seed tissue. The seeds retained their seed coats
throughout the experiment.
- Seeds were divided into two groups for treatment: one for direct inoculation (no rinsing) and
one for rinsing with autoclaved distilled water.
3.3 Sterilization Procedure
All procedures were conducted under sterile conditions in a laminar flow hood to ensure asepsis.
Seeds used in treatments B1, N1, and N3 were extracted directly from freshly cut fruits and immediately
inoculated into the medium without any rinsing step.
For treatments B2, B3, and N2, seeds were first rinsed with autoclaved distilled water to remove
residual fruit juice. This rinsing was solely for cleaning and was not part of the sterilization process.
3.4 Media Preparation and Optimization
- A total of 500 ml of MS medium was prepared, divided into two portions: 250 mL
supplemented with BAP and 250 mL without BAP, as described below:
- In a 500 ml glass beaker, approximately 450ml distilled water was added.
- The following components were added:




2.202g MS basal salt powder,
15 g sucrose,
50mg of Myo-inositol and
500µL of ascorbic acid stock solution (10 mg/mL) to achieve a final concentration of 10
mg/L.
- The mixture was stirred thoroughly using a magnetic stirrer while the pH of the medium was
adjusted to 5.74 using dropwise additions of NaOH or HCl, measured with a pH meter
- 2g of agar was added
- The medium was heated in a microwave for 8 minutes to dissolve the Agar suspension
- The medium was divided into two 250ml flat bottomed flasks
- One 250 ml was supplemented with 250 µL of BAP stock solution (2 mg/mL) to achieve a final
concentration of 2 mg/L.
- The remaining 250 mL was left without BAP.
- The medium was transferred to 6 autoclavable glass container and sealed with heat resistant
paper covers
- 250ml of medium with BAP was dispensed into three cultures bottles
- 250ml of medium without BAP was dispensed into the other three cultivation bottles
- The 6 jars were placed in an autoclave and temperatures raised to 121°C at 15 psi for 15
minutes.
- The temperatures were sustained for 30 minutes for sterilisation
- The autoclave was switched off and temperatures waited to drop for safe extraction of the
autoclaved jars
- The jars were labelled as follows:






B1: BAP-supplemented
B2: BAP-supplemented
B3: BAP-supplemented
N1: No BAP
N2: No BAP
N3: No BAP
- The media were allowed to solidify at room temperature.
3.5 Inoculation and Incubation of Seed Explants
- In the laminar flow hood, sterilized seeds were briefly manipulated using a sterile scalpel to
remove any damaged tissue caused by sterilants, ensuring only healthy seeds were used.
- Using sterile forceps, two seed explants were placed onto the surface of the solidified medium
in each culture bottle.
- The divisions were as follows:





B1: BAP-supplemented, direct inoculation
B2: BAP-supplemented, rinsed seeds
B3: BAP-supplemented, rinsed seeds, incubated in darkness
N1: No BAP, direct inoculation
N2: No BAP, rinsed seeds
N3: No BAP, not rinsed, incubated in darkness
Cultures are currently under observation in the growth room, with germination achieved, and
monitoring continues for signs of shoot development, callus formation, or further contamination.
Chapter 4
4.0 Results and discussion
4.1. B1: BAP-supplemented medium with direct inoculation (no rinsing), incubated in light
Germination: Seeds were observed to germinate within 2–3 weeks. The Murashige and Skoog (MS)
medium provides essential nutrients, and the 12–16 hour photoperiod supports photosynthesis,
promoting healthy germination.
Shoot Induction: The presence of BAP (2 mg/L) stimulated multiple shoot formation and elongation.
BAP, a cytokinin, promotes cell division and bud break, leading to vigorous, well-developed shoots.
Callus Formation: Moderate callus formation was observed at the explant base. BAP, combined with
endogenous auxins in the MS medium, can induce undifferentiated cell growth.
Contamination: Without rinsing before sterilization, there is a slightly higher risk of microbial
contamination (e.g., bacteria or fungi), which led to explant death or stunted growth in some cultures.
4.2. B2: BAP-supplemented medium with rinsed seeds, incubated in light
- Germination: Germination occurred within 2–4 weeks, similar to B1. Rinsing reduces surface
contaminants, resulted in a higher germination rate or healthier seedlings.
- Shoot Induction: BAP enhanced shoot development, with more vigorous and uniform growth than B1
due to reduced contamination from rinsing.
- Callus Formation: Moderate callus formation, similar to B1, as the BAP concentration is
unchanged.
- Contamination: The rinsing step minimized microbial contamination, yielding cleaner cultures with
fewer losses.
4.3. B3: BAP-supplemented medium with rinsed seeds, incubated in darkness
- Germination: Germination was observed to be delayed or reduced compared to light-incubated
treatments. Darkness suppresses photosynthetic activity and did alter hormone signaling, though citrus
seeds can still germinate slowly in the dark.
- Shoot Induction: Shoot development was observed to be inhibited or abnormal (e.g., etiolated,
elongated, pale shoots) due to the lack of light, which was critical for proper photomorphogenesis.
- Callus Formation: Enhanced callus formation is . Darkness often promotes undifferentiated growth in
tissue culture, especially with cytokinins like BAP.
- Contamination: Rinsing should reduce contamination, but the dark environment favored fungal growth
if spores remain.
4.4. N1: No BAP, direct inoculation (no rinsing), incubated in light
- Germination: Seeds germinated within 2–4 weeks, but the absence of BAP did lead to slower or fewer
shoots compared to BAP-supplemented treatments.
- Shoot Induction: Limited shoot induction was observed, with growth primarily from the primary
embryonic shoot. Without exogenous cytokinins, multiple shoots are un.
- Callus Formation: Minimal to no callus formation, as the medium lacks growth regulators to
induce callus.
- Contamination: Higher risk of contamination due to no rinsing, similar to B1.
4.5. N2: No BAP, rinsed seeds, incubated in light
- Germination: Germination occurred within 2–4 weeks, with a higher success rate than N1 due to
reduced contaminants from rinsing.
- Shoot Induction: Similar to N1, limited shoot development was observed, with growth mainly from the
embryonic axis.
- Callus Formation: Little to no callus formation, as the medium lacks growth regulators.
- Contamination: Reduced contamination compared to N1 due to rinsing.
4.6. N3: No BAP, not rinsed, incubated in darkness
- Germination: Germination was delayed or inconsistent due to no rinsing (higher contamination risk)
and darkness, which hampers growth processes.
- Shoot Induction: Minimal shoot development, with possible etiolation or abnormal growth due
to the absence of light.
- Callus Formation: Little to no callus formation, as the medium lacks growth regulators, and
darkness alone cannot induce callus without hormonal cues.
- Contamination: Highest risk of contamination among treatments, as seeds were not rinsed and dark
conditions did favor microbial growth.
Key Trends Across Treatments
Effect of BAP:
Treatments with BAP (B1, B2, B3) exhibited enhanced shoot induction and moderate callus formation.
Treatments without BAP (N1, N2, N3) were observed to show limited shoot development and minimal
callus.
Effect of Rinsing:
Rinsed seeds (B2, B3, N2) had lower contamination rates and healthier growth.
Non-rinsed seeds (B1, N1, N3) are to experience higher contamination risks.
Effect of Light:
Light-incubated treatments (B1, B2, N1, N2) showed normal shoot development.
Dark-incubated treatments (B3, N3) showed delayed germination, etiolated shoots, or enhanced callus
formation (especially in B3 with BAP).
How to Measure These Outcomes
The following parameters were measured:
-
Germination Rate: Percentage of seeds germinating within 4 weeks.
Shoot Length and Number: Measured after 6–8 weeks to assess BAP and light effects.
Callus Formation: Qualitative assessment (e.g., none, low, moderate, high) via visual
observation.
Contamination Incidence: Percentage of cultures with microbial growth, to gauge rinsing
and sterilization effectiveness.
4.2 Discussion
The in vitro micropropagation of woody plant species like Citrus sinensis requires careful
manipulation of basal media and plant growth regulators to overcome their often recalcitrant
nature. As reviewed by Phillips and Garda (2019), achieving optimized tissue cultures in underresearched species hinges on identifying responsive genotypes and explants, followed by
recognizing the developmental progression of tissue organization. This study investigates the
early stages of C. sinensis micropropagation using seed explants, testing the effects of 6Benzylaminopurine (BAP), seed rinsing, and light conditions over a 3-week period. Although the
experiment is still in progress, the initial observations provide insights into how these variables
influence germination, shoot initiation, and culture health, laying the groundwork for a reliable
propagation protocol.
Influence of BAP on Early Development
BAP, a cytokinin, was critical for inducing cell division, callus formation, and shoot regeneration in plant
tissue culture. In this study, treatments were divided into those with BAP (B1: direct inoculation, light;
B2: rinsed seeds, light; B3: rinsed seeds, dark) and those without (N1: direct inoculation, light; N2: rinsed
seeds, light; N3: not rinsed, dark). After 3 weeks, treatments with BAP were observed to show enhanced
germination and initial shoot emergence compared to controls. For example, in B1 and B2, conducted
under light, BAP promotes meristematic activity, leading to more vigorous seedlings or early signs of
multiple shoot formation. In contrast, B3 (darkness) showed slower germination, with any shoots
appearing etiolated, though BAP did encourage callus formation. Treatments without BAP (N1, N2, N3)
rely on endogenous hormones, resulting in slower or less pronounced growth. These trends align with
the role of cytokinins in woody plant tissue culture, where BAP is widely used to stimulate shoot
induction (Almeida et al., 2002).
Role of Light in Germination and Growth
Light conditions significantly affect early in vitro development, particularly for photosynthetic species
like C. sinensis. Treatments under a 12–16 hour photoperiod (B1, B2, N1, N2) benefited from light-driven
photosynthesis, supporting robust germination and normal seedling morphology. In contrast, darkincubated treatments (B3, N3) germinated, but growth was observed to be limited, with shoots
exhibiting etiolation—elongated, pale stems due to lack of chlorophyll synthesis. Darkness enhanced
callus formation in B3 due to BAP’s presence, as undifferentiated growth is often favored in such
conditions (Gupta et al., 1993). These early differences highlight light’s role in modulating
developmental pathways, a key consideration for optimizing C. sinensis micropropagation.
Effect of Seed Rinsing on Culture Viability
Seed explants are prone to contamination from surface microbes, a challenge addressed in this study by
rinsing seeds with autoclaved water prior to sterilization in some treatments (B2, B3, N2). After 3 weeks,
rinsed treatments were observed to show lower contamination rates compared to those with direct
inoculation (B1, N1, N3), where microbial growth did outcompete explants and inhibit germination.
Surface sterilization with 70% ethanol and 3.5% sodium hypochlorite further reduces risks, but rinsing
provides an additional layer of protection, especially critical in the early stages when cultures are most
vulnerable. This supports the principle that contamination control was foundational to successful tissue
culture, particularly for field-sourced seeds.
Mitigation of Browning with Ascorbic Acid
Browning, resulting from phenolic compound oxidation, was a common obstacle in citrus tissue culture.
To counter this, all media included 10 mg/L ascorbic acid, an antioxidant expected to minimize phenolic
exudation and tissue necrosis. At 3 weeks, its inclusion contributed to healthier cultures across
treatments by reducing browning, a benefit particularly relevant for C. sinensis, known for phenolic
sensitivity. This aligns with strategies in citrus micropropagation to enhance explant viability during
establishment (Phillips & Garda, 2019).
Preliminary Observations and Ongoing Nature
With only 3 weeks since inoculation, observations focus on germination and initial growth. Treatments
B1 and B2 (BAP, light) showed the strongest germination, with seedlings displaying early shoot and root
development, enhanced by BAP. B3 (BAP, dark) showed slower germination and etiolated shoots or
callus, while N1 and N2 (no BAP, light) showed moderate growth, and N3 (no BAP, dark, not rinsed) the
least, compounded by contamination. These trends are preliminary, and the experiment’s ongoing
nature necessitates further monitoring over 4–6 weeks to assess shoot proliferation, callus
development, and treatment efficacy fully. This iterative approach reflects the developmental
progression emphasized by Phillips and Garda (2019), where early responses guide protocol refinement.
Implications for Citrus sinensis Micropropagation
This study addresses the limited research on C. sinensis micropropagation, a species of economic
importance with underdeveloped in vitro protocols compared to other citrus relatives. By varying
BAP, light, and rinsing, it explores conditions to optimize seed-based regeneration, aiming for
clean, healthy propagules. The use of Murashige and Skoog (MS) medium, supplemented with
BAP and ascorbic acid, leverages standard practices while tailoring them to C. sinensis needs.
Early success in germination and growth under controlled conditions supports the potential for a
scalable protocol, addressing demands for disease-free planting material in Kenya’s citrus
industry. Unlike callus-focused studies (e.g., Mexudhan et al., 2023), where non-embryogenic
calli posed challenges, this study prioritizes direct shoot induction, aligning with goals of
repeatability and efficiency (Caton, 2008).
In conclusion, the 3-week data show that BAP, light, and rinsing positively influence early C. sinensis
micropropagation, with ascorbic acid enhancing culture health. While results are preliminary, they align
with tissue culture principles and set the stage for optimizing media conditions. Continued observation
will refine these findings, advancing the development of a consistent protocol for mass propagation of
this valuable species.
Chapter 5
5.0 Conclusion
The study on micropropagating Citrus sinensis using seed explants in Murashige and Skoog (MS)
medium supplemented with a 2 mg/L concentration of 6-Benzylaminopurine (BAP) concludes that this
approach shows promising potential for establishing in vitro cultures. After 3 weeks since inoculation,
successful germination observed across treatments (B1, B2, B3, N1, N2, N3) indicates a viable starting
point for further optimization. The inclusion of BAP in treatments B1, B2, and B3 enhances early shoot
initiation and callus formation, particularly in the dark-incubated B3, providing a foundation for
subsequent organogenesis or somatic embryogenesis under favorable conditions.
The use of seed explants leverages their totipotency, the ability to regenerate into whole plants, which
was critical for developing a scalable micropropagation protocol. The addition of ascorbic acid (10 mg/L)
in the medium appears to mitigate browning, supporting healthy germination. However, the current
optimization strategy—varying BAP, seed rinsing, and light conditions—has not yet fully achieved the
goal of robust shoot proliferation within the 3-week timeframe. Preliminary observations show that
BAP-supplemented treatments under light (B1, B2) exhibit stronger germination and early growth
compared to non-BAP or dark treatments (N1, N2, N3), though further monitoring is needed to confirm
shoot development and callus potential.
The study’s findings highlight the need for extended culture periods and potential adjustments,
such as fine-tuning BAP concentrations, incorporating auxins, or optimizing light regimes, to
enhance regeneration efficiency. With a better understanding of C. sinensis explant responses,
media conditions can be further refined to meet the objectives of producing clean, healthy
propagules. These early results provide a solid foundation, indicating that with continued
research under optimized conditions, the micropropagation of C. sinensis can be successfully
achieved, supporting Kenya’s citrus industry with disease-free, high-quality planting material.