i

PSZ 19:16 (Pind. 1/97)

UNIVERSITI TEKNOLOGI MALAYSIA

BORANG PENGESAHAN STATUS TESIS ♦ JUDUL : TISSUE CULTURE OF Nicotiana plumbaginifolis.

SESI PENGAJIAN : 2006/2007 Saya : TING CHIN YEE

(HURUF BESAR) mengaku membenarkan tesis (PSM/Sarjana/Doktor Falsafah)* ini disimpan di Perpustakaan Universiti Teknologi Malaysia dengan syarat-syarat kegunaan seperti berikut : 1. Tesis adalah hakmilik Universiti Teknologi Malaysia. 2. Perpustakaan Universiti Teknologi Malaysia dibenarkan membuat salinan untuk tujuan

pengajian sahaja. 3. Perpustakaan dibenarkan membuat salinan tesis in sebagai bahan pertukaran antara institusi

pengajian tinggi. 4. **Sila tandakan (� )

SULIT ( Mengandungi maklumat yang berdarjah keselamatan atau kepentingan Malaysia seperti yang termaktub di dalam AKTA RAHSIA RASMI 1972) TERHAD ( Mengandungi maklumat TERHAD yang telah ditentukan oleh organisasi/badan di mana penyelidikan dijalankan) TIDAK TERHAD

Disahkan oleh

________________________________ ______________________________ (TANDATANGAN PENULIS) (TANDATANGAN PENYELIA) Alamat Tetap : P.O.Box 3, Dr. Fahrul Zaman Huyop 96507, Bintangor, Sarawak. Nama Penyelia Tarikh : 7 May 2007 Tarikh : 7 May 2007

CATATAN : * Potong yang tidak berkenaan.

** Jika tesis ini SULIT atau TERHAD, sila lampirkan surat daripada pihak berkuasa/ organisasi berkenaan dengan menyatakan sekali tempoh tesis ini perlu dikelaskan sebagai SULIT atau TERHAD.

◆♦ Tesis dimaksudkan sebagai tesis bagi Ijazah Doktor Falsafah dan Sarjana secara penyelidikan, atau disertasi bagi pengajian secara kerja kursus dan penyelidikan, atau Laporan Projek Sarjana Muda (PSM).

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Tissue Culture of Nicotiana plumbaginifolis

This thesis is submitted in fulfillment of the requirements of

the Bachelor of Science degree in Industrial Biology, Faculty

of Science, University Technology Malaysia

By

TING CHIN YEE

2007

i

Abstract

Tissue Culture of Nicotiana plumbaginifolis

Ting Chin Yee and Fahrul Zaman Huyop

Nicotiana species is a type of plant species commonly used in plant science research,

including research of plant genetics. The main objective of this research is to grow

successfully Nicotiana plumbaginifolis through tissue culture technique. Seeds of

Nicotiana plumbaginifolis were obtained from Malaysian Palm Oil Board. Seeds

were surface sterilized using 20% hypochlorite solution. Sterilized seeds were placed

on the surface of Murashige and Skoog (MS) medium without plant growth

regulators (hormones). Young leaves started to grow from seed after two weeks.

Young leaves were taken and placed on fresh MS medium with addition of

appropriate concentration of hormone benzylaminopurine (BAP) and

naphthaleneacetic acid (NAA). Based on initial finding, 1.0 mg/L BAP for shoot

initiation and 0.1 mg/L NAA for root induction were the best concentration used to

initiate callus. When callus regenerated, callus was transferred to fresh MS medium

containing hormones (1.0 mg/L BAP and 0.1 mg/L NAA) until shoots were formed.

ii

Abstrak

Kultur Tisu Tumbuhan Nicotiana plumbaginifolis

Ting Chin Yee dan Fahrul Zaman Huyop

Spesies Nicotiana ialah salah satu spesies tumbuhan yang sering kali diggunakan

dalam kajian sains tumbuhan, termasuk kajian tentang genetik tumbuhan. Objektif

utama dalam kajian ini ialah untuk menanam Nicotiana plumbaginifolis dengan

menggunakan teknik kultur tisu. Biji benih Nicotiana plumbaginifolis diambil

daripada Malaysia Palm Oil Board (MPOB). Biji benih disterilkan dengan

menggunakan 20% larutan hypochlorite. Biji benih yang steril diletakkan atas

permukaan agar kultur tisu, iaitu Murashige dan Skoog (MS) medium tanpa

penggunaan hormon. Daun muda mula bertumbuh daripada biji benih selepas dua

minggu. Daun muda dipetik dan diletakkan dalam agar kultur tisu yang baru. Hormon

tumbuhan yang spesifik iaitu benzylaminopurine (BAP) dan naphthaleneacetic acid

(NAA) dengan kepekatan yang tertentu ditambahkan ke dalam agar. Berdasarkan

kajian yang dijalankan, didapati 1.0 mg/L BAP (pembentukkan pucuk) dan 0.1 mg/L

NAA (pembentukkan akar) adalah kepekatan yang paling sesuai untuk pembentukkan

kalus daripada explant. Selepas kalus terbentuk, kalus dipindahkan ke agar kultur tisu

yang baru yang mengandungi hormone (1.0 mg/L BAP dan 0.1 mg/L NAA) sehingga

pucuk terbentuk.

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BAP - Benzylaminopurine

CaCl2.2H2O – calcium chloride 2-hydrate

CoCl2.6H2O – cobalt chloride

CuSO4.5H2O – cupric sulfate

ddH2O – deionized distilled water

2, 4-D - 2, 4-dichlorophenoxyacetic acid

FeSO4.7H2O – ferrous sulphate

H3BO3 – Boric acid

IBA - indole 3-butyric acid

KH 2PO4 – potassium dihydrogen

phosphate

KI – potassium iodide

KNO 3 – Potassium nitrate

L – liter

M - Mol

Abbreviations

mg/L - milligram per liter

ml - milliliter

MgSO4.7H2O – magnesium

sulfate-7-hydrate

MnSO4.4H2O – maganase sulphate

MPOB – Malaysia Palm Oil Board

MS - Murashige and Skoog

NAA - 1-naphthylacetic acid

Na2.EDTA.2H2O –

ethylenediaminetetraacetic acid

Na2MoO4.2H2O – sodium molybdate

NH4NO3 – ammonium nitrate

spp – species

UV - ultraviolet

v/v – volume per volume

ZnSO4.7H2O – zinc sulphate

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“I hereby would like to state that I have read this thesis and in my opinion this thesis

fulfils the scope and quality to be awarded a bachelor degree in Industrial Science

(Industrial Biology)”.

Signature : ………………………………

Supervisor : Dr. Fahrul Zaman Huyop

Date : 7 May 2007

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“I declared that this thesis entitled ‘Tissue Culture of Nicotiana plumbaginifolis’ is the

result of my own research except as cited in references”.

Signature : …………………………..

Name of candidate : TING CHIN YEE

Date : 7 May 2007

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Specially dedicated to our mighty God, my beloved family,

my loving father and mother…

Thanks for everything from you.

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Acknowledgements

First of all, I wish to express my special thanks and gratitude to my supervisor,

Dr. Fahrul Zaman Huyop for his invaluable supports, guidance and encouragement in

completing this project. I greatly appreciate his helpful comments, suggestions and

corrections.

Besides, I am indebted to all lecturers in biology department, all laboratory

staffs of Microbiology Laboratory, Plant Tissue Culture Laboratory and also post-

graduated students for their support and guidance.

I also wish to express my special thanks to my aunt, my friends, and all my

fellow coursemates who have directly or indirectly helped and shared information with

me in finishing my project.

Last but not least, I wish to extend my appreciation to my beloved parents, Mr.

and Mrs. Ting and family for their loving and moral support in completing my thesis.

viii

Table of contents

Contents Page No.:

Abstract i

Abstrak ii

Abbreviations iii

Supervision’s certification iv

Author Declarations v

Dedication vi

Acknowledgements vii

Table of contents viii

List of Tables x

List of Figures x

CHAPTER 1 INTRODUCTION

1.1 Background of in vitro plant regeneration 1

1.2 Aseptic technique 2

1.3 Medium 3

1.4 Plant growth regulators (Hormones) 4

1.5 Callus formation 5

1.6 Research species 6

1.6.1 Importance of tissue culture of Nicotiana

Plumbaginifolis 8

1.7 Research objective 9

CHAPTER 2 MATERIALS AND METHODS

2.1 Aseptic technique 10

2.2 Agar media preparation

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2.2.1 Stock solution preparation 11

2.2.2 Complete MS medium agar media preparation 12

2.3 Surface sterilization and seedling 13

2.4 Explant regeneration 13

2.5 Hormone treatment 14

CHAPTER 3 RESULTS AND DISCUSSION

3.1 Sterilization technique 15

3.2 Seedling 16

3.3 Hormone treatment 18

3.4 Explant regeneration and callus formation 23

3.5 Shoot formation 25

CHAPTER 4 CONCLUSION 28

CHAPTER 5 FUTURE WORK 30

REFERENCES 32

APPENDIX 35

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LIST OF TABLES Page No.:

CHAPTER 2

Table 2.1. List of components in MS medium and the concentration

in stock solution 11

Table 2.2. Different concentration of hormone treatment was designed 14

CHAPTER 3

Table 3.1. Total explants regenerated for each type of hormone

concentration 21

LIST OF FIGURES ` Page No.:

CHAPTER 1

Figure 1.1. Structural formula of some auxins and cytokinins 4

Figure 1.2. Nicotiana spp 7

CHAPTER 3

Figure 3.1. Seeds were sown on the surface of MS agar media without

addition of hormones 17

Figure 3.2. Young plants were developed from seedling 17

Figure 3.3. A graph shows the total callus formed in every types of

combination of hormone concentration 21

Figure 3.4. Different sizes of callus formed based on varies hormone

concentration after hormone treatment for three weeks 22

Figure 3.5. Explant started to regenerate after three weeks under

0.5 mg/L BAP 24

Figure 3.6. Callus was formed when explant was regenerate further 24

xi

Figure 3.7. Plants with complete young leaves (around 4.30cm) were

formed from shoot 27

Figure 3.8. Plant was died; the color has change from green to black 27

1

CHAPTER 1

Introduction

1.1 Background of in vitro plant regeneration

Nowadays, tissue culture technique (including tissue culture of cells, tissue or

organ) has become an important technique in plant research. This tissue culture

technique has been made practical and has been applied for commercial purpose.

Plant tissue culture is also called micropropagation, which was a technique

consisting of taking a piece of a plant (such as a stem tip node, meristem, embryo, or

even a seed) and placing it in a sterile nutrient medium where it multiplies. Plant tissue

can be described as plasticity and totipotency. Plasticity, allows plants to alter their

metabolism; growth and development to best suit their environment. While totipotency

can be defined as the ability of each living cell of a multicellular organism to develop

independently if provided with proper external conditions. A totipotent cell is one that

is capable of developing by regeneration into whole organisms, and this term was

probably coined by Morgan (1901).

The first experiment to culture isolated, fully differentiated plants cells in vitro

on an artificial medium was originated by Haberlandt (Dodds and Roberts, 1995). The

2

cultured cells able to survived for several months, but they were incapable of

proliferation. As a result for failure to obtain cell division, tissue culture was being

continued by others researcher, and lately being proven to be a success by White (1934).

White (1934) had successfully to culture a whole plant by using root of tomato. Further

researches had been done by a group of researchers that is Gautheret et al (1939) that

showed cells can be cultured continuously and go through differentiation step. These

finding has encouraged more research being done on plant tissue culture, from 1940 to

1960.

1.2 Aseptic technique

The importance of maintaining a sterile environment during the culture of plant

tissues was necessary. A few simple precautions to avoid contamination will save

valuable time in not repeating experiments.

The main factor for aseptic technique was selecting a right working area and

using the aseptic tools. The purpose for selecting a suitable working area was to prevent

the possible flow of unfiltered air over the disinfected working area. Most tissue culture

procedures were conducted in sterile operations, such as laminar flow cabinet. Besides

the special design of gentle flow of sterile air in cabinet, aseptic cabinet is also

equipped with germicidal lamp emitting ultraviolet (UV) light. This type of radiation is

useful in eliminating airborne contaminants and for surface disinfection.

Glassware and all the tools used for tissue culture process can also cause

contamination. It is extremely necessary to autoclave all the material before using it, so

that all the microbial contaminants are destroyed.

3

1.3 Medium

Nutritional requirements for optimal growth of a tissue in vitro may vary with

the species. Even tissues from different parts of a plant may have different requirements

for satisfactory growth (Murashige and Skoog, 1962). No single medium can be

suggested as being entirely satisfactory for all types of plant tissues and organs. When

starting with a new system, it is essential to work out a medium that would fulfill the

specific requirements of that tissue.

The components of a plant tissue culture medium included macronutrients,

micronutrients, a separate iron supplement, vitamins, a carbon source, and usually plant

growth regulators. Amino acids and various nitrogenous compounds may be present in

the vitamin mixture. Macronutrients were nitrogen, phosphorus, potassium, calcium,

magnesium, and sulfur. These macronutrients are also called inorganic chemicals, and

were essential elements required in relatively large amounts. Micronutrients, were

traces of certain elements required by all plant cells. Micronutrient elements include

iron, manganese, zinc, boron, copper, molybdenum, iodine, cobalt and chlorine.

Meanwhile, vitamins have catalytic functions in enzyme systems and were required

only in trace amounts.

The need to culture diverse tissue and organs has led to the development of

several recipes of nutrient medium. White (1943) has created a new medium that is low

in salt and free of ammonium ion. White’s medium was the earliest control medium

that consists of all needed nutrients, and applied widely for root cultivation. On the

other hand, Murashige and Skoog (1962) have developed MS medium that consist of

ammonium, nutrient and others mineral includes inorganic nutrient that needed by plant

to establish growth. Until now, the most known mediums that researchers used in plant

tissue culture were Murashige and Skoog medium (1962) that is high salt or B5

(Gamborg et al., 1968).

4

1.4 Plant growth regulators (Hormones)

In addition to the nutrients, it was generally necessary to add one or more

growth substances, such as auxins, and cytokinins, to support good growth of tissues

and organs. However, the requirement for these substances varies considerably with the

tissue (Bhojwani & Razdan, 1992).

Auxins, a class compounds that stimulate shoot cell elongation, and are useful

to stimulate the formation of adventitious roots, inhibit bud formation, and to play a

role in embryogenesis. Cytokinins, promote cell division in plants tissues under certain

bioassay conditions and only in the presence of auxin. Cytokinins stimulate bud

proliferation and inhibit rooting. Auxin-cytokinin supplements are instrumental in the

regulation of cell division, cell elongation, cell differentiation, and organ formation.

Some auxins commonly applied were IBA (indole-3-butyric acetic acid), α-NAA

(naphthaleneacetic acid) and 2, 4-D (dichlorophenoxyacetic axid). Meanwhile; the most

widely used cytokinins in media were kinetin (furfurylamino purine) and BAP

(benzylamino purine).

Figure1.1: Structural formula of some auxins and cytokinins.

a) Indole-3-acetic acid b) Naphthaleacetic acid

c) 2,4-dichlorophenoxyacetic acid d) Adenine

5

Plant growth regulators can be subdivided into natural occurring and synthetic

types. BAP, NAA, 2, 4-D were some example of synthetic growth regulators. These

synthetic growth regulators have several advances feature. They help to increase yields,

to make uniform ripening and facilitate harvesting, increased resistance against some

unfavorable factors (drought, frost), and regulated rooting, fruit setting (Sebanek, 1992).

In ornamental horticulture, they were used for regulation of plant habit, time of

flowering, and size. Another advance of using synthetic growth regulator is it would not

be degraded easily. Natural occurring hormones were rapidly degraded by light and

enzymatic oxidation (Dodds and Roberts, 1995).

1.5 Callus formation

Callus refers to a disorganized proliferated mass of actively dividing cells. A

callus consists of an amorphous mass of loosely arranged thin-walled parenchyma cells

arising from the proliferating cells of the cultured plants. The most important function

of callus was that it has the potential to develop normal roots, shoots, and embryoids

that can form plants. In addition, it can be used to initiate a suspension culture (Dodds

and Roberts, 1995). For callus formation, pieces of cotyledon, hypocotyls, stem, leaf, or

embryo were usually used. Sinnott (1960) had described some of the early observations

on wound callus formation. The stimuli in the initiation of wound callus were the

endogenous hormones auxin and cytokinin. Using tissue culture techniques, callus

formation can be induced in numerous plant tissues and organs that do not usually

develop callus in response to an injury (Street, 1969).

Establishment of a callus from an explant was divided into three developmental

stages: induction, cell division, and differentiation. Induction phase was metabolism

stimulated prior to mitotic activity. The length of induction phase depends on the

physiological status of the explant cells as well as the cultural condition (Dodds &

Roberts, 1995). Cell division phase, a phase of active cell division as the explants cells

revert to a meristematic state. The third phase involves the appearance of cellular

6

differentiation and the expression of certain metabolic pathways that lead to the

formation of secondary products. It becomes possible to subculture the callus to a fresh

medium, after the callus had been grown for a while in association with the original

tissue.

The first successful prolonged cultures of experimentally induced callus were

achieve in 1939 almost simultaneously at the research laboratory of Gautheret in Paris,

Nobecourt in Grenoble, and White in Princeton. These cultures were originally derived

from explants of cambial tissue of carrot and tobacco. Callus appears as yellowish,

white, green, or pigmented with anthocyanin.

1.6 Research species

Nicotiana plumbaginifolis is related to garden vegetables, flowers, weeds and

poisonous herbs. It is also refers to a genus of broad-leafed plants of the nightshade

family. The family of plant is Solanaceae, and the genus of plant is Nicotiana. There is

about 100 species of Nicotiana. Nicotiana spp has huge green leaves and very sweet-

scented flowers with variety colors. The flowers are hermaphrodite, which has both

male and female organs. Pollination can be done through insects such as bees, moths,

and butterflies. The plant prefers sandy, loamy and clay soils and requires well-drained

soil.

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Figure 1.2: Nicotiana spp

Nicotiana spp origin from North and South America, and it was first discovered

by South Americans. Nicotiana spp, is believed to be first used by native American by

chewing or snorting the leaves for medicinal purpose.

As it is from genus Nicotiana, this plant contains nicotine. Nicotiana tabaccum

and Nicotiana rustica having high nicotine content, and this makes them suitable as

cigarettes or cigars. Nicotiana spp has a long history in medical field. The leaves are

antispasmodic, discutient, diuretic, emetic, expectorant, irritant, narcotic, sedative and

sialagogue. The leaves were mostly used to treat rheumatic swelling, skin diseases and

scorpion stings to relieve the pain. Besides, nicotine can also be extracted and used as

an insecticide, which the leaves being take and dried. The dried leaves remain effective

for 6 months. The juice of the leaves can be rubbed on the body as an insect repellent.

The leaves have been dried and chewed as an intoxicant.

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1.6.1 Importance of tissue culture of Nicotiana plumbaginifolis

In the research, Nicotiana spp was commonly used as the first model to study

variety aspect of plant, such as genomic transformation, metabolism, or adaptation of

plants to environmental changes. Life cycle for Nicotiana spp is about 4-5 month.

Although the life cycle is about twice as long as that of Arabidopsis thaliana (2 month)

and large growing space required, the larger size of Nicotiana reproductive structures is

an advantage for some expression and manipulation studies (McDaniel, 1999).

For example recombinant human tissue transglutaminase produced into tobacco

suspension cell cultures (Sorrentino, 2004). The cell was active and recognizes

autoantibodies in the serum of celiac patients. The recombinant enzyme was

successfully expressed in different plant cell compartments.

Another advance characteristic of Nicotiana species was the stable chromosome

numbers. As cell suspension cultures have been used frequently for mutant isolation

and protoplast fusion, the establishment of cell suspension cultures with stable

chromosome number is an essential factor for regeneration of stable haploid and diploid

plants (Evans and Gamborg, 1982). Leaf tissue of diploid tobacco has been shown to

have only diploid cells and this tissue is preferable for the initiation of cell cultures.

In previous research, several species of Nicotiana have been combined with

cultivated tobacco using sexual hybridization to introduce useful agricultural traits into

the tobacco. This had resulted in the development of several cultivated tobacco varieties

that contain genes derived from the wild species. As routine techniques were available

for plant regeneration from isolated leaf mesophyll protoplasts of numerous Nicotiana

species, more effort has been directed toward production of interspecific Nicotiana

somatic hybrids (Evans et al, 1982). New variety of plants with new characteristic had

been discovered. For example transgenic plant, developing resistant plant, plant with

high commercial value and others.

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An example for transgenic plant using tobacco was a chimeric green fluorescent

protein gene as an embryogenic marker in transgenic cell culture of Nicotiana

plumbaginifolia (Chesnokov, 2001). In the tissue culture process, only a limited

number of cells from the in vitro culture actually undergo the transition of somatic into

embryogenic cells. The problem was that no clear evidence about the morphogenetic

changes can be observed. Therefore, specific markers with the ability to distinguish

precisely between embryogenic and non-embryogenic cells were required in order

separate out the cells. Insertion of the marker gene into transgenic Nicotiana has been

proven successful.

Nicotiana spp has also been used in the study of several enzyme activities.

Investigation on Nicotiana plumbaginifolis has been done through RNA hybridization

to distribute the differences level of enzyme production according different part of the

plant.

1.7 Research objectives

The main objective of this research was:

1. To establish plant tissue culture of Nicotiana plumbaginifolis.

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CHAPTER 2

Materials and methods

2.1 Aseptic technique

Before starting the experiment, it is necessary to ensure all the material and

tools used are sterile. Three most important aseptic techniques that need to be done

were autoclaving, filter sterilization, and surface sterilization.

All glassware, tools and chemical materials that heat resistant were autoclaved

at temperature 121°C for 20 minutes. Microfiltration was used for media components

that were not heat resistant, such as plants growth regulators and vitamins. The working

area was generally disinfected with either ethanol or isopropanol (70% (v/v)), and also

UV light.

Surface sterilization was done before seedling. The seeds were first immersed in

20% (v/v) hypochlorite for 10 to 15 minutes. Then the seeds were rinsed in four

changes of sterile distilled water. After rinsing, the seeds were poured on a sterile filter

paper to dry off the unwanted water.

11

2.2 Agar media preparation

2.2.1 Stock solution preparation

Stock solutions were prepared as described in Table 2.1.

Table 2.1: List of components in MS medium and the concentration in stock solution.

Constituents Amount (mg l-1)

Macronutrients (Stock 1)

NH4NO3

KNO3

CaCl2.2H2O

MgSO4.7H2O

KH2PO4

33000

38000

8800

7400

3400

Micronutrients (Stock 2)

KI

H3BO3

MnSO4.4H2O

ZnSO4.7H2O

Na2MoO4.2H2O

CuSO4.5H2O

CoCl2.6H2O

166

1240

4460

1720

50

5

5

Iron (Stock 3)

FeSO4.7H2O

Na2.EDTA.2H2O

5560

7460

Vitamin (Stock 4)

Inositol

Nicotinic acid

Pyridoxine HCl

Thiamine HCl

Glycine

20000

100

100

100

400

12

To prepare stock solution of stock 1, stock 2 and stock 3, 400 ml deionized

distilled water was first added to a 1 L beaker. Each constituent was being weighed and

dissolved in a beaker by using a magnetic stirrer. The solution was then transferred to a

1 L volumetric flask, and adds DDH2O to the final volume (until 1 L). All of the stock

1, 2 and 3 was store under refrigeration.

For iron stock, FeSO4.7H2O was dissolved in 450 ml of warm ddH2O in a 1 L

beaker. In a seperate beaker, Na2.EDTA.2H2O was dissolved in 450 ml of warm ddH2O.

The two solution were then mixed and transferred to a 1 L volumetric flask. pH should

adjusted to 5.5. ddH2O was added to the final volume. A precaution about iron stock is

that it should be protected from light by storing the solution in an amber bottle or

wrapped with aluminium foil. The solution was stored at room temperature since

precipitation may occur at chiling temperatures.

2.2.2 Complete MS agar media preparation

To prepare a ready use agar media, 50 ml of stock 1, 5 ml of stock 2, 5 ml of

stock 3, and 5 ml of stock 4 were poured into a 1 L beaker. All the stock solutions and

800 ml of ddH2O were added in the beaker. Magnetic stirrer was used to mix the

solution well. Next, 30 g of sucrose (carbon source) was added to the solution. pH of

the solution was adjusted to pH 5.5. Then 8 g of agar powder was added in. The

solution was transferred to a 1 L volumetric flask, and ddH2O was added to the final

volume (1 L). The solution was autoclaved at 121°C for 20 minutes. Caution about agar

media preparation was that media firmness might be expected to fluctuate in response

to changes induced by autoclaving and pH. Finally, hormones BAP and NAA were

added to liquid solution according to different concentration. Note that hormones were

added after medium sterilization, as high temperature will denature the hormones and

makes it lose its function, as hormone is not resistance to heat.

13

Then, liquid solution was poured to a suitable container, such as petri dishes,

culture tube, marjenta jar, or wide mouth conical flask. These tools were being

sterilized before use. After pouring, the containers were left in a sterile laminar flow

cabinet, to let the agar media to solidify. Next, the container closed tightly and wrapped

using parafilm. The solidified agar was then placed on a suitable place for storage.

2.3 Seedling

The original source of plant was the seeds of Nicotiana plumbaginifolis. The

seeds were obtained from MPOB. The sizes of seeds were very fine. Seedling, is a

process to sown the seeds and grow the desired plant. Sterile seeds were used to grow

young plants. By using a forceps, the seeds were sown carefully on the surface of MS

agar media that does not containing any plant growth regulators. Each Petri dish can be

sown with 30 seeds. The Petri dishes were closed tightly using parafilm to prevent any

cross contamination.

The Petri dishes were then placed on tray and let it grow under fluorescence

light until young plants and leaves developed. After young plants with young leaves

were developed, the leaves were taken as explant for regeneration.

2.4 Explant regeneration

Explant, is an excised fragment of plant tissue or organ used to start a tissue

culture, and it can also be called as primary explant. During this research, leaves were

used as explant. The leaves were cut from young plants. As the leaves were cut from

the plant free from contaminant, the leaves do not require surface disinfection. The

leaves were transferred to new MS agar media supplemented with plant growth

regulators. The petri dishes were closed tightly using parafilm and left on tray. The

explant were left to regenerate until callus is formed from explant.

14

2.5 Hormones treatment

To enhance plant growth, hormone auxin and cytokinin were added to the media

agar. Type of auxin used is NAA, and type of cytokinin used is BAP. To prepare BAP

and NAA solution, both hormones required addition of several drops of 1M NaOH as

solvent. Then it was later diluted by using ddH2O until the final volume.

Table 2.2: Different concentration of hormone treatment was designed

BAP

NAA

0 mg/L 3 plates x

10 explant

3 plates x

10 explant

3 plates x

10 explant

3 plates x

10 explant

0.1 mg/L 3 plates x

10 explant

3 plates x

10 explant

3 plates x

10 explant

3 plates x

10 explant

1.0 mg/L 3 plates x

10 explant

3 plates x

10 explant

3 plates x

10 explant

3 plates x

10 explant

Combination of different NAA and BAP concentration (shown in Table 2.2)

was added to explants to determine the effect of hormone towards explant regeneration.

3 plates of explants were prepared for every type of concentration, and each plate of

agar media consists of 10 small explants. After three weeks, total amount of callus

formed from the explant that undergoes hormone treatment in each plate was counted.

0.5 mg/L 1.0 mg/L 2.0 mg/L 4.0 mg/L

15

CHAPTER 3

Results and discussion

3.1 Sterilization technique

Sterilization is an important element to ensure every thing was sterile when

doing the research of tissue culture.

In the experiment, hypochlorite was used to sterilize the seed’s surface. Seeds

were dipped in the hypochlorite solution for 10 to 15 minutes, as this is the suitable

time range. Dipping the seeds in hypochlorite solution for a long time may spoil the

seed surface, and kill the cell inside the seeds. As a result, long exposure time of seeds

with hypochlorite caused less seeds generation. Meanwhile short sterilization time was

not enough for a total elimination of contamination or microorganism. After dipping the

seeds in hypochlorite, seeds were washed with sterilized distilled water repeatedly to

wash off hypochlorite that was stuck on seed’s surface. Excessive amount of

hypochlorite within the seed’s surface give more harm than good, as it will

continuously break and kill the cells of seed.

Plant tissue culture media, which contain a high concentration of sucrose,

support the growth of many micro-organisms, such as bacteria and fungi. These

16

microbes generally grow faster than the cultured tissue and finally kill it. Apart from

that, the contaminants may also give out metabolic waste which is toxic to plant tissues.

To minimize the possibility of contamination, several steps should be done with caution,

such as hands, wrists, and forearms should be cleaned, and should not pass the hand or

arm directly over a sterile exposed surface, such as open agar plate. All sterilize open

surfaces should be placed as far back in the hood as conveniently possible. When

pouring sterile liquids, the bottle should be grasping at the base, and hands should be

kept as far as possible from the open tube or petri dishes receiving the liquids.

3.2 Seedling

Sterile seeds were firstly sown on the surface of MS agar that does not

containing plant growth regulators. The media components were described in table 2.1.

A plate of MS media with seeds sown on the surface was shown in figure 3.1. After two

weeks, young plants started to form from seeds after it were sown for two weeks.

Young plants continue to grow, until it develops young leaves as shown in figure 3.2.

Young plants used the nutrient supplement from MS agar media as their growth factor.

The purpose to do seedling was to produce young plants with young leaves.

Young leaves were needed as explant to start the tissue culture process. The uptake of

water is the beginning of seed germination. The main factor for a plant to germinate

was the rate of water penetration into the seed, which depends on the permeability of

the cell walls of the testa or pericarp. Seeds of Nicotiana plumbaginifolis need seven

days to break down the testa layer. The seeds germination capacity can be increased if

seeds are kept at lower temperatures and in high humidity (Sebanek, 1992). Embryo

grows when all conditions were ensured for the swollen of seeds, and enzymatic

activity increase. Cotyledons were the primary sources of necessary substances. As

seeds of Nicotiana plumbaginifolis were dicotyledons, so the seeds go on epigeal

germination, which the cotyledons were brought above ground by hypocotyls, and then

turned green to become the first assimilation organs, that is leaf.

17

Figure 3.1: Seeds were sown on the surface of MS agar media without addition of

hormones.

Figure 3.2: Young plants were developed from seedling after two weeks.

18

3.3 Hormone treatment

This experiment was carried out to determine the suitable concentration

of hormone (shown in table 2.2) to induce callus and shoot-bud formation. In the

previous research done by other researchers; different hormone and different

concentration were used. The use of hormone for a plant species may vary under

influence of different environment, such as the weather. So, this is the purpose for us to

redo the hormone test, as plant might need different hormone concentration that varies

from what the research done before. In a paper written by Sun and Kang (2003), 1.2

mg/L BAP was used. The amount of NAA used by researchers was in the range

between 0.2 to 1 mg/L. By referring to these previous researches, a new hormone

treatment was designed. The concentration of BAP tested was 0.5 mg/l, 1 mg/l, 2 mg/l,

and 4 mg/l, while the concentration of NAA used was 0 mg/l, 0.1 mg/l, and 1.0 mg/l.

Explant (young leaves) was taken from young plant and grown on fresh MS

media containing hormones. After the explants were grown in different concentration

of hormones for four weeks, the observation was done. The number of callus formed

from explant was counted. Callus was the intermediate structure before shoot initiation.

Hormone capable of inducing shoot formation, also induce the callus formation. These

means any hormone that induces or increase the time to form callus, can also give the

same effect for shoot initiation (Sebanek, 1992). The result was shown in table 3.1.

Based on the result in table 3.1, the result was converted into figure 3.3, which is a

graph that gives a better view and analysis of the result.

The parameters used to determine the result were the amount of callus

regenerated for different set of combined BAP and NAA, and the time range needed for

explant to regenerate to form callus and the size of callus formed. Based on the

hormone treatment result, it shows that explants regenerated more when concentration

of BAP was 1.0 mg/L, and NAA was 0.1 mg/L, whereby all explants were regenerated.

The shortest time to form callus from explant was three weeks. The explant was first

regenerated at the cutting site, whereby small clumps were formed. The clumps were

19

regenerate continuously, until a big size clump in green color was formed. In this stage,

the explant loses its origin structure.

In the research, the NAA concentration used was not more that 1 mg/L as high

concentration of auxin can suppress morphogenesis. Auxin is always added in a small

quantity to affect both cell division and cellular expansion. According to the “acid

growth theory”, auxins may directly stimulate the early phase of cell elongation by

causing responsive cells to actively transport hydrogen ions out of the cell, thus

lowering the pH around the cells. This acidification of the cell wall region activates

enzymes known as expansins, which break bonds in the cell wall structure, making the

cell wall less rigid. When the cell wall is partially degraded by the action of auxins, this

now-less-rigid wall is expanded by the pressure coming from within the cell, especially

by growing vacuoles. From figure 3.3, we know that 0.1 mg/L of NAA (auxin) always

shows the best result when compared to other NAA concentrations used based on the

sizes of callus formed and time range needed for callus formation. Besides, explant

shows 100% growth when induce with 0.1 mg/L NAA. From here, it is clearly know

that 0.1 mg/L NAA was suitable for induce Nicotiana plumbaginifolis growth.

Explant was also regenerated using other hormone concentrations, but was less

efficient. High BAP concentration, such as 4.0 mg/L was unsuitable as plant growth

regulator because it suppresses shoot initiation. Another reason to explain the

unsuitable concentration of hormone that reduced the explant growth was the hormone

may have a specific activity, including binding to a receptor, which will inhibit enzyme

activity in explant.

During the four weeks observation, it can be concluded that the speed of growth

or shoot initiation was different according to different hormones concentration. The

fastest callus initiation was when the concentration of BAP was 1.0 mg/L, while the

concentration of NAA was 0.1 mg/L, as 100% explant regenerated. Figure 3.4 shows

the sizes of callus formed based on different hormone concentration. The observation

was done after treating the explant with hormone for three weeks. It was clear that the

20

callus treated with 1.0 mg/L BAP and 0.1 mg/L NAA had the biggest callus compared

to others. This means the explants generated faster under this specific hormone

concentration. On the other hand, 4.0 mg/L BAP had shown a clearly different result. It

can be seen that those explant treated with 4.0 mg/L BAP have small size callus, as it

just start to regenerate. This means longer time was needed for callus to regenerate

when unsuitable hormone concentration was used.

21

BAP

NAA

0 mg/L 93% explant

regenerated

90% explant

regenerated

83% explant

regenerated

33% explant

regenerated

0.1 mg/L 97% explant

regenerated

100% explant

regenerated

87% explant

regenerated

63% explant

regenerated

1.0 mg/L 87% explant

regenerated

93% explant

regenerated

87% explant

regenerated

47% explant

regenerated

Table 3.1: Total percentage of explants regenerated for each type of hormone

concentration.

Total callus formed in every combination of hormone concentration

9390

83

33

97100

87

63

87

93

87

47

0

20

40

60

80

100

120

0.5 1 2 4

BAP concentration (mg/L)

Per

cen

tag

e (%

) o

f ca

llus

form

ed

0

0.1

1

Figure 3.3: A graph shows the total callus formed in every types of combination of

hormone concentration

1.0 mg/L 4.0 mg/L 0.5 mg/L 2.0 mg/L

NAA (mg/L)

22

Figure 3.4: Different sizes of callus formed based on varies hormone concentration

after hormone treatment for three weeks.

0.5 mg/L 1.0 mg/L 2.0 mg/L 4.0 mg/L

0 mg/L

0.1 mg/L

1.0 mg/L

BAP

NAA

23

3.4 Explant regeneration and callus formation

From the result of hormone treatment, it can be concluded that the most suitable

hormones concentration to induce explant regeneration were 1.0 mg/L BAP and 0.1

mg/L NAA. By using both concentrations, the process of explant regeneration becomes

faster, which was three weeks compared to other hormone concentrations, whereby four

weeks were needed. Pictures about the beginning of explant regeneration and then

formation of callus were shown in figure 3.5 and 3.6.

In nature, stem, leaf, and root pieces are able to differentiate shoots and roots

leading to the establishment of new individuals. So, any plant tissue can be used as an

explant. In this research, leaf has been chosen as explant. Leaf has large surface, and is

easier to handle.

The gel in a solidified tissue culture medium can influence explant or callus

growth and morphology (Cameron, 2006). The early phases of tissue culture, the initial

stage-often favor proliferation over differentiation, and rapid growth tends to be best in

moderate or low gel concentration. The lateral cultural stages of differentiation are

promoted by higher gel concentration.

24

Figure 3.5: Explant started to regenerate after three weeks under 0.5 mg/l BAP.

Figure 3.6: Callus (a mass of undifferentiated cell) was formed when explant was

regenerated further.

25

3.5 Shoot formation

When the callus was allowed to continue its growth, the callus soon initiate

shoot. To induce shoot, the callus was grown in fresh MS media that containing plant

growth regulators, where the concentrations were the same as used in callus

regeneration, 1.0 mg/L BAP and 0.1 mg/L NAA. It is important to ensure a sufficient

supply of nutrient all along the plant growing process. The plant needs to be transferred

to fresh MS agar media every two weeks. Growth on the same media agar for an

extended period will lead to depletion of essential nutrients and gradual desiccation of

the gelling agent. Following callus initiation, shoot bud started to form after three

weeks in culture.

Chemical factor and physical factor are the main factors that affect shoot bud

differentiation in tissue culture. Chemical factor, which means the effect of hormone,

has been discussed further previously in section 3.3. Meanwhile physical factor

includes the medium used, light, temperature, physiological state of the donor plant,

and the others. High light intensity has been shown to be inhibitory for shoot-bud

formation in Nicotiana spp (Bhojwani and Razdan, 1992). The quality of light also

influences organogenic differentiation. Blue light promote shoot bud differentiation

whereas red light stimulates rooting in Nicotiana spp (Bhojwani and Razdan, 1992).

Shoot will continue to grow until complete young leaves were formed. It is

important to ensure complete young leaves were formed from shoot. When this step

was achieved, it indicates that the plant was ready to develop certain bioprocess to

maintain its growth. For example, the plants started to utilize solar energy by the leaves

for transpiration, and also photosynthesis. In figure 3.7, it shows that plants with

complete young leaves (4.30 cm height) were formed from shoot.

However, plants died in the process to continue its growth. Plants died when it

was transferred to fresh MS media with addition of hormones. Plant which died

changed color from green to brown or black.

26

The possible reasons that can explain the plant death, is a correlative

phenomenon called senescence. Senescence may be accelerated by lack of nutrients,

especially of nitrogen, kalium, phosphorus, and magnesium. The beginning of cell

senescence is the loss of its capacity to divide. In the cell, the velocity of RNA and

protein degradation increases, the synthesis of ribonucleases, and chlorophyll-

degrading enzyme increases, degeneration of chloroplasts begins, followed by

endoplasmic reticulum, tonoplast, mitochondria, and nucleus (Sebanek, 1992). Another

possible reason is that when the plants were transferred to new agar media, it does not

adapt to new environment, and the growth deplete. It is suggested the media used was

not suitable, and the possible media should be use is MS free media. As a conclusion,

an intensive care is needed when doing the tissue culture research as technical problem

and carelessness may affect the result.

27

Figure 3.7: Plants with complete young leaves were formed from shoot.

Figure 3.8: Plant died, the color changed from green to black.

28

CHAPTER 4

Conclusion

As with any plant transformation studies, a regeneration protocol must first be

established in order to regenerate transformed plants. Therefore, the first approach in

plant research is to carry out tissue culture experiments and figure out the most suitable

condition for plant growth.

Before starting plant tissue culture, it is evident to know well the tissue culture

process and the characteristic of the research plant. Literature reviews on tissue culture

and research on plant help gain more knowledge on the research to be done, and

prevent mistakes.

For tissue culture of Nicotiana plumbaginifolis, sterilization technique suitable

to be used was to immerse the seed in hypochlorite for 10 to 15 minutes.

The best hormone concentrations to grow the explant were at 1.0 mg/L BAP

and 0.1 mg/L NAA. Suitable hormone concentrations promote the optimum growth of

explants and also induce the callus and shoot formation. Suitable hormone

concentrations also shorten the time for plant to initiate callus and shoot.

29

Callus start to form after three weeks with the addition of plant growth

regulators. Shoot or young leaves began to form after three weeks of callus initiation.

Proper sterilization technique and experiment procedures handling can prevent the

contamination of culture.

30

CHAPTER 5

Future work

It is undeniable that tissue culture and genetic engineering goes hand in hand in

the molecular improvement of plant species. Therefore, the first step in the molecular

improvement of plant is the establishment of tissue culture technique.

Apart from leaf explant, other tissue sources can be used for tissue culture. Here,

a suitable source is through somatic organogenesis. Somatic embryos provide a ready,

long term source of tissues for transformation. It is also advantageous since somatic

embryos could arise from single cell, and this could minimize somaclonal variation.

Therefore, future studies can be focused on the establishment of somatic embryogenesis.

Manipulation of culture medium and condition must be further looked into.

Different content of culture media such as macronutrient, micronutrient, and carbon

source may give a different result of plant development. Besides, varieties of plant

growth regulator should also be test to study the effect on plant development. Study on

the possible factors and condition that can cause plant death also can be conducted.

Last but not least, with the tissue culture development for Nicotiana

plumbaginifolis, more genetic studies of this species can be conducted. As Nicotiana

31

spp allow the transformation of Agrobacterium easily, this gives the benefits to having

more studies on genetic transformation.

32

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