ESTABLISHMENT OF AXENIC EXPLANTS AND CALLUS CULTURE OF CLINACANTHUS NUTANS

(RUMPUT BELALAI GAJAH)

Ng Li Ying

QK 769 NS76 Bachelor of Science with Honours 2013 (Plant Resource Science and Management)

2013

I

----~-~

Pus at Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

Establishment of Axenic explants and Callus Culture of

Clinacanthus nutans (rumput belalai gajah)

F'.KHIDMAT MAKLUMAT AKADEMIK

11111 1IIIIfiiiilili III III 1000246800

NG LI YING (27241)

This report is submitted in partial fulfilment of the requirement for the degree of Bachelor of Science with Honours in Plant Resource Science and Management

Plant Resource Science and Management

FACULTY OF RESOURCE SCIENCE & TECHNOLOGY UNIVERSITY MALAYSIA SARA WAK

94300 KOTA SAMARAHAN

DECLARATION

I declare that this thesis entitle "Establishment of Axenic explants and Callus Culture

of Clinacanthus nutans (rumput belalai gajah)" is the result of my own research

except as cited in the references. The thesis has not been accepted for any degree and

is not concurrently submitted in candidature of any other degree.

Signature: ~ Name: lin 11 Y/Nh

Date: / . 1-, dO/~

ACKNOWLEDGEMENT

Firstly, I would like to dedicate my sincere thanks to my supervisor, Prof. Dr. Hamsawi

Sani for giving me the opportunity to work on this project and his intellectual support

throughout this study. His generous, sincere guidance and advice enable me to complete

this project on time.

Nevertheless, my appreciation is extended to my senior and master students from Tissue

Culture Laboratory, FRST, UNIMAS: Fong Yin Mei, Chen Mei Yin and Cherrie Lim for

their invaluable time and advice to support me in completing my laboratory work in these

two semester. Our sweet memories always on my mind forever.

Last but not least, my warmest gratitude to my beloved family and my soulmate Chia

Choon Hiung. Their continuous support, care, love and understanding have built me to a

better person in my campus life. Thanks for being with me always.

Pusat Khidmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS

TABLE OF CONTENTS ii

APPENDICES iv

LIST OF ABBREVIATION v

LIST OF FIGURES vi

LIST OF TABLES viii

ABSTRACT/ABSTRAK ix

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 4

2.1 The plants 4

2.2 Botanical description 4

2.3 The economic importance of Clinacanthus nutans

3.3.1 Medicinal uses of Clinacanthus spp. . . . . . . . . . . . . . . . . . . . . . . .. 8

2.4 Micropropagation of medicinal plants

2.4.1 Selection of explants ........................................... 11

2.4.2 Establishment of axenic explants .......................... ... 12

2.4.3 Plant growth regulator and media............................ 14

2.5 Callus culture............................................................... 16

2.6 Acclimatization.. ............................................ .... . .......... 17

ii

CHAPTER 3 MATERIALS AND METHODS

3.1 Development of an effective surface sterilization regime for the

explants ................................................................. . ... 18

3.2 Establishment of axenic explant .... .................................... 20

3.3 Induction of callus formation through indirect organogenesis. ..... 21

3.4 Shoot regeneration from callus tissue ............. .. ................... 23

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Establishment of axenic explant

4.1 .1 Effect of different sodium hypochlorite (Clorox®)

concentration ................................................... 25

4.1.2 Effect of Sodium hyochlorite (Clorox®)

concentrations with benomyl ............................ :.... 32

4.2 Induction of callus fonnation through indirect organogenesis

4.2.1 Induction of callus fonnation using 2,4-D..... . ............. 34

4.2.2 Induction of callus fonnation using Picloram ............... 35

4.2.3 Induction of callus fonnation using BAP ................. ... 36

4.3 Shoot regeneration from callus tissue

4.3.1 Shoot regeneration by BAP

and in combination with IBA ........ .. ........ . ...... . ....... 41

CHAPTERS CONCLUSION AND RECOMMENDATION 43

REFERENCES

iii

APPENDICES

Page

Appendix A Murashige and Skoog (1962) medium fonnulation 51

Appendix B Number of axenic, contaminated and damaged leaf 52 explants in half MS soft agar after surface sterilization in 10% of Clorox® concentration at day 14 after initiation of culture

Appendix C Number of axenic, contaminated and damaged internode 53 explants in half MS soft agar after surface sterilization in 10% of Clorox® concentration at day 14 after initiation of culture

Appendix D Number of axenic, contaminated and damaged shoot tip 54 explants in half MS soft agar after surface sterilization in 10% of Clorox® concentration at day 14 after initiation of culture

Appendix E Number of axenic, contaminated and damaged leaf explants 55 in half MS soft 'agar after surface sterilization in 15% of Clorox® concentration at day 14 after initiation of culture

Appendix F Number of axenic, contaminated and damaged internode 56 explants in half MS soft agar after surface sterilization in 15% of Clorox® concentration at day 14 after initiation of culture

Appendix G Number of axenic, contaminated and damaged shoot tip 57 explants in half MS soft agar after surface sterilization in 15% of Clorox® concentration at day 14 after initiation of culture

iv

BAP

Benomyl

B5

rnA MS

NAA

Picloram

PPMTM

WPM

2,4- D

LIST OF ABBREVATIONS

6-benzyl amino purine

Methyl [l-[(butylamino )carbonyl]-l H-benzimidazol-2-yl]carbamate

Gamborg B5 medium

Indole-3-butyric acid

Murashige and Skoog medium

Naphthalene acetic acid

4 Amino-2,5,6-trichloropicolinic acid

Plant preservative mixture

Wood Plant Medium

2,4- dichlorophenoxyacetic acids

v

LIST OF FIGURES

Figure No. Page

2.1 The Snake Grass (Clinacanthus nutans) leaves 6

2.2 The Snake Grass (Clinacanthus nutans) seedling 7

4.1 Percentage of axenic explants with response to 10% Clorox® 26 concentration in 10 minutes after 14 days

4.2 (a) Axenic leaf explants after culturing for 14 days in halfMS 27 medium

(b) Fluffy and whitish callus formed at the cut edge of internode explants after culturing for 14 days in half MS medium

(c) Axenic shoot tip explants after culturing for 14 days in half MS medium

(d) Damaged leafexplants after culturing for 14 days in halfMS medium

4.3 Percentage of axenic explants with response to 15% Clorox® 29 concentration in 10 minutes after 14 days

4.4 (a) Leaf explants contaminated with pinkish bacteria 31 (b) Internode explants contaminated with whitish fungi (c) Shoot tip explants contaminated with whitish fungi and

bacteria

4.5 Comparison between percentage of axenic explants with response 32 to 10% Clorox® concentration with and without the addition of 0.1 % of Benomyl fungicide in 10 minutes after 14 days

4.6 (a) Internode without any sign of regeneration in MS medium 35 supplemented with different concentration of2,4-D

(b) Shrunk and yellowish brown callus formed at the cut edges of internode explant

4.7 Percentage of internode explants producing callus with response 37 to different BAP concentration after 2 months of cultures

vi

4.8 (a) A light callusing fonned at the cut edges of internode explant after one week of culturing in MS medium supplemented with 0.5 mg/l BAP

39

(b) A light callusing fonned at the cut edges of internode explant after one week of culturing in MS medium supplemented with 1.0 mg/l BAP

(c) Creamy white and fluffy callus fonned at the cut edges of internode explant after four weeks of culturing in MS medium supplemented with 1.5 mg/l BAP

(d) Creamy white and fluffy callus fonned at the cut edges of internode explant after four week of culturing in MS medium supplemented with 2.0 mg/l BAP

4.9 (a) Yellowish brown callus fonned at the cut edges of internode explant after 2 months of culturing in MS medium supplemented with 0.5 mgll BAP

40

(b ) Yellowish brown callus formed at the cut edges of internode explant after 2 months of culturing in MS medium supplemented with 1.0 mg/l BAP

(c) Creamy white and fluffy callus fonned at the cut edges of internode explant after 2 months of culturing in MS medium supplemented with 2.0 mg/l BAP

(d) Creamy white and fluffy callus fonned at the cut edges of internode explant after 2 months of culturing in MS medium supplemented with 2.0 mg/l BAP

4.10 (a) No any sign of shoot regenerated from the callus derived from explant after two weeks of culturing in MS medium supplemented with 2.0 mg/l BAP and 1.0mg/l IBA

42

(b) Died callus without any shoot induced after four weeks of culturing in MS medium supplemented with 2.0 mg/l BAP and 1.Omg/l IBA

vii

LIST OF TABLES

Tables No.

4.1

4.2

4.3

Page

Chi-squared comparison between different types ofexplants

with response to 10% Clorox® concentration

28

Chi-squared comparison between different types of explants

with response to 15% Clorox® concentration

30

Chi-squared comparison between different BAP on

explants producing callus

38

viii

Establishment of Axenic explants and Callus Culture of

Clinacanthus nutans (rumput belalai gajah)

Ng Li Ying

Plant Resource and Management Programme Faculty of Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Clinacanthus nulans (Burm.f.) Lindau (snake grass) of family Acanthaceae is known as a herb, shrub or ornamental grass used for medicinal purposes. Clinacanthus nlltans is a famous tropical herb and have been used widely in Southeast Asia especially in Sabah, Malaysia as an anti- inflammatory drug for the treatment of wound, disinfection and cancer. The purpose of this study is to establish an effective surface sterilization protocol and to develop an in vitro propagation of Clinacanthlls nutans using plant hormone. The study started with the establishment of axenic explants culture. Shoot tips, internode and leaves from grown seedling of snake grass were as the ex plants. Resulted showed most of the explants were found damaged by treatment of 15% 'Clorox®' . The protocol below would result in 93% contamination-free internodes explant in culture. The protocol involves dipping explants first in 70% ethanol for 30 seconds followed by agitating the explants in 10% 'Clorox®' for 10 minutes, rinsed at least three times with distilled water and then dipped in 0.1 % of benomyl fungicide for one hour, rinsed for three times before culture in half MS medium. Study on callus inducljon was carried out quite successful. Result showed that BAP alone at 2.0 mg/I is effective in the induction of callus formation. It was found that usingthe plant growth regulators such as 2,4-D and picloram could not be used for callus induction of this plant species. The study of BAP and IBA or NAA effect on shoot induction at different concentration was conducted but no any shoot produced in about six weeks after culturing the callus induced explant in the shoot induction medium. Such 'protocol needs to be further refined to imporove the efficiency and modified for other types of plant growth regulator.

Key words: Clinacanthus nutans (Burm.f.) Lindau, callus culture, surface sterilization, shoot induction.

ABSTRAK

Clinacanthus nulans (Burm.f) Lindau (rumput ular) merupakan species herba, pokok renek alau hiasan rumpul yang digunakan untuk tujuan perubatan dan dik/asifikasikan da/am keluarga Acanlhaceae. C/inacanthus nlltans ada/ah herba tropika yang terkena/ dan te/ah digunakan secara me/uas di Asia Tenggara lerulamanya di Sabah, Malaysia sebagai ubat anti-radang untuk rawalan luka, pembasmian

kuman dan kanser. Tujuan kajian ini ada/ah untuk mewujudkan prolokol penslerilan permukaan berkesan dan unluk membangunkan in vilro penyebaran Clinacanlhus nutans menggunakan hormon tumbuhan. Kajian ini bermula deJlgan penubuhan budaya eksplanl axenic. Tips pucuk, ruas daun dan dari anak benih dilanam rumpul adalah seperli ular yang eksp/ant. Akibal menunjukkan kebanyakan eksp/anl didapali rosak o/eh rawalan sebanyak 15% 'Clorox ®. Prolokol di bawah akan menghasilkan 93% pencemaran bebas internodes eksp/an da/am budaya. Prolokol ini melibatkan mencelup eksp/anl perlama dalam 70% etano/ selama 30 saal diikuli o/eh agitating yang eksplant dalam 10% 'Clorox ®' selama 10 minit, dicuci sekurang­kurangnya liga ka/i dengan air suling dan kemudian dice/up da/am 0.1% daripada racun ku/al Benomyl selama salujam, dicuci se/ama tiga kali sebe/um budaya di separuh medium MS. Kajian induksi ka/us telah dijalankan agak berjaya. Kepulusan menunjukkan bahawa BAP sahaja pada 2.0 mg / I ada/ah berkesan

dalam induksi pembenlukan kalus . la telah mendapali bahawa bahagian atas perkakas dahl/lu pengawal selia perlumbuhan tumbuhan seperti 2,4-D dan pic/oram tidak bo/eh digunakan untuk induksi kalus ini spesis lumbuhan. Kajian BAP dan IBA atau kesan NAA pada induksi menembak pada kepekatan yang berbeza lelah dijalankan telapi tiada apa-apa penalti yang dihasilkan kira-kira enam minggu selepas pengkulluran eksplan kalus disebabkan dalam medium induksi menembak. Protokol itu perlu diperhalusi lag; unluk imporove kecekapan dan diubahsuai untuk lain-lain jenis pengawal selia pertumbuhan lumbuhan.

Kala kunci: Clinacanlhus nulans (Burm.f) Lindau, in vitro ku/lur, permukaan penslerilan, induksi kalus.

CHAPTER 1

INTRODUCTION

Natural organisms, especially plants have been used for traditional medicine or treatment of

various diseases since thousand of years (Rao et ai, 2004). The World Health Organization

(WHO) reported that herbs and various forms of traditional medicine for disease treatment are

currently used by about three quarters of the world's population. Nowadays, plants was found

to be a main sources of highly effective conventional medicine that could be used for the

treatment of various types of cancer (Cragg & Newman, 2004). Cragg et ai. (2005) reported

that over 60% ofcurrently used anti-cancer agent are produced from natural resources.

The discovery of new compound from plants in the era of globalization. has been developed

and stimulating interest in clinical studies. Many medicinal plants that reported to have

therapeutic potential was investigated for plant-derived bioactive compound. For example,

Jatropha curcas Linn, a multipurposes plants was found to have antioxidant activity and anti­

inflammatory agent with the extraction of leaf, root and latex of the plants (Oskoueian et ai.,

2010). Curry leaf tree (Murraya koenigii), a medicinal plant traditionally used in many

important uses in traditional system of medicine in Eastern Asia (Ningappa & Srinivas, 2008).

It had been proven to have antimicrobial, anti diarrhea, antiulcer activity by verifying the

efficacy of the plant through scientific biological screening (Bonde et ai., 20 II).

Acanthaceae, the Acanthus family consists of 250 genera and about 2500 species is a taxon of

dicotyledonous flowering plants. This family mostly are tropical herbs, shrub and some of

them are epiphytes. At family level, they can be easily recognized morphologically by their

simple, opposite, decussate, entire leaves, zygomorph flowers and their superior ovary. C.

nutans is a well known medicinal herb of Acanthaceae that have long been traditionally used

in the treatment of insect, snake bites and herpes infection. Its common is 'Sabah Snake Grass'

in Sabah, Malaysia, Drooping Clinacanthus in English, 'rumput belalai gajah' in Malay and

'You Dun Cao' in Mandarin, 'Pyaya yo' in Thailand. It is widely grown in tropical Asia and

has been used as important medicinal herb in China, Thailand and Malaysia (Yuann et aI.,

2012).

C. nutans is a medicinal plants in Thailand with its alcoholic fresh leaves which has been used

externally for the treatment of skin infection, insects and snake bites, herpes simplex virus

(HSV), and vavicella- zostervirus (VSZ) lesion (Sookmai et aI., 2011). According to

Thawaranantha et al. (1992), c. nutans leaves extract was observed to have an inhibitory

effect on vavicella- zostervirus (VSZ) by using both the DNA hybridization technique and

plaque reduction assay. Sakdarat et ai. (2009) identified three chlorophyll derivatives or pure

compound which isolated from. the chloroform extract of C. nutans leaves. These 3

compounds were namely as 132 -hydroxy-(l32 -R)-phaeophytin b, 132 -hydroxy-(132 -S)­

phaeophytin a and 132 -hydroxy-( 132 -R)-phaeophytin a.

2

Clinacanthus nutans was found to have other properties. For instance, the extract of C. nutans

was purified as medicinal cream to treat genital herpes suspected patients (Jayavasu et ai.,

1992). Recently, C. nutans or Sabah snake grass has been utilized by locals in Malaysia as a

cure for cancers. However, its effectiveness has not yet been proven scientifically and it could

be taken as an alternative treatment for cancer patients. The method of consuming Sabah

snake grass was introduced by blending the leaves of the grass and drink as normal juice. It

has created a fast growing market for herbal based products such as Sabah snake grass tonic

nowadays.

Recently, the demand for medicinal plant is increasing and it may reduce the sustainable

supply of the medicinal herbs in the future. Avoid endangered species, over-collecting,

unsustainable planting practices, pollution are important in the sustainable management of

valuable meclicinal plants. Therefore, plant tissue culture techniques or in vitro culture

method could be an alternative technique for the production of compounds that have medicinal

properties. This study is proposed to examine the possibility of mass propagation of this

species using in vitro culture technique for the production of bioactive compound. Hence, the

specific objective of this study are:

1. To establish an effective surface sterilization protocol for producing C. nutans

via micropropagation technique.

2. To develop an in vitro propagation of C. nutans using plant hormone.

3

CHAPTER 2

LITERATURE REVIEW

2.1 The plant

Acanthus is a membemof the Acanthaceae family which comprises of 250 genera and about

2500 species and it is also a taxon of dicotyledonous flowering plants. This family mostly are

tropical herbs shrub and some of them are epiphytes. At family level, they can be easily

recognized morphologically by their simple, opposite, decussate, entire leaves, zygomorph

flowers and their superior ovary. C. nutans (Burrn.f.) Lindau has various common names. It is

known as Dandang gendis (Indonesia), Belalai gajah (Peninsular Malaysia), Phaya yor

(Thailand), You Dun Cao (China), Sha Ba She Cao (Sabah, Malaysia). Th~ common English

name is Snake plant or snake grass.

Snake grass has various synonyms, they are Clinacanthus angustus, Clinacanthus burmanni,

Clinacanthus pirei, Clinacanthus siamensis (Nees, 1847). These four species of Clinacanthus

are used traditionally as herbs in Southeast Asia and China.

2.2 Botanical description

The taxonomic classification and nomenclature of Clinacanthus nutans is as follow:

Kingdom: Plantae

Phylum: Magnoliophyta

4

t Khidmat Maklumat Akademik Pusa MALAYSIA SARAWAKUNlVERSm

Class: Magnoliopsida

Subcla ss: Asteridae

Order: Lamiales

Family: Acanthaceae

Genus: Clinacanthus

Species: nutans- Lindau

Scientific name: Clinacanthus nutans Lindau

Clinacanthus nulans is a shrub or perennial herbs that can grow up to 1 m in height with

pubescent branches. The stem is torete, striate and glabrescent. The leaves are simple, opposite,

narrowly elliptic-oblong or lanceolate (2.5- 13 cm long x 0.5- 1.5 cm wide) (Panyakom, 2006).

The leaves have apex acute or acuminate and exsculptate; dentate or subentine margins. Both

surfaces of leaves are pubescent when young then glabrescent. The leaf base are cuncate,

obtuse rounded or truncate; often oblique. Petiole 3-15mm long (Hu & Daniel, 2011).

5

Figure 2.1 The Snake Grass (Clinacanthus nutans) leaves

The flowers are sordidly yellow or greenish yellow as recorded by Panyakom (2006). They are

in dense cymes at the top of branches and branchlets; always covered with 5-alpha cymules.

The calyx of flower about 1 cm long with grandular-pubescent. Corolla dull red with green

base, about 3- 4.2 cm. The stamen are exserted from the throat of corolla. The ovary is

compressed into two cell and each cell having two ovules. The styles are filiform with shortly

bidentate. Capsule is oblong basally wrapped into 4-seeded short stalk (Backer & Bakhuizen,

1965).

6

Figure 2.2 The Snake Grass (Clinacanthus nutans) seedling

2.3. The Economic Importance of Clinacanthus nutans

C. nutans is an importance medicinal plant. It provides various medicinal uses and products.

The plant can be used to produce medicinal cream, lotion, tea, drig and secondary metabloites

products. Apart from that, an extracts from leaves can be used as cream and drinks for disease

treatment as well . The dried leaves is extracted with alcohol as cream and serves as medicine

in relieve pain and healing herpes infection (Sangkitppom et aI., 1993).

7

2.3.1 Medicinal uses of Clinacanthus spp.

C nutans has been used traditiona111y and is important herbal medicine especially in Thailand.

In Thailand, typical C. nutans products are commonly being used as the replacement of

tropical acyclovir for the treatment of herpes simplex and herpes zoster in many hospitals

(Sangkitppom et aI, 1993). Acyclovir is the anti-viral drug to treat the herpes simplex virus

(HSV) infection and the consumption of this medicine in Thailand is increasing at

approximatelly 20% per year (Jayavasu, 1998). The extracts from C. nutans has been proven

that this herb exhibit anti-viral properties to fight with HSV (Thawaranantha et aI., 1992) and

varicell zoster virus (VZV) (Jayavasu et aI., 1992). For instances, Clinacanthus nutans cream

bas been clinically shown to be efficiacy in relieving pain and healing herpes simplex and

herpes zoster without burning sensation and side effect (Sangkitpporn et a1.; 1993).

Moreover, the extracts from C. nutans had been used as tropical cream or lotion to treat

anti-inflammatory of skin and insect bites (Satayavivad et aI. , 1996). For example, the ability

of ethanol extract of C. nutans leaves was reported to inactivate HSV-2 compared with that of

acyclorir in the treatment of genital herpes patient.

Jayavasu (1998) further reported that the patients who used C. nutans could develop crust

lesion within 3 days and the lesion healed within 2 days. The time taken for crusting in the

placebo- treated group was in 4- 7 days and 7- 14 days for healing time. This showed that C.

nutans have a good efficacy in short infection time and decrease harshness of diseases.

8

Mkropropagation of medicinal plants

.00000m>J)agiaticm or plant tissue culture is an advanced plant propagation technique that being

pupa:u to improve the plant genetic and to produce disease- free plants in a short duration of

and space (Zobayed & Saxena, 2003). Chaturuedi et at. (2007) reported that in vitro

although there are only a few complete medicinal plants have been propagated and some

been regenerated in the field. Species of some Acanthaceae families such as Justicia

Por Justicia gendarussa, a medicinal' shrub in the Acanthaceae, BAP was used to induce

fbrmation of shoots from nodal segment derived from potted plants. Shoot mUltiplication were

rained on nodal explant of J. gendarussa using MS medium supplemented with 1.0 mg/L

SAP alone with 10% coconut milk (CoM) (Janarthanam & Sumathi, 2010). This indicated that

a direct regeneration may be obtained in C. nutans, another species in Acanthaceae family.

ost of the medicinal plant in the acanthaceae family could be propagated using

micropropagation techniques. For example, Basu and Yogananth (2011) reported that the

multiple shoot (4- 6 shoots) were produced from nodal explants of Andrographis paniculata

(Bwm.f.) using 4.0 mg/L BAP and 0.5 mg/L NAA.

9

t the same time tissue culture technique had been utilized for the production of secondary

metabolites. Commercilization of secondary metabolites has created a great interest in

ndary metabolism especially alternatives to the production bioactive plant metabolites by

ue culture technology (Vanisree et aI., 2004). The technology of tissue culture in the

production of bioactive metabolites may provide continuous, reliable source of plant

pharmaceuticals and large scale of plant cell cultures for the extraction of metabolite. In the

advancement in cell culture area, the production of medicinal compound has resulted in the

possibility of a wide variety of phannaceuticals such as alkaloids, terpenoids, steroids,

saponiods, phenolics, flavanoids and amino acids (Siahsar et aI., 2011).

. For instances, Angelica dahurica or called 'Bai-Zhi' in China is a valuable medicinal herb

which had been studied on its cell suspension cultures for the production of imperatorin

(bioactive component) as secondary metabolite products. The imperatorin component as the

major secondary metabolites in the plant has been extracted for curing skin diseases, headache

and psoriasis in China (Zhou et aI., 1988 ; Zhou, 1980). The callus was induced from petiole

explants from Angelica dahurica on a medium by using 1.0 mg/L 2,4-D and 0.5 mglL Kinetin

in establishing the cell suspension culture. The secondary metabolite can be increased by

adding BAP into the medium (Vinterhalter et aI., 2008). By adding 0.5 - 1.0 mglL of BAP to

the culture medium, it increased the imperatorin production in cell suspension culture (Tsay et

aI. 1994).

10

There are four stages of micropropagation of most plants. The first phase is the establishment

ofaxenic explants for producing aseptic culture. This stage is to ensure contamination- free of

Jccted explants which will be used for the next stage of shoot production. The second phase

is the shoot multiplication, the developed multiple shoots will be separated and subcultured

several times to new media for good quality of shoot production. The shoot produced in phase

2 will be used for root fonnation at third phase. The induction of roots could be achieved

either in vitro or in vivo. The final stage is acclimatization of regenerated plants. The in vitro

cultured plantlets is transferred to soil or glasshouse for better survive (Pierik, 1997).

Various factors are known to influence the success of plant tissue culture, including the choice

of plant growth regulator and their concnentration, the age, state and part of the plant from

hich the original explants were taken. he genotype and environmental condition of the

mother plants, the culture medium, timing, duration of exposure and subsequent culture

condition also influence the success of plant tissue culture (Robert & Dennis, 2010).

1A 1. Selection of explants

proper selection of explant is a necessary condition in micropropagation. In shoot culture,

the shoot formation usually can be obtain successfully by using actively growing young shoots.

The concentration of plant hormone that supplemented into the medium always control the

activation of explants either it is less or more active explants. The size of explant, health of

shoot, nodal segment, petiole taken and dormancy condition must be measured in the selection

ofexplant.

11

The achievement of micropropagation of plant specIes is dependent for the condition of

lants and often depends on season, climate and environment of the original plants, as these

condition can affect the growth and morphogenesis of the tissue explant in culture (Miller et

2 .2. Establishment of axenic explants

An axenic explants are the main requirement for a success of establishment and maintenance

of plant tissue culture. The explants selected from outdoor or green house are usually of

concern because the air may be highly contaminated with microorganisms. Usually the

contaminants outgrow the explants and resulted in the death of explants. These contaminants

or micoorganism must be removed by establishing sterile or aseptic condition to minimize the

possibility of microorganism remain or enter the cultures. There are some factor affect the

pes ibility of contamination includes the explants, culture medium, apparatus and materials,

cmvironment of explant transfer area, technicians and tissue culture room (Dodds & Roberts,

1995).

A rapid production of axenic explant is the main objective in the plant tissue culture process.

Contaminants usually obtained externally such as fungi, bacteria, molds and other

microorganism which are present everywhere either in the air or many surface of the plants,

tables hands and so on (Hartmann et aI., 1990). Normally, the explants that obtained from

field-grown plants, diseased specimen or plant parts which are located close to or below the

soil may be impossible to be sterilized due to the presence of endophytic and epiphytic

microbes (Leifert et aI. , 1994).

12