GENETIC STABILITY OF WHITE KELAMPAYAN (NEOLAMARCKIA CADAMBA) PLANTLETS DERIVED FROM CALLUS AS

EXPLANT USING ISSR MARKERS

Ngo Yi Lei

Bachelor of Science with Honours (Resource Biotechnology)

2016

iusat Khidmat MakJuDlat Akadtmik UNlVERSITl MALAYSIA SARAWAK

P. KHIDI1AT I1AKLUI1AT AKADEI1IK

UNI"AS

11111 1111111111 n1~I 1000272695

GENETIC STABILITY OF WHITE KELAMPAYAN (NEOLAMARCKIA CADAMBA) PLANTLETS DERIVED FROM CALLUS AS EXPLANT USING ISSR MARKERS

NGOYILEI

This thesis is submitted in partial fulfillment of the requirements for the degree of Bachelor of

Science with Honours (Resource Biotechnology)

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2016

I

I

ACKNOWLEDGEMENTS

For my accomplishment, I would like to express my deepest appreciation and sincerest

gratitude to those who supported me throughout my degree studies. I would foremost to thank

my supervisor, Assoc. Prof Dr. Ho Wei Seng and my co-supervisor, Ms. Maslini Japar Ali, for

their faith in me while giving me the helpful guidance and advice throughout my research

project at any point of time. Their kind motivation, constructive suggestions, and immense

knowledge have been great value in this study.

I would also like to extend my many thanks to the post-graduate senior of Forest

Genomics and Informatics Lab (fGilab), Mr. Lai Nan Keong for his invaluable guidance and

excellent assistance throughout the conduct of molecular works in the laboratory. The research

was also made smoothly with the kind help and cooperation from the laboratory assistant, Ms.

Kamaliawati in the fGilab. Her valuable support in providing the laboratory materials were

highly appreciated.

Last but not least, my special thanks to my family and friends for their constant moral

support and encouragement from the beginning until the end of the project. Without their

continuous assistances and patience, this study would not have succeeded on time.

I

- -----

UNIVERSITI MALAYSIA SARA WAK

Grade:

Please tick tV) Final Year Project Report

Masters

PhD

DECLARATION OF ORIGINAL WORK

This declaration is made on the 2ih April 2016.

Student's Declaration:

I, Ngo Yi Lei hereby declare that the work entitled genetic stability of white kelampayan

(Neolamarckia cadamba) plantlets derived from callus as explant using ISSR markers is my

original work. I have not copied from any other students' work or from any other sources except where due reference or acknowledgement is made explicitly in the text, nor has any part been written for me by another person.

~.

Date submitted Name of the student (Matric No .)

~O 'I,' L( i (4 ).."'1l )

Supervisor's Declaration:

I Assoc. Prof Dr. Ho Wei Seng hereby certifies that the work entitled genetic stability of white kelampayan (Neolamarckia cadamba) plantlets derived from callus as explant using ISSR markers was prepared by the above named student, and was submitted to the "FACULTY" as a * partiaVfull fulfillment for the conferment of Bachelor of Science with Honours in Resource Biotechnology and the aforementioned work, to the best of my knowledge, is the said student's work.

Date~ ~ J.o-t.{,Received for examination by:

11

I declare that ProjectlThesis is classified as (Please tick (;/)):

oCONFIDENTIAL (Contains confidential information under the Official Secret Act 1972)*

DRESTRICTED (Contains restricted information as specified by the organisation where research was done)*

6ENACCESS

Validation of Projectffhesis

I therefore duly afftrm with free consent and willingly declare that this said ProjectlThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows:

• This ProjectlThesis is the sole legal property ofUniversiti Malaysia Sarawak (UNIMAS).

• The Centre for Academic Information Services has the lawful right to make copies for the purpose ofacademic and research only and not for other purpose.

• The Centre for Academic Information Services has the lawful right to digitalise the content for the Local Content Database.

• The Centre for Academic Information Services has the lawful right to make copies of the ProjectlThesis for academic exchange between Higher Learning Institute.

• No dispute or any claim shall arise from the student itself neither third party on this ProjectlThesis once it becomes the sole property ofUNIMAS.

• This Project/Thesis or any material, data and information related to it shall not be distributed, published or disclosed to any party by the student except with UNIMAS permission.

~. Student signature ____--:----:-___ Supervisor signature:

(Date: ;).'1b/l-O Ib )

Current Address: Fakulti Sa ins dan Tekoologi Sumber, Assoc. Prof. Dr. Ho Wei Seng

Proaramme CoordinatorUniversiti Malaysia Sarawak, DepartmmtofM~ Biology

94300 Kota Sarnarahan, FICUIty ofResource Science and Technology Sarawak. Univeniti Malaysia Snwak

Notes: * If the Projectrrhesis is CONFIDENTIAL or RESTRICTED, please attach together as annexure a letter from the organisation with the period and reasons of confidentiality and restriction.

III

I

Table of Contents

Page

ACKNOWLEDGEMENT

DECLARATION ii

TABLE OF CONTENTS iv

LIST OF ABBREVIATIONS vi

LIST OF FIGURES vii

LIST OF TABLES viii

ABSTRACT 1

CHAPTER I INTRODUCTION 3

CHAPTER II LITERATURE REVIEW 6

2.1 Neolamarckia cadamba 6

2.2 Inter-simple sequence repeat markers 10

2.3 Polymerase Chain Reaction 12

2.4 Plant tissue culture 13

2.5 Previous studies related to genetic stability using ISSR markers 18

CHAPTER III MATERIALS AND METHODS 20

3.1 Plant materials 20

3.2 Shoot regeneration from callus via direct organogenesis 20

3.3 Subculturing of regenerated plantlets 21

3.4 Genomic DNA extraction 23

3.5 Polymerase Chain Reaction amplification 24

3.5.1 PCR condition 24

IV

3.5.2 ISSR-PCR optimization 25

3.5.3 ISSR-PCR analysis 25

3.6 Agarose gel electrophoresis and PCR product visualization 26

3.7 ISSR Data analysis 26

CHAPTER IV RESULTS AND DISCUSSION 29

4.1 Shoot regeneration from callus via direct organogenesis 29

4.2 Subculturing of regenerated plantlets 35

4.3 Genomic DNA extraction 41

4.4 ISSR-PCR optimization 42

4.5 ISSR-PCR analysis 44

4.6 ISSR Data analysis 49

4.6.1 Data scoring 49

4.6.2 Analysis of genetic stability of micropropagated plantlets 51

CHAPTER V CONCLUSIONS 60

REFERENCES 62

APPENDIX A 69

APPENDIX B 70

APPENDIX C 71

APPENDIX D 74

APPENDIX E 77

APPENDIX F 80

APPENDIX G 85

v

List of Abbreviations

AFLP BAP bp cm CTAB CW DAC DNA GA3

I lAA ISSR ISSR-PCR loglO M m mg mm ml mM P PCR POPGENE RAPD rpm SSR TAE UV UPGMA v/v V w/v °c % Jll

Amplified Fragment Length Polymorphism 6-Benzylaminopurine base pair centimeter Cetyl-Trimethyl Ammonium Bromide Coconut water Days after culture deoxyribonucleic acid Gibberellins Shannon's diversity Index indole-3-acetic acid Inter Simple Sequence Repeat Inter Simple Sequence Repeat-Polymerase Chain Reaction log to the power of 10 molar meter milligram minutes milliliter millimolar Polymorphic loci Polymerase Chain Reaction population genetics Randomly Amplified Polymorphic DNA revolutions per minute Simple Sequence Repeat Tris-Acetate-EDTA ultraviolet unweighted pair group mean average volumer per volume volt weight per volume celcius percent microliter

VI

List of Figures

Figures Page

2. 1 Morphological characteristics of Kelampayan 8

2.2 A schematic representation of genome amplification region targeted by [SSR 11

2.3 Three phases of organogenesis 15

2.4 Callus with different morphogenic potential isolated from a single explant 17

3. 1

3.2

3.3

4.1

4.2

Maintenance of Kelampayan cultures on plant tissue culture racks installed with LED plant grow light Cultured shoot tip and nodal segments of Kelampayan on Oamborg's B5 medium with 1.0 mglL BAP

Overview of in vitro regeneration of Kelampayan plantlets and determination of their genetic stability.

Development of organogenesis from cotyledon derived callus of Kelampayan Direct organogenesis from Kelampayan nodal segments

21

22

28

31

38

4.3 Effect of subculture on shoot multiplication for 4 Kelampayan mother plants 39

4.4

4.5

4.6

4.7

Quantitation agarose gel showing highly resolved high molecular bands with

a concentration of 476.8 nglJ.lI by comparing with the Lambda DNA HindIII marker Electrophoresis of PCR products of primer (OTO)6 on 1.5 % agarose gel at 110V

ISSR amplification pattern obtained for 4 different mother plant samples using primer (OTO)6 ISSR amplification pattern obtained for DNA of 4 mother plant and their

long-term micropropagated Kelampayan plantlets corresponding to first and

41

43

44

45

second subculture using primer OT06 4.8 Dendrogram generated using UPOMA analysis showing

between Kelampayan mother tree MPO 1 and their sub-clones 4.9 Dendrogram generated using UPOMA analysis showing

between Kelampayan mother tree MPOI and their sub-clones 4. 10 Dendrogram generated using UPOMA analysis showing

between Kelampayan mother tree MPO 1 and their sub-clones

4.11 Dendrogram generated using UPOMA analysis showing between Kelampayan mother tree MPO 1 and their sub-clones

relationships 53

relationships 54

relationships 55

relationships 56

VB

I I

List of Tables

Table Page

3.l PCR components for 25 Jil reaction mixture 24

3.2 Thermal cycling profile for ISSR-PCR amplification 25

4.l Effect of BAP, N AA and coconut water for shoot regeneration from the 30 leaf cotyledon derived callus of Kelampayan on full-strength Gamborg's B5 after 60 DAC (days after culture)

4.2 Details analysis of regenerated Kelampayan plantlets at each cycle after 40 36 DAC

4.3 Average regeneration percentage, shoot number and days required for one 36 cycle ofeach stock plants after three subculturing cycle.

4.4 Optimization of the ISSR-PCR reaction parameters for (GTG)6primer 42

4.5 Optimum PCR ingredients for 25 III reaction mixture of (GTG)6 primer 42

4.6 DNA fragment size at each locus of ISSR primer. 49

4.7 Details of ISSR analysis for 77 plant samples of Kelampayan 52

Vlll

Genetic stability of white Kelampayan (Neolamarckia cadamba) plantlets derived from

callus as explant using ISSR markers

Ngo Yi Lei

Resource Bioteclmology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Neolamarckia cadamba or generally known as white Kelampayan has been perceived as one of the potential fast growing tree species suitable for forest plantation establishment in Sarawak, Malaysia due to its multipurpose and high commercial values. Hence, micropropagation is a prevalent method to obtain sufficient amount of unifrom plants. In the present study, in vitro propagation of Kelampayan was attempted through multiple shoot regeneration from both callus and nodal segments cultured on Gamborg's B5 medium supplemented with various combinations of BAP, NAA and CWo 1.0 mg/L BAP was found to be the most effective for shoot regeneration from callus explants (9.09%) and produced multiple shoots (1.6) per explant. Repeated subculturing of newly fonned nodal parts after each harvest up to third passage, yielded higher number of shoots (1.97) per explant was obtained. Inter Simple Sequence Repeats (ISSR) marker was used to detennine the genetic stability of micropropagated and stock plants of Kelampayan. ISSR primers, (GTG)6 had generated an average of 6.5 clear bands, of which 36.11 % of the loci were polymorphic among 77 Kelampayan samples. All ISSR profiles from micropropagated plants were closely monomorphic and similar to the mother plants, while low variation was induced in the second subculture cycle. The genetic diversity, estimated by Shannon's index, was 0.1862, revealing a low level of genetic variation among them. The results indicated that genetic instability was obtained among the micropropagated Kelampayan plantlets when subcultured to second cycle.

Keywords: genetic stability, Neolamarckia cadamba, Inter simple sequence repeats (ISSR), micropropagated plantlets, callus.

1

Genetic stability of white Kelampayan (Neolamarckia cadamba) plantlets derived from

callus as explant using ISSR markers

Ngo Yi Lei

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRAK

Neolamarckia cadamba atau dikenali sebagai Kelampayan telah diiktiraf sebagai salah salu daripada spesies pokok yang membesar dengan cepat dan sesuai unluk penubuhan ladang hulan di Sarawak, Malaysia disebabkan oleh ia mempunyai pelbagaifungsi dan nilai komersial yang linggi. Oleh itu, mikropropagasi adalah salu kaedah yang lazim untuk mendapatkan pengeluaran komersil yang berskala besar. Dalam kajian ini, in vitro mikropropagasi untuk Kelampayan telah dihasilkan daripada kedua-dua kalus dan segmen nod dengan menggunakan medium Gamborg B5 dicampur dengan pelbagai hormon kombinasi iaitu BAP, NAA dan CW 1.0 mg / L BAP lelah didapali paling berkesan unluk perlumbuhan semula daripada pucuk (9.09%) dan memperbanyakkan pucuk (1.6) bagi seliap eksplan. Kullur pucuk yang berulang sehingga kali ketiga lelah meningkalkan nombor pucuk (1.97) bagi seliap eksplan. Inler Simple Sequence Repeals (ISSR) penanda lelah digunakan untuk menganalisis kestabilan genelik anak pokokdan tumbuhan kawalan Kelampayan. ISSR primers, (GTG)6 telah menghasilkan purata 6.5 band DNA yang jelas, di mana 36.11% daripada lokus adalah polimorfik antara 77 Kelampayan sampel. Semua ISSR profil telah menunjukkan corak yang dekat monomorjik dan serupa, manakala variasi genetik yang rendah telah muncul pada tahap multiplikasi yang kedua. Kepelbagaian genetik yang dianggarkan oleh Shannon indeks, adalah 0.1862, ini telah mendedahkan variasi genetik adalah pada tahap yang agak rendah dalam kalangan anak pokok dan tumbuhan kawalan Kelampayan. Keputusan kajian ini telah menunjukkan bahawa kepelbagaian genetik adalah rendah antara anak pokok Kelampayan yang dilahirkan dengan teknik mikropropagasi.

Kata kunci: kestabilan genetik, Neolamarckia cadamba, Inter Simple Sequence Repeals (ISSR)

penanda, kultur tisu, kalus.

2

CHAPTER I

INTRODUCTION

Neolamarkcia cadamba which is commonly known as white Kelampayan, belongs to

Rubiaceae family. It is an endemic wood species in Sulawesi, Celebes and the Moluccas

islands (Cahyono et al., 2015). It grows naturally in a large range of area such as Philippines,

Indonesia, India, Vietnam and Australia (Krisnawati et ai., 2011). Kelampayan has been used

for multiple purpose ranging from wood carvings to light construction materials and furniture.

Recently, Kelampayan has been widely promoted as one of the potential fast growing tree

species suitable for forest plantation establishment in Sarawak (Tiong et al., 2014). They are

seen as ideal investment choices for timber plantations or community forestry.

However, vegetative propagation of Kelampayan by traditional methods is not successful

due to high sensitivity of nodal segments against mechanical injury, poor rooting, easily

susceptible to pests in the early growth stage especially nematodes and disease known as

'sudden death of cadamba' (Gupta & Dalal, 1973; Gibson & Nylund, 1976). Besides, seed

propagation of Kelampayan is limited due to lack of viable seed production thus leading to

low germination rate (Bose & Chaudhary, 1991). Therefore, advanced biotechnology

approaches including micropropagation are required for large scale production of this species

since the distribution of this valuable tree has becomes very limited.

Micropropagation of woody plants constitutes a noteworthy achievement in the

commercial use of in vitro cultures (Leva et al., 2012). The ability to maintain the genetic

stability of regenerated plants in relation to mother plant has become a prominent aspect to be

considered during plant tissue culture. Plant tissue culture involves cultivating plants outside

3

the natural growth environment and requirements by optimizing various kind of physical,

chemical and environment factors for growth (Bairu et al., 2011). However, the application of

this alternative technique still restrained by some developmental and physiological problems

although improved and meticulous efforts have been made in the plant tissue culture (Bairul et

al., 2011).

As mentioned by Singh (2005), the chance of genetic instability and polymorphism may

be emerged within a species due to several factors such as somaclonal variation, mutation and

recombinant of chromosome during plant tissue culture. The species may not reflect the true

genetic composition among individuals although there are no significant visual differences in

morphological appearance. Due to similar morphology with the interested trait of plants,

cuJ tivation of undesired trait may be happened (Singh, 2005). These genetic variations among

the micropropagated plants should be identified and monitored periodically in order to

produce true-to-type plants with the desired genotype (Rani & Raina, 2000).

One of the early methodologies that can be done is by screening the genetic variability by

using molecular DNA markers since the morphological markers are unable to reveal the

genetic diversity and relationships among the regenerated plants (Karp et al., 1997). Therefore,

molecular markers that have a few protrude characteristic over the conventional phenotypic

markers are widely used to determine and characterize the generic variation at molecular DNA

level. They are often used to point out the genetic variability of plant genome since they are

detectable and unaffected in each development stages (Karp et al., 1997).

According to Verstrepen et al. (2005), ISSR is generally known as a genome region

between the microsatellite loci. This simple and rapid PeR-based technique permits

4

"usat Khidmat Maklumat Akademik UNJVERSITl MALAYSIA SARAWAJ(

characterization of polymorph isms in inter-microsatellite loci by targeting the simple sequence

repeats that are abundant in the eukaryotic genome (Nagaoka & Ogihara, 1997; Zhou et al.,

2008). This technique involved a designated primer from either dinucleotide or trinucleotide

simple repeats sequence to produce a large number of DNA fragments per primer. The stable,

reliable and reproducibility features of ISSR markers allowed them to be widely used for

phylogenetic studies, population genetics, gene tagging, DNA fingerprinting. Besides, they are

also known as an effective tool to study the genetic diversity and detect the genetic similarities

within species levels (Zhou et al., 2008).

Understanding the genetic stability of regenerated woody speCIes is prominent for

conservation management and genetic advancement for forest plantation establishment. This

can be achieved at the seedling stage thus resulting in a better economic return due to lower

time and cost are needed for the production of good quality planting materials. Hence, the

present study was aimed to decipher the genetic stability of micropropagated plantlets derived

from callus as explant by using an efficient molecular marker, ISSR marker.

The objectives of the research are as following:

i) To regenerate the Kelampayan plantlets via organogenesis using callus as explant.

ii) To determine the genetic stability of micropropagated Kelampayan plantlet using

ISSR marker.

5

CHAPTER II

LITERATURE REVIEW

2.1 Neolamarckia cadamba

Neolamarckia cadamba is a timber species which endemic in Sulawesi, Celebes and Moluccas

islands where it belongs to Rubiaceae family (Cahyono et at., 2015). This fast growing tree

species has been developed aggressively in China, Thailand, India, Indonesia, Vietnam,

Myanmar, Australia, Filipina, Sri Lanka, Papua New Guinea and Malaysia especially Sarawak

(Krisnawati et al., 2011). Neolamarckia cadamba is also known as Anthocephalus chinensis,

Nauclea cadamba and Anthocephalus macrophyllus (Krisnawati et al., 2011).

2.1.1 Taxonomy

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Rubiales

Family: Rubiaceae

Genus: Neolamarckia

Species: Neotamarckia cadamba (Roxb.) Bosser

Source: GBIF Backbone Taxonomy (2013)

6

2.1.2 Morphological characteristics

Kelampayan is a huge tree with a wide umbrella-shaped crown and long straight bole

(Krisnawati et al., 2011). According to Soerianegara and Lemmens (1993), this wood species

has straight trunk with clear round, tube shaped boles and small diameter of sdf-pruning

branches. This timber possesses smooth, grey colour bark which distinct from the red

Kelampayan. The leaves are glossy green and oval to elliptical shape. The fruit occur in small

and plump capsules which packed firmly together to form a fleshy yellow-orange

infrustescence containing more or less 8000 seeds when mature (Na'iem et al., 2014). The

trees may achieve a height of 45 m with a stem diameter of 100-160 cm with a small buttress

of up to 2 m. (Soerianegara & Lemmens, 1993). The height and diameter increments by 3 m

and 7 cm individually per year (Krisnawati et al., 2011). It can be harvested in 5 years when

the widths reach 30-40 cm (Mansur & Tuheteru, 2010). Unlike other fast growing tree species

which have been suggested for forest plantation and community forestry, these species are

easier to adapt to unfavorable conditions and resistant to pests (lrawan & Purwanto, 2014).

7

Figure 2.1 Morphological characteristics of Kelampayan. (A) Grey and smooth stem bark, (B) glossy green leaves which usually elliptical in the length of 15-50 cm and width of 8-25 cm and (C) mature fruit of Kelampayan (Adapted source from Krisnawati et al., 2011).

8

I

!

2.1.3 Usage

Kelampayan has several advantages over other types of tree. High adaptability and economic

profitability of these fast-growing species are greatly favorable by most of the local

communities (Irawan & Purwanto, 2014). It has been selected as one of the important

plantation tree species suitable for forest rehabilitation program in Malaysia, especially

Sarawak (Ho et al., 2014).

Kelampayan is classified as light hardwood that produces timber for pulp and light

construction (Soerianegara & Lemmens, 1993). With fine and smooth texture, it is often used

as the raw material for plywood, furniture and some accessories. Moreover, the tree can be

grown as an ornamental and shade along the roadsides (Krisnawati et al., 2011). As stated by

Jeker (2000), the leaves, roots, barks and fruits are widely used in medical applications since

various parts of the plant have many bioactivities such as being hypoglycemic, hypolipidemic,

antioxidant, antibacterial and antimicrobial. Additionally, it also possibly be used as one of the

renewable resource of raw materials for bioenergy production such as cellulosic biofuels in the

near future (Jeker, 2000). Therefore, this tropical timber tree is considered as a good choice

for research activities. Due to its outstanding characteristic, they are seen as ideal investment

choices for timber plantations or community forestry.

However, limited propagation and cultivation information has constrained the success of

farmers' Kelampayan seedling production efforts. To further improve the quality of this

timber species, it is necessary to examine this wood quality periodically to improve its

utilization (Irawan & Purwanto, 2014).

9

-

2.2 Inter-simple sequence repeat markers

To date, advanced molecular biology have introduced a series of DNA markers which have

been proved valuable in the studies of crop breeding, especially genetic diversity and gene

mapping (Reddy et a/., 2002; Vijayan, 2005). Several molecular markers have been used to

study the genetic variation in micropropagated plants such as amplified fragment length

polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), simple sequence

repeat (SSR) and inter-simple sequence repeat (ISSR) markers (Leva et a/., 2012).

However, Gupta and Varshney (2000) as well as Staub et a/. (1996) stated that the most

commonly used DNA marker such as AFLP, RAPD and SSR marker are still restrained by

several factors. For example, high expense of AFLP, low reproducibility of RAPD and the

need of previous knowledge about the flanking sequences for SSR. Hence, the polymerase

chain reaction based DNA marker, ISSR has been developed to overcome most of these

limitations (Meyer et a/., 1993; Wu et a/., 1994; Zietkiewicz et aI., 1994; Gupta & Varshney,

2000).

ISSR technique relies on the amplification of DNA fragments closely between two

identical and inversely oriented microsatellite repeat regions (Reddy et a/., 2002). This method

uses a single primer which constituted of a short peR reaction microsatellites sequence,

usually in the length of 16 to 25 base pair to amplify primarily different sizes of inter-SSR

sequences by targeting multiple genomic loci . The primers can be either dinucleotide,

trinucleotide tetranucleotide or pentanucleotide (Reddy et a/., 2002). According to Meyer et

a/. (1993), Wu et at. (1994), Zietkiewicz et al. (1994), Gupta and Varshney (2000), the

primers used can be designed as either unanchored or anchored at 3' or 5' end with 1 to 4

degenerate bases that extended into the flanking sequences. ISSR becomes highly reproducible

10

I

due to utilization of long primers (16-25 bp). The longer primers allowed high annealing

temperature usually between 45-60 °C for better fixation of primers.

s

PrirrerI~G\~CICICICICICICICIC;;;CI

'~______ ArrpIified proctJCt

Figure 2.2 A schematic representation ofgenome amplification region targeted by ISSR. (Adapted source from Reddy et aI., 2002).

2.2.1 Applications of ISSR markers

ISSR markers have many applications In plant genome analysis attributed to the above

mentioned advantages. For example, studies have shown that ISSR markers are widely used to

characterize the elite gennplasm and identify the closely identical of cultivars (Korbin el al.,

2002; Reddy et al., 2002; Arolu et al., 2012), to estimate the degree of genetic diversity at

inter and intra-specific level in variety crop species such as wheat (Nagaoka & Ogihara, 1997),

sweet potato (Huang & Sun, 2000) and rice (Joshi et aI., 2000). In addition, ISSR is a useful

marker system which has high potential to detect somaclonal variations in micropropagated

plants, to screen the distribution of microsatellites in the plant genome (Bolibok et al., 2006)

and to detennine genetic identity and genetic stability in many propagated plants such as Musa

spp. (Lu el aI., 2011), Garcinia spp. (Mohan el al., 2012) and Stevia rebaudiana Bertoni

(Soliman el al., 2014).

11

2.3 Polymerase Chain Reaction

peR is an advance technique developed by Kary Mullis in 1983 (Mullis, 1993). It is a

powerful in vitro method used for amplification of a particular segment of a nucleic acid

(Stephenson, 2012). A DNA fragment can be replicated by over a million-fold in a relatively

short time. DNA polymerase is used in peR method to synthesize a new DNA strand by

adding a complementary nucleotide onto a preexisting 3'-OH end of the targeted template

strand (Stephenson, 2012). A short, single-stranded DNA will act as primer anneals to the

complementary sequences on the opposite strands of the template DNA by flanking the target

region. They serve as initiation point for the addition of incoming nucleotide bases by DNA

Polymerase (Saiki, 1990).

Each peR cycle involved three main steps: denaturation, annealing and elongation (Saiki,

1990). Two oligonucleotide primers will flank the interested DNA fragment to amplify it.

DNA amplification begins with repeated cycles of heat denaturation of the DNA strand,

annealing of the primers to the complementary sequences and elongation of the annealed

primers with the help of DNA polymerase. At the end of the peR reaction, the specific

sequence will be multiplied in billions of copies since each successive amplification cycles

double the amount of the target DNA synthesized in the previous cycle (Saiki, 1990).

According to Stephenson (2012), peR has found popularity in molecular biology and

served for a wide range of applications including gene cloning, DNA-based phylogeny, DNA

fmgerprinting, and DNA sequencing due to its great sensitivity. In this study, a simple and

rapid ISSR-PCR technique was used to determine the genetic stability of micropropagated

Kelampayan plantlets. Amplifications of ISSR marker are multi locus and polymorphic since

the microsatellites are randomly spread along the genome (Zietkiewicz et at., 1994). The long

12

primers (16-25 bp) are resulting in higher stringency (Reddy et al., 2002). The amplified

products are usually 200-2000 bp long and amenable to detection by both agarose or

polyacrylamide gel electrophoresis systems.

2.4 Plant tissue culture

2.4.1 Conventional vegetative propagation

According to Neumann et al. (2009), conventional propagation is the usual method of plant

multiplication using plant tissues such as seeds and stems under outdoor conditions. Although

traditional propagation done by cutting, pulling and division of seeds have several benefits as

a means of propagation (Neumann et al., 2009), but George et al. (2008) stated that the plants

produced from seeds may not provide a practical way of making new field plantings. Besides,

it is often affected by some factors, such as seasonal constraints and lack of viable seeds

production (Smith, 2013).

Therefore, the alternative plant tissue culture technique has overcome the conventional

method and enonnously used in many applications such as molecular breeding, gennplasm

preservation and mass production of genuine planting materials through micropropagation

method (Smith, 2013).

2.4.2 Micropropagation

Plant tissue culture or technically known as micropropagation is the science of maintaining

and growing plant cells, tissues or organs extracted from the donor plant on artificial media in

aseptic or in vitro culture (Raven et al., 1999). Gottlieb Haberlandt is the one who proposed

this theoretical basis for plant tissue culture in 1902 which is later known as the father of plant

tissue culture (Smith, 2013).

13

.-----------~------------------------~---------

Through micropropagation, plant can be regenerated by manipulating only a small amount

of sterile plant material ranging from single cells to stem segments under conditions favorable

to the plant. It has been proven to be the most reliable and cost-effective approach for large

scale propagation of identical offspring in both herbaceous and woody perennial species (Leva

et 01.,2012; Smith, 2013). Study has been proved that micropropagation has the potential to

increase the multiplication rate of elite genotypes and to produce improved cultivars (Leva el

01.,2012).

On the other hand, Bouiamrine el 01. (2012) found that one major drawback of tissue

cultured-derived plantlets is somaclonal variation and polymorphism may arise among the

micropropagated plantlets which might affect the quality of regenerated plantlets considerably.

Therefore, it is necessary to check the genetic stability of micropropagated plantlets

thoroughly before grown under field conditions.

2.4.3 Direct and indirect organogenesis

Regeneration of plants by micropropagation can be achieved from organ primordia that

existing in shoot tips and axillary bud (Mathur & Koncz, 1998). Organogenesis involved in

vitro fonnation and development of organs such as shoots and roots either directly from an

explants or indirectly via callus differentiation through wounded parenchyma. According to

Christianson and Warnick (1985), in vitro propagation through organogenesis usually involves

three major stages which are dedifferentiation, induction and differentiation.

14