THE EFFECTS OF SUCROSE CONCENTRATIONS ON SOMATIC

EMBRYOGENESIS OF LIMAU MANIS (Citrus sinensis L.)

SITI NURHAZLINDA BINTI KAMARUDIN

Bachelor of Science with Honours

(Plant Resource Science and Management Programme)

2012

Faculty of Resource Science and Technology

THE EFFECTS OF SUCROSE CONCENTRATIONS ON SOMATIC

EMBRYOGENESIS OF LIMAU MANIS (CITRUS SINENSIS L.)

SITI NURHAZLINDA BINTI KAMARUDIN

This report is submitted in partial fulfillment of the requirements for the degree of

Bachelor Science with Honours in

Plant Resources Science and Management

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2012

APPROVAL SHEET

Name of candidate: Siti Nurhazlinda binti Kamarudin

Title of dissertation: The effects of sucrose concentrations on somatic embryogenesis of

Limau

Manis (Citrus sinensis L.)

________________________

(Dr. Rebicca Edward)

Supervisor

________________________

(Dr. Siti Rubiah Zainudin)

Coordinator

Plant Science and Resource Management

Department of Plant Science and Environmental Ecology

Universiti Malaysia Sarawak

DECLARATION

No portion of the work referred to in this report has been submitted in support of an application for

another degree qualification of this or any other university or institution of higher learning

……………………………………..

(SITI NURHAZLINDA BINTI KAMARUDIN)

Plant Resource Science and Management

Department of Plant Science and Environmental Ecology

Universiti Malaysia Sarawak

ACKNOWLEDGEMENT

Alhamdulillah,

First and foremost, I would like to thanks Allah for the blessings and miraculous strength

along the project.

I would like to thank my supervisor, Dr. Rebicca Edward for the valuable guidance and

advice. She helpful and inspired me greatly in this project. Her willingness to motivate me

contributed tremendously in this project.

Besides, I would also extend my gratitude to master student, Mr. Zulhelmy for his endless

patience and endless efforts to guide along my research.

I would like to express greatest gratitude to my mum, Madam Hjh. Zawiah binti Senan and

sister, Siti Nurhidayah binti Kamarudin who gave me endless support and encouragement

during my studies and completing my project.

Last but not least, an honourable mention goes to my best friend, Nurul Atiqah binti Abd.

Rahman, Nurul Afiqah, Siti Zakiah, Nurul Amiera, Nurul Shakirah, Nurul Nazirah, Nuraishah

and Nurnadia all my friends for their understanding and supports in completing this project.

Thank you.

TABLE OF CONTENTS

1.0 Introduction

1.1 Problem Statement

1.2 Objective

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2

2.0 Literature Review

2.1 Citrus sinensis

2.2 Diseases of Citrus sinensis

2.3 Tissue culture in Citrus

2.4 Somatic embryogenesis

2.5 Sucrose in tissue culture

2.6 Coconut water in culture media

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3.0 Material and method

3.1 Laboratory work

3.2 Study resources

3.3 Surface sterilisation for axenic culture

3.4 In vitro germination of Limau Manis seeds

3.5 Induction of Limau Manis callus

3.6 Preparation of sterilised coconut water

3.7 Induction of Limau Manis somatic embryos

3.8 Data collection

3.9 Data analysis

4.0 Result

4.1 The germination percentage of Limau Manis seeds

4.2 Induction of Limau Manis callus

4.3 Induction of Limau Manis somatic embryogenesis

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Page

Acknowledgement

Table of contents

Abbreviation

List of tables

List of figures

Abstract

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II-III

IV

VI

5.0 Discussion

6.0 Conclusion and recommendations

7.0 References

8.0 Appendices

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ABBREVIATION

MS Murashige and Skoog

NA N6 benzyl adenine

NAA 1-napthaleneacetic acid

IADA Kementerian Pertanian dan Industri Asas Tani

Samarahan

BAP Benzylaminopurine

LIST OF FIGURES

Figure 1: Limau Manis fruit obtained from Limau Manis Project, Kampung Kerian,

Kilometre

16, Jalan Kuching-Padawan, Kuching

Figure 2: Stages for general formation of somatic embryogenesis

Figure 3: The chemical structures of Glucose, Fructose and Sucrose molecule

Figure 4: (A)The germinated explants after 1 month of cultured in MS media containing 2.0

mg/L BAP

(B) The Limau Manis explants in MS media containing 1.0 mg/L BAP

Figure 5: The different callus performance regarding to different sucrose concentration

Figure 6: The embryogenic callus formed in Media A (0 g/L sucrose)

Figure 7: The non embryogenic callus in Media G (60g/L sucrose)

Figure 8: (A) The contamination in leaf explant

(B) The contamination in node explants

Figure 9: The callus in embryos induction media (50g/L)

LIST OF TABLES

Table 1: Diseases in Limau Manis

Table 2: Different concentrations of sucrose

Table 2: Mean of number of shoots and explants length (cm)

Table 4: The callus response, mean and standard deviation for each sucrose concentration in

Experiment 1

Table 5: The callus response, mean and standard deviation for each sucrose concentration in

Experiment 2

1

The effects of sucrose concentrations on somatic embryogenesis of Limau Manis

(Citrus sinensis L.)

Siti Nurhazlinda binti Kamarudin

Plant Resource and Management Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Citrus sinensis or Limau Manis belongs to the family of Rutaceae.It is a species among most cultivated

worldwide. This species can be cultivated sexually or asexually. Despite of it, the reproduction by seeds for this

species leads to many problems. The species is susceptible to pathogens and diseases too. Somatic embryogenesis

is one of the efficient methods in plants tissue culture for mass propagation within a short period of time. Limau

Manis seeds were successfully germinated axenically in 2.0 mg/L BAP. The node and leaf segments were

purposely used in this experiment. Seven different sucrose concentrations were used to identify its effects

towards the development of callus and somatic embryos. Limau Manis callus were gave different growth

performance towards callus induction media, 20% coconut water, 2.0 mg/L of 2,4-D and different sucrose

concentration. No somatic embryo Was developed when callus were cultured on embryos induction media, 1.5

mg L-1 Kinetin + 0.1 mg L-1

NAA even after 3 months of the culture established.

Keywords: Citrus sinensis, somatic embryogenesis, callus, sucrose concentration, somatic embryo

ABSTRAK

Citrus sinensis atau Limau Manis berasal dari keluarga Rutaceae.Ia adalah spesies yang paling popular ditanam

di seluruh dunia. Spesies ini boleh dibiak melalui seksual atau aseksual. Pembiakan melalui biji benih membawa

kepada banyak masalah. Spesies ini terdedah kepada patogen dan penyakit. Embriogenesis somatik adalah salah

satu kaedah berkesan dalam kultur tisu tumbuhan untuk pembiakan dalam tempoh masa yang singkat. Benih

Limau Manis telah berjaya bercambah dalam 2.0 mg / L BAP. Segmen nod dan daun digunakan dalam

eksperimen ini. Tujuh kepekatan sukrosa yang berbeza telah digunakan untuk mengenal pasti kesannya ke arah

pembangunan embrio kalus dan somatik. Limau Manis kalus telah memberikan prestasi pertumbuhan yang

berbeza terhadap media induksi kalus, 20%air kelapa, 2.0 mg / L 2,4-D dan kepekatan sukrosa yang berbeza.

Tiada embrio somatik telah tumbuh apabila kalus dikulturkan pada media embrio induksi, 1.5 mg L-1 Kinetin +

0.1 mg L-1 NAA selepas 3 bulan dikulturkan.

Kata kunci: Citrus sinensis, somatic embryogenesis, kallus, kepekatan sukrosa,somatic embrio.

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1.0 INTRODUCTION

Citrus belongs to the family of Rutaceae. It becomes the first fruit crop in the world leaving

behind grapes, bananas and apples (Ladaniya, 2008). Citrus species is grown in more than 80

countries (Chang, 1992). Most popular species are oranges and tangerins (Germana, 2005).

The oranges constitute about 60 percent of the total citrus production worldwide and the

countries that produce the largest production of citrus are Brazil, followed by the United States

and China (Ladaniya, 2008). Malaysia is also one of the oranges production countries. In

Sarawak, an estimated 28.35 metric tons of fresh oranges exported in 2005 (Eng, 2007).

Davies & Albrigo (1994) stated that the C. sinensis or Limau Manis is the most widely

distributed and it is the greatest production of all commercial citrus species.

Citrus species are propagated sexually through seeds, while most of the commercial varieties

are propagated by various asexual methods (Chaudary, 1994). Citrus species propagate

including cuttings, air layering, grafting and also tissue culture (Davies & Albrigo, 1994).

Despite of it, the reproduction by seeds lead to much problems such as the species require

more time to produce fruits, more difficult to keep true to type, tend to produce more thorns

than grafted varieties and the fruits is harder to pick (Davies & Albrigo, 1994). In addition,

Mukhtar et. al (2005) stated that slow growth, long juvenility, insects, pests, diseases, alternate

bearing, pre and post harvest losses, large number of seeds per fruit, short season of supply

and short storage life are the problems facing by citrus species too.

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Among different types of citrus propagation, in vitro propagation is the most effective method

for citrus regeneration. The citrus species can be arising from a single somatic cell or a group

of somatic cells by the somatic embryogenesis process. Furthermore, somatic embryogenesis

is one of the efficient methods in plants tissue culture for mass propagation the plant within a

short period of time. This process requires different sets of hormones and growth medium to

develop successfully (Khalil, 2011). As reported by Mukhtar (2005), the somatic

embryogenesis is a choice to be used as a tool in citrus improvement study. However, the

research of Citrus sinensis somatic embryogenesis is not widely studied (Kiong, 2008).

The study was conducted to address the following objectives:

1. To develop an in vitro propagation of Limau Manis seeds for explants material.

2. To optimize the production of somatic embryos from Limau Manis explants.

3. To observe the growth of somatic embryogenesis of Limau Manis in different sucrose

concentrations.

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2.0 LITERATURE REVIEW

2.1 Limau Manis

Limau Manis (Citrus sinensis) belong to genus Citrus of Rutaceae family. The common names

in Malays are Limau Langgat, Choreng, and Chula. The Limau Manis originated in Southern

China (Davies & Albrigo, 1994). However, it is distributed in all subtropical areas including

Northeastern India, Upper Burma and Southern China (Kale & Adsule, 1995). Limau Manis is

adaptable to a wide range of climate and cultivars which is available to many growing regions.

Mukhopadhyay (2005) reported that the characteristics of Limau Manis are medium to large

trees; the leaf apex is bluntly pointed with distinct aroma, the petiole is narrowly winged. The

seeds are oblong, ovoid, numbers of seeds varies from 2 to 25 and whitish cotyledons. The

fruits are spherical, orange colored, tight skinned with solid central core and smooth orange

peel. It is also generally low to moderate acidic fruits.

The fruit composed of three distinct morphology parts that are:

i. Flavedo

The epicarp is consists of the colored portion of the peel

ii. Albedo

The portion is under the epicarp. It is known as mesocarp too.

iii. Pericarp

The morphology is a combination of albedo and flavedo

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There are many uses of Limau Manis. The fruits are eaten raw and the flavour of the fresh

fruit and juice products are acceptable to people worldwide. Ladaniya (2008) stated that in

Asian countries like Malaysia, citrus fruits are consume fresh and are supplied by the local

citrus industries. The fresh fruits consumption is higher in Limau Manis due to convenience as

they have less seed and small. Besides, Limau Manis can be extracted to make juices. The

fruit rind and leaves often used as flavoring and spices. Jam, jelly, and marmalade are

commonly prepared from this fruits too. Besides, Limau Manis are utilized in making teas,

cosmetics, soap and also perfumes. Limau Manis can be for medicinal purposes too.

Figure 1. Limau Manis fruit obtained from

Limau Manis Project, Kampung Kerian, Kilometre 16,

Jalan Kuching-Padawan, Kuching

© siti nurhazlinda

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2.2 Diseases of Limau Manis

Timmer et al. (2003) stated that there are four citrus diseases which are diseases of fruit and

foliage, root and trunk diseases, postharvest decays and systemic diseases. Diseases of fruit

and foliage do not cause direct loss of trees and can be healed without replacing the trees. The

root disease is caused by Phytophthora spp which can lead to substantial losses. These

pathogens attack every part of the citrus tree including seedlings to harvested fruit. Besides,

the postharvest decays mostly by pathogens that is less aggressive. It causes less or no damage

to the tree. Systemic pathogens diseases are the most severe of all citrus diseases and result in

tree losses.

According to Eng (2007), a disease known as citrus greening disease (CGD) or

Huanglongbing was discovered in Samarahan, Sarawak. The disease was first discovered in

China, and was named yellow shoot or Huanglongbing. The disease, which is caused by the

bacterium Candidatus Liberibacter asiaticus, causes dieback of the citrus trees. These bacteria

spread vegetatively through marcotting and bud grafting. An infected tree will display leaves

with symptoms similar to nutrient deficiency, especially zinc and manganese deficiencies.

Leaves sometimes show mottling, and can remain small and become leathery and curling

backwards. In a severe infection, fruit remain small and seeds become aborted. Sometimes, the

fruit also become distorted. Severe infection leads to the death of the tree.

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The diseases faced by Citrus sinensis also has been discussed by several authors (Table 1):

Table 1. Diseases in Limau Manis

Authors Diseases

Begeman & Wright, 2009 1. Physiological disorders

Fruit splitting

Root rot

Nutrient deficiencies

2. Diseases caused by pathogens

Melanose

Greasy spot

Scab

Black spot

Citrus canker

Greening

Triteza

Psorosis ringspot

Nematodes

Manner et al., 2006 Citrus diseases are divided into 3 categories:

1. Problems with fruits

Fruits drops prematurely

Dry juice Sacs (granulation)

Sunburn

Fruit splitting

Misshapen fruit

Holes

Yellow fruit turns in to green colour

Black fungus inside fruit

Lopside fruit with aborted seed

2. Problems of leaves

Leaf curling

Bleached out or yellow patches

Leaf streaking and distortion

3. Problems of branches, trunk or entire trees

Stunting entire trees

Patches of pealing or scaling Bark

Lesions on lower section of trunk

Die back

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2.3 Tissue culture in Citrus

Biotechnology options are being extensively discovered to develop different fruit varieties.

The main purpose of the studies is to make citrus species can tolerate biotic and abiotic

stresses and produce better quality fruit too. Modern technologies basically on in vitro cell,

protoplast, tissue culture and gene introduction are been studied to improve citrus species

quality.

Tissue culture is in vitro culture of sterilize plant cells, tissues or organs on a nutrient medium.

The plant cells are totipotenty, define as each cell has the capacity to regenerate the entire

plant. Micropropagation is the regeneration of whole plants from small pieces of plant

material. These small pieces known as explants are grown on sterile media and the plants

produced can be planted in soil and transferred to the field. Various parts of a plant can be

cultured have been regenerated from leaves, stems, roots, meristems, flowers and even pollen

or ovules.

This technique involves callus induction from explants, morphogenesis, shoot development

and finally root development to regenerate into a complete somaclone. In vitro

micropropagation technologies help to overcome some constraints to citrus improvement and

cultivation, and can increase fruit quality and resistance to diseases and environmental stresses

(Rathore et al., 2007). Many studies of citrus species have been carried out by using different

approaches.

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Khalil et al. (2011) had conducted a research of in vitro regeneration of plantlets from

unpollinated ovary culture in Limau Manis (Citrus sinensis). The study utilized unopened

floral buds that are cultured on Murashige and Skoog (MS) medium supplemented with

different concentrations and combinations of N6 benzyl adenine (BA), 1-napthaleneacetic acid

(NAA) and 2,4-D. The result showed that 1 mg/l BA with 0.5 mg/l NAA is the most effective

in callus induction and proliferation.

Sharma et al. (2009) conducted in vitro propagation of citrus rootstocks. The explants used are

shoot tips and nodal segments were from Pectinifera (Citrus depressa Hayata), Troyer citrange

(Poncirus trifoliata (L.) Raf. x Citrus sinensis Osbeck), Rough lemon (Citrus jambhiri Lush.)

and Cleopatra mandarin (Citrus reshni Hort. ex. Tan.). The explants are cultured on MS basal

medium supplemented with different concentrations of hormones which are BAP, Kinetin,

NAA, and 2, 4-D. The result showed that shoot tip explants are found better than nodal

segment for callus induction. The maximum callus formation with treatment 0.5mg/l Kin,

2.0mg/l NAA and 2.0mg/l 2, 4-D was observed in all the rootstock.

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2.4 Somatic embryogenesis

Source:

Figure 2. Stages for general formation of somatic embryogenesis

Vicient (1998) reported that somatic or asexual embryogenesis is the production of embryo-

like structures from somatic cells without gametes fusion. The somatic embryos arise from in

vitro cultured cells are known as indirect somatic embryogenesis. This process requires the

induction of embryogenic competence. The usual method to induce somatic embryos is to

expose the explants to a high auxin concentration for a certain period. Then, it will transfer to

auxin-free medium. The stage of somatic embryogenesis is started with a single cell. It is then

followed by repeated cell divisions and leads to the cells aggregates progressively. In addition,

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it will pass through globular, heart and torpedo stages and formed plantlets after all. According

to Kohlenbach (1978) these sources of cultured diploid cells may arise in vitro from:

1. Vegetative cells of mature plants.

2. Reproductive tissues other than the zygotes.

3. Hypocotyls and cotyledons of embryos and young plantlets without any callus

development.

Somatic embryogenesis from embryos developed has been extensively investigated in Citrus

species. Mukhtar et al. (2005) reported, a research on producing somatic embryos by using

Citrus aurantifolia and Citrus sinensis species. The result showed that the callus formation is

the highest when it was cultured on Murashige and Skoog (MS) media supplemented with 2,

4-D and coconut water. El-Sawy et al. (2005) has performed an experiment on somatic

embryogenesis and plant regeneration from the undeveloped ovules of citrus. In their research,

they used several species of citrus which are C. sinensis, C. limon, C. paradise, and C.

auranticum. The undeveloped ovules were cultured on MS media. After the somatic embryos

has produced, the somatic embryos were transferred to media supplemented with 50 g/L

sucrose and 1mg/L Gibberellic acid. The C. sinensis showed the highest percentage of embryo

development after 2 to 3 months of culture.

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2.5 Sucrose in tissue culture

Figure 3. The chemical structures of Glucose, Fructose and Sucrose molecule

Among the culture medium components, carbohydrates have the strongest importance in

inducing somatic embryogenesis and embryo culture (Ricci, 2002). In plant tissue culture,

sugar serves as a carbohydrate supply to provide an optimum culture condition for cell.

Sucrose is a disaccharide sugar molecule that comprised of glucose and fructose molecule.

Sucrose has been widely used as a sole carbohydrate source of plant culture medium (Marcia

et al., 2000). The other sugars such lactose and maltose can be utilized for culture media but it

showed poor results for citrus. Besides that, the concentration of the carbohydrate is also

influence the plant growth (Ricci, 2002).

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Ricci had conducted a study to evaluate the effect of different carbohydrates in somatic

embryogenesis by using calli from Ponkan mandarin (Citrus reticulata, Blanco), Cravo

mandarin (C. reticulata), Itaborai sweet orange (C. sinensis L. Osbeck.), Valencia sweet

orange (C. sinensis) and Kinnow mandarin (C. nobilis Loureiro x C. deliciosa Tenore). The

culture medium used was Murashige and Tucker (MT) supplemented with sucrose, galactose,

glucose, maltose or lactose with the following concentrations of 18, 37, 75, 110, and 150 mM.

The culture medium used for the maturation of somatic embryos had 0, 15, 29, 44, 58 and 73

mM of sucrose. Sucrose concentrations of 58 and 73 mM generated a higher number of

plantlets from mature embryos of Ponkan mandarin and Valencia sweet orange.

2.6 Coconut water in culture media

Cocos nucifera or coconut is utilized in tissue culture protocol. The part of coconut being used

is coconut water, the colorless liquid endosperm. The coconut water is complex combination

of compounds. It contains a number of amino acids, organic acids, nucleic acids, several

vitamins, sugars and sugar alcohols, plant hormones, minerals, and other unidentified

substances (Molnar et al., 2011). None of this element is totally responsible to promote

qualities alone. Coconut water added to a medium containing auxin, inducing plant cells to

divide and grow rapidly. Molnar et al. (2011), explained that liquid has been found to be

beneficial for inducing growth of both callus and suspension cultures and for the induction of

morphogenesis. The amount of these substances required for successful culture varies with the

species and genotype (Thorpe et al. 2008).

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There are few studies on the effect of coconut water in tissue culture. Al-Khayri et al. (1992)

reported that studied the Spinach tissue culture was improved with the incorporation of

coconut water. They had discovered that an addition of 15% of coconut water into the medium

can improved the callus growth, shoot regeneration, and shoot growth in spinach leaf disk

cultures. Spinach shoot regeneration developed within 4 to 5 weeks in media containing

coconut water compared to the growth rate which was about 12 weeks in coconut water free

media. Nasib et al. (2008) studied on optimizing and improving method for the in vitro

propagation of kiwi fruit by using coconut water. Maximum shoot length, number of shoots

and nodes achieved in MS media contained 20% coconut water with 2.0 mg/L of BAP. The

explants grown on the media without coconut water showed less growth as compared to the

explants grown with the coconut water. It was also observed that the explants, grown without

coconut water were turned brown after four weeks in culture media.