effects of different basal media and … of different basal...declaration i declare that this thesis...

EFFECTS OF DIFFERENT BASAL MEDIA AND COMPLEX ADDITIVES INTERACTION ON IN VITRO SEED GERMINATION

AND SEEDLING GROWTH OF WHITE KELAMPAYAN (NEOLAMARCKIA CADAMBA)

Ling Wan Ting

Bachelor of Science with Honours QK (Resource Biotechnology) 50

2015L755 2015

Pusat Khitlmat Maldu ruatkadclU1 UN E SJn MLYSf A ARAWA

EFFECTS OF DIFFERENT BASAL MEDIA AND COMPLEX ADDITIVES

INTERACTION ON IN VITRO SEED GERM INA nON AND SEEDLING GROWTH OF

WHITE KELAMP A Y AN (Neolamarckia cadamba)

Ling Wan Ting

This project report is submitted in partial fulfilment of the requirements for the degree of

Bachelor of Science with Honours

(Resource Biotechnology)

~

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2015

ACKNOWLEDGMENTS

First and foremost I would also like to show my sense of gratitude to the God for the

healthiness and well being that were necessary to complete this thesis

I am also grateful to Dr Ho Wei Seng and Miss Maslini Binti Japar Ali my supervisor

and co-supervisor in the Department of Molecular Biology I am extremely thankful and

indebted to them for sharing expertise and sincere and valuable guidance and encouragement

extended to me

I take this opportunity to express gratitude to all the members of the Forest Genomics

and Infonnatics Laboratory (fDiLab) for their help and support I also thank my parents for the

unceasing encouragement support and attention

I am greatly thankful to one and all who directly or indirectly have lent their hand in

this project J

0

II

t

DECLARATION

I declare that this thesis entitled Effects of Different Basal Media and Complex Additives

Interaction on In Vitro Seed Gennination and Seedling Growth of White Kelampayan

(Neolamarckia cadamba)is my original research work Wherever contributions of others are

involved every effort is made to indicate this clearly with due reference to the literature and

acknowledgement of collaborative research and discussions The work was done under the

guidance of supervisor Dr Ho Wei Seng and co-supervisor Miss Maslini Binti Japar Ali at

the Forest Genomics and Informatics Laboratory (fGiLab) UNlMAS

1i~ Date 12062015

Ling Wan Ting

Resource Biotechnology Programme




III

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PUS~l bullbull1 lhakhilUlh An t rT UNJVERSlll MALAYSIA SARAAl

TABLE OF CONTENTS

ACKNOWLEDGMENTS II

DECLARATION III

TABLE OF CONTENTS IV

LIST OF ABBREVIATIONS VI

LIST OF TABLES VII

LIST OF FIGURES VIII

ABSTRACTABSTRAK IX

CHAPTER I INTRODUCTION 1

CHAPTER II LITERATURE REVIEW 3

21 Neolamarckia cadamha (Roxb) Bosser 3

211 Distribution 3

212 Uses ofNeolamarckia cadamba 4

213 Seed Gennination Stage and Seedling Growth 6

22 In vitro Seed Gennination and Seedling Growth 8

23 Factor Affecting in vitro Seed Germination and Seedling 8

Growth

231 Seed Maturity 8

232 Selection of Basal Medium 9

233 Nutritional Components - Complex Additive 10

CHAPTER III MATERIALS AND METHODS 12

IV

r

II I

I

t

31

32

33

34

35

36

37

38

CHAPTER IV

41

42

43

44

45

CHAPTER V

REFERENCES

APPENDICES

Plant Material 12

Pre-treatment of Seeds 12

Surface Sterilisation 12

Culture Medium Preparation 12

Culture Condition 13

Complex Additive Preparation 14

Experimental Design 14

Data Collection and Statistical Analysis 14

RESULTS AND DISCUSSION 16

In vitro Seed Germination 16

Effect of Different Basal Media on in vitro Seed 19

Germination

Effect of Different Complex Additives Concentrations on 21

in vitro Seed Germination

Effect of Different Basal Media on Seedling Growth 23


Seedling Growth

CONCLUSIONS AND RECOMMENDATIONS 35

36

40

v

LIST OF ABBREVIATIONS

MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI

Murashige and Skoog medium (1962)

Gamborgs B5 medium (1968)

Coconut water

Banana homogenate

Potato homogenate

Plant growth regulators

Sarawak Forestry Corporation

Revolutions per minute

Sodium hydroxide

Hydrochloric acid

Pounds per square inch

Completely Randomised Design

Analysis of Variance

Duncans Multiple Range Test

milligram per liter

Days after inoculation

VI

6

LIST OF TABLES

Table Page Number

21 Arabidopsis growth stages for the plate-based phenotypic analysis

platform

41 Effect of basal media on in vitro seed germination of N cadamba at 19

30 days after inoculation (OAI)

42 Effect of different complex additives concentrations on in vitro seed 21

germination ofN cadamba at 30 days after inoculation (OAI)

43 Effect of basal media on seedling growth of N cadamba at 60 days 24

after inoculation (OAI)

44 Effect of different complex additives concentration on seedling 29

growth ofN cadamba at 60 days after inoculation (OAI)

45 The composition of natural phytohormones in coconut water 34

0

VII

LIST OF FIGURES

Figure Page Number

21 Characteristic ofN cadamba 5

22 Arabidopsis germination 6

31 Seedling with two leaves and one root 13

41 Seed germination ofNcadamba 17

42 In vitro seed germination growth stages of N cadamba 18

43 Effect of basal media on the seedling growth according to DAI (days 23

after inoculation)

44 Effect of different banana homogenate concentration on seedling 26

growth according to DAI (days after inoculation)

45 Effect of different potato homogenate concentration on seedling 27


46 Effect of different coconut water concentration on seedling growth 28

according to DAI (days after inoculation)

47 Effect of coconut water concentrations on seedling growth according 30

to DAI (days after inoculation)

VIII

Effects of Different Basal Media and Complex Additives Interaction on In Vitro Seed Germination and Seedling Growth of White Kelampayan (Neolamarckia cadamba)

Ling Wan Ting

Resource Biotechnology Faculty of Resource Science and Technology


ABSTRACT

Neolamarckia cadamba or white kelampayan is a tropical tree species important for both industrial plantation and forest plantation in Malaysia Yet its seed production is time-limited and its growth period of seedling is also quite long In this study a protocol for the effects of different basal media and complex additives interaction on in vitro seed germination and seedling growth of white kelampayan was established The effects of different complex additives such as CW BH and PH at different concentrations (5 10 and 15) on seed germination and seedling growth were investigated The results obtained in the study showed tbat Y2 B5 medium was the most suitable basal madia for in vitro seed germination with 4567plusmn119 of seed germination Y2 B5 medium and 10 CW were the best combination medium for seedling growth

Keywords Neolamarckia cadamba basal media complex additives in vitro seed germination and seedling growth

ABSTRAK

Neolamarckia cadamba atau kelampayan putih merupakan spesies pokok tropika penting bagi ladang dan hutan tanaman industri di Malaysia Namun masa penghasilan biji benih amat lerllad dan tempoh pertumbuhan anak benih juga agak panjang Dalam kajian ini protokol telaII ditubuhkan untuk mengaji kesan interaksi antara media asas yang berbeza dan bah an tambahan kompleks di in vitro percambahan biji benih dan pertumbuhan anak benih dari kelampayan putih Kesan bah an tambahan kompleks yang berbeza seperti CW BH dan PH pada kepekatan yang berbeza (5 10 dan 15 ) ke atas percambahan biji benih dan pertumbuhan anak benih telah dikaji Keputusan yang diperolehi dalam kajian ini menunjukkan bahawa medium lhB5 adalah terbaik untuk in vitro percambahan benih dengan 45 67x1 19 percambahan biji benih lh B5 dan 10 CWadalah kombinasi medium yang terbaik untuk pertumbuhan anak benih

Kala kunci Neolamarckia cadamba media asas bahan tambahan kompleks in vitro percambahan biji benih dan pertumbuhan anak benih

IX

CHAPTER I

INTRODUCTION

The deforestation of Malaysia rain forests has increased rapidly in the past few years due to

industrial logging large-scale oil palm plantations and agribusiness road and dam

construction A survey of deforestation and forest degradation area indicated that increased

rate of deforestation by 150 in Malaysia and it was ranked in the top 2 after Bolivia (162)

during the flrst quarter of year 2014 Besides Malaysia had been recorded a total lost of 144

offorest area from the year 2000 to 2012 which is the worlds highest rate (Yong et al 2014)

In Sabah and Sarawak approximately 80 of the land area was used by undocumented

activities logging or clearing operations Yet only 3 of Sarawaks forest is undisturbed

under the protected areas (Yong et al 2014) The decline in the number of harvested native

fore ts leads to the industrial wood prices rapidly increasing over the years So the high

yielding forest plantations are recommended to deal with these problems It indicates high

wood productivity which is 25-30 m3halyr while the natural forest is only 20-25 m3halyr

Forest plantation ensures that the timber is sustainable for wood-based industries in Sarawak

Besides natural forests will not be targeted for deforestation so the roles of natural forest in

protection and conservation can be maintained (Samwak Forestry Corporation 2013)

Neolamarckia cadamba (Roxb) Bosser has been selected as one of the important

plantation tree species in Malaysia either for industrial plantation or for reforestation and

afforestation due to its fast-growing characteristics its silvicultural characteristics and free

from serious pests and disease problem (Krisnawati et al 2011) It also called as

1

Anthocephalus cadamba Roxb or commonly known as white kelampayan m Malaysia

(Krisnawati et aI 2011) N cadamba belongs to the family of Rubiaceae It is widely

distributed from India and Nepal to Thailand and Indo-China and eastward in Malaysian

Archipelago to Papua New Guinea (Jeker 2000) It is a multipurpose tree species that mainly

used as plywood light construction materials medicine ornamental use and others

(Krisnawativ et al 2011)

The superiority of N cadamba has led to the increase demand of this species yet its

seed production is time-limited This tree species will only flowering from June to September

and fruiting from September to February (Krisnawati et al 2011) Besides the growth of its

seedling is also time-consuming (Bedell 1998) Efforts have been focused on in vitro culture

fo r speeding up the growth of N cadamba In vitro culture is a rapid and cost effective

technique to improve germination and seedling growth since it provides favourable culture

condition specific basal medium supplemented with plant growth regulators (PGRs) (Aileni et

al 2014) There is no available information discussed on in vitro seed germination and

seedling growth of N cadamba In this study a cost-effective protocol for in vitro seed

germination and seedling growth ofNeolamarckia cadamba will be developed

The objectives of this study are

1 To determine the effect of different basal media-and complex additive interaction on in

vitro seed germination and seedling growth ofN cadamba

2 To enhance the in vitro seed germination and seedling growth of N cadamba by

identifying the most suitable combination of basal media and complex additive

3 To develop a cost-effective protocol for in vitro seed germination ofN cadamba

2

CHAPTER II

LITERATURE REVIEW

21 Neolamarckia cadamba (Roxb) Bosser

Neolamarckia cadamba (Roxb) Bosser also called as Anthocephalus cadamba (Roxb) Miq

or commonly known as white kelampayan in Malaysia (Krisnawati et al 2011) N cadamba

belongs to the family of Rubiaceae It is a woody plant with a broad conical crown and straight

cylindrical bole Its branches spread horizontally and arranged in tiers (Figure 21 A) The

bark of young trees is grey smooth and very light but it becomes rough and longitudinally

fissured once it developed into old trees (Figure 21 B) Its leaves are characteristically by

glossy green opposite simple sessile to petiolate ovate to elliptical (Figure 21 C) The fleshy

yenow~orange infructescence formed by many small and fleshy capsules is the N cadambas

fruits It consists of about 8000 trigonal or irregular shaped seeds (Figure21 D) (Krisnawati et

al 201 1) It is a seasonal tree that only flowering within June to September and fruiting

within September to February (Krisnawati et al 2011)

211 Distribution

N catamba is generally found in Australia China India Indonesia Malaysia Papua New

Guinea Philippines Singapore and Vietnam Besides it has also become exotic species in

Costa Rica Puerto Rico South Africa Surinam Taiwan Province of China and Venezuela

(Orwa el al 2009) N cadamba grow on deep damp alluvial sites normally in the

secondary forests along the riverbanks and in the translational zone between moist areas

3

~bull

(Krisnawati et al 2011) It grows at below 1000 m altitude within its natural distribution with

more than 1500 nun rainyear However it also can be found in dry areas with approximately

200 nun rainyear (Krisnawati et al 2011)

212 Uses of Neolamarckia cadamba

Neolamarckia cadamba is a multipurpose tree species as its wood leaves bark and fiuits can

be utilised in different field The wood is used for plywood light construction materials

flooring beams and rafters pencils and others (Soerianegara amp Lenunens 1993) Besides it

also can be made dug-out canoes and furniture The medium quality of paper can be produced

through mixing the pulp with the long-fibred materials (Krisnawati et al 2011) The leaves

are utilised as cattle fodder or plates and serviettes and its extract has been used as mouth

gargle The dried bark can be applied in medicine field as it can relieve fever and consume as

a tonic (Orwa et ai 2009) In addition the root bark can be processed to produce a yellow dye

that can be used as tannin or dyestuff (Soerianegara amp Lenunens 1993) The whole tree can

even serve as ornamental plants and plantation trees involved in reforestation and afforestation

programmes (Krisnawati et al 2011) Moreover N cadamba is also a medicinal plant for

treatment of fever anaemia diabetes uterine and liver complaints menorrhagia blood and

skin diseases diarrhoea colitis stomatitis dysentery and semen quality improvement using its

leaf bark and root (Zayed et aI 2014)

4

Pusat Kh middotdm1 MakJumar k~atr UNIVERSm MALAY IA ARAW

Figure 21 Characteristic of N cadamba (A) Four year old N cadamba trees (B) The grey and smooth stem bark of young N cadamba trees (C) Glossy green leaves of N cadamba (D) N cadamba fruits (Krisnawati et at 2011)

5

213 Seed Germination Stage and Seedling Growth

Figure 22 Arabidopsis germination (Bar=1 mm) (Modified from (Boyes et al 2001raquo

Table 21 Arabidopsis growth stages for the plate-based phenotypic analysis platform (Boyes et al 2001)

Stage Description Principal growth stage 0 Seed gennination Ol Seed imbibition 05 Radicle emergence 07 Hypocotyl and cotyledon emergence Principal growth stage 1 Leaf development 10 Cotyledons fully opened 102 2 rosette leaves gt 1 nun 104 4 rosette leavesgt 1 nun Stage R6 More than 50 of the seedlings have primary roots ~6 cm in

length

6

Seed gennination stage of N cadamba is similar to Arabidopsis species (refer to Figure 21)

Studies had been conducted to evaluate the growth stages of Arabidopsis based on BBCH

scale (Boyes et al 2001) (refer to Table 21)

The germination pattern of Kelampayan seed is classified under epigeal germination

because cotyledons are pulled above the ground After the seed is inoculated in the soil it will

absorb the water and swelling that result in increased moisture content Next the radicle of

seed emerges via the seed coat and subsequently developed into primary root in the soil The

elongation of radicle will lead to lateral roots emergence and growth of root hairs (Rea

Hybrids 2014)

After five to ten days of inoculation the new seedling is developed The hypocotyl

starts to elongate and grow upward via the surface of soil together with cotyledon The

hypocotyl may be broken and lead to the death of seedling due to hardness the soil The

cotyledon provides the temporary source of stored food to the seedling It will fully open and

turn green due to light and synthesis sugar via photosynthesis process The epicotyl

containing small leaves buds and the growing point is revealed in following germination

process After formation of the first set of rosette leaves the cotyledons will drop off (Rea

Hybrids 2014)

7

22 In vitro Seed Germination and Seedling Growth

Seed gennination and development defmed as the process of new plant developed from the

seed However the seed production of woody species is irregular particularly forest trees in

vitro techniques is vital to forest plantation programmes for continuous seedling production

(Wang et al 1993) In vitro techniques is used to optimize gennination since it provides

favourable culture condition specific basal medium supplemented with plant growth

regulators (PGRs) that are vital for the growth of seeds (Aileni et al 2014) In vitro seed

gennination and seedling growth is an alternative method to propagate plants that are required

in high demand and having problem in seed viability through conventional plant breeding

method (Aileni et al 2014)

23 Factor Affecting in vitro Seed Germination and Seedling Growth

231 Seed Maturity

Seeds ought not to be collected before they are fully ripened Immature seeds of many species

may result in no gennination and easily contaminated with fungi when placed on a moist

substrate such as Pinus cembra Furthermore immature seeds of some species such as

Helianthus annuus and Zea mays cannot undergo desiccation because they will die due to its

low desiccation tolerance In some cases the immature seeds can obtain higher gennination

percentage as compared to mature seeds just as noted in Juniperus oxycedrus (Baskin amp

Baskin 2014) On the other hand mature seeds of some species had been recorded no

gennination due to seed coat impermeability to water For these hard seeded species such as

Gymnocadus dioicus treatments are required to make them permeable to water (Baskin amp

Baskin 2014) In the cases of N cadamba the mature seeds will be collected when the fruits

8

appear dark brown colour The higher gennination rate had been indicated on seeds stored in

cool and airtight container for 2S months as compared to fresh seeds with only 2S

(Krisnawati et al 2011)

232 Selection of Basal Medium

Basal media playa significant role in detennining the success or failure ofplant tissue culture

It provides water nutrients vitamins and growth regulators for the plant as well as place for

gases exchange and removal of metabolite wastes (Mather amp Robert 1998)

A widely known basal medium such as Murashige amp Skoog basal medium (MS)

(Murashige amp Skoog 1962) always be used to start the preparation of most appropriate

medium for a new system so it is selected in the present study (Chiruvella et al 2014) The

MS medium was designed for tobacco explants based on its mineral compounds in previous

studies (Jha amp Ghosh 200S) Half strengths MS medium had been determined to optimize in

vitro seed germination of other species such as Withania somnifera (Pandey et al 2013) and

Pterocarpus marsupium (Mishra et al 2013) MS medium containing high amounts of both

macro- and micro- nutrients can increase gennination and seedling development (Pandey et al

2014)

On the other hand Gamborgs BS medium (Gamborg et al 1968) was designed for

callus or suspension culture but it also works as a basal medium for plant regeneration

(Mather amp Robert 1998) BS medium was proven that increased seed germination ofJatropha

curcas (Warakagoda amp Subasinghe 2009) In the cases of Rhyncostylis retusa BS medium

was shown the maximum root length as compared to the other media Besides BS medium

was known as the most likely medium influencing the expression of all the seedling

9

parameters such as leaf root length and breadth fresh weight and dry weight (Basu et al

2010) However the factors contributed to these results have yet to be evaluated For the half

strength B5 medium it was indicated to be the most suitable medium for in vitro seed

germination of Commiphora wightii (Am) Bhandari (Burseraceae) (Kant Prajapati amp

Parmar 2010)

233 Nlltritional Components - Complex Additive

A suitable complex additive can contribute to the establishment of an efficient and economical

protocol for plant tissue culture (Bakar et aI 2014) It is obvious that a variety of substances

are able to promote gennination and growth and they are not restricted to any species Certain

growth factor(s) is possible to be found in additives as some complex additives used may

contain little or no sugar or mineral (Arditti 2009)

Coconut water (CW) was first utilized in tissue cultures by enhancing the growth of

Datura stramonium embryos (Van Overbeek et aI 1941) Previous studies had shown that the

CW that contains carbohydrate vitamin amino acid organic acid organic ion and enzyme

had played an important role in plant cell development (Ali et al 2011) It also increases seed

germination and seedling growth of many orchid species such as Paphiopedilum villosum var

oncidioides (Long et al 2010) and Encyclia aff Oncidioides (Znaniecka et al 2005)

Similar to CW potato homogenate (PH) had been evaluated its role in enhanced seed

gennination and seedling growth ofDim orph ics lowii (Bakar et aI 2014) It contains sucrose

dipotassium phosphate potassium chloride ferrous sulphate nitrogen source protein

carbohydrate Vitamin Band C that had resulted in improved seed gennination (Bakar et aI

2014) Banana homogenate (BH) had been proven that enhanced orchid seed germination and

10

seedling growth It contains growth factors such as cytokinins auxins and gibberellins which

can act as a buffer in culture medium (Manawadu et ai 2014) Nonetheless no previous

studies had been conducted on N cadamba

11

CHAPTER III

MATERIALS AND METHODS

31 Plant Material

Seeds of N cadamba were obtained from the Sarawak Forestry Corporation (SFC) Sarawak

Malaysia

32 Pre-treatment of Seeds

Collected seeds were immersed in 42degC of sterile distilled water for 30 minutes Then the

water was removed and seeds were air-dried at room temperature

33 Surface StenUsation

Pre-treated seeds were immersed in 15 bleach solution containing 1 to 2 drops of Tween 20

for 15 minutes and agitated gently Then the seeds were rinsed three times with sterile

distilled water followed by deep immersed in 70 ethanol for 30 seconds

34 Culture Medium Preparation

Three basal media used in this study were half strength Murashige and Skoog (MS) medium

and halfand full strength Gamborgs (85) media The culture medium was adjusted to pH 55

to 57 with IN NaOH or HCI before the addition of 85 gr of agar powder The glass bottle

containing culture medium was then autoclaved at 121degC and 15 psi for 20 minutes Culture

12

medium was poured into sterilised petri dish and each petri dish was sealed with a single layer

ofparafilm to avoid desiccation

35 Culture Condition

Two separate cultures were maintained the first for seed germination and the second one for

seedling growth For seed germination culture the surface-sterilised seeds were inoculated on

the culture medium either alone or supplemented with complex additives After 30 days the

seedlings with two leaves and one root were subjected to standardisation in B5 medium for a

week Seedlings were inoculated on the culture medium either alone or supplemented with

complex additives Cultures were placed in the laboratory and allowed to grow at 25plusmn2degC

under 16 hours light 8 hour dark photoperiod

Figure 31 Seedling with two leaves and one root (Bar = 2mm)

13

36 Complex Additive Preparation

Three complex additives were added into the selected basal medium such as coconut water

(CW) (vv) banana homogenate (BH) (wv) and potato homogenate (PH) (wv) with different

concentration such as 5 10 and 15 CW was obtained directly from young coconut and

flltered to remove the impurities For the banana and potato the skin of samples was peeled

off and cut into fme pieces then weighed and blend in a blender

37 Experimental Design

The experiments were carried out in a Completely Randomised Design (CRO) A number of

surface-sterilised seeds were dispersed uniformly on the surface of three basal medium half

strength MS medium and half and full strength B5 medium were used either alone or

supplemented with three types of complex additives coconut water (CW) banana homogenate

(BH) and potato homogenate (PH) with concentration of 5 10 15 Each treatment was

carried out in three replicates

38 Data Collection and Statistical Analysis

The data were collected and calculated at 20 days interval up to 30 days on percentage of seed

germination

Number of germInated seeds Germination() = T 1 b f d x 100ota num er 0 see s

The data of seedling development were collected and calculated at 20 days interval up to 60

days by evaluation of height of plant and the number of leaves shoots and roots Statistical

analysis was carried out using one and two way Analysis of Variance (ANOVA) by SPSS

14

~

Pusat Khitlmat Maldu ruatkadclU1 UN E SJn MLYSf A ARAWA

EFFECTS OF DIFFERENT BASAL MEDIA AND COMPLEX ADDITIVES

INTERACTION ON IN VITRO SEED GERM INA nON AND SEEDLING GROWTH OF

WHITE KELAMP A Y AN (Neolamarckia cadamba)

Ling Wan Ting

This project report is submitted in partial fulfilment of the requirements for the degree of

Bachelor of Science with Honours

(Resource Biotechnology)

~




2015

ACKNOWLEDGMENTS






extended to me





this project J

0

II

t

DECLARATION








1i~ Date 12062015

Ling Wan Ting





III

ibull


TABLE OF CONTENTS

ACKNOWLEDGMENTS II

DECLARATION III



LIST OF TABLES VII


ABSTRACTABSTRAK IX




211 Distribution 3





Growth

231 Seed Maturity 8




IV

r

II I

I

t

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32

33

34

35

36

37

38

CHAPTER IV

41

42

43

44

45

CHAPTER V

REFERENCES

APPENDICES

Plant Material 12











Germination





Seedling Growth


36

40

v


MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI



Coconut water

Banana homogenate

Potato homogenate




Sodium hydroxide

Hydrochloric acid





milligram per liter


VI

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

ACKNOWLEDGMENTS






extended to me





this project J

0

II

t

DECLARATION








1i~ Date 12062015

Ling Wan Ting





III

ibull


TABLE OF CONTENTS

ACKNOWLEDGMENTS II

DECLARATION III



LIST OF TABLES VII


ABSTRACTABSTRAK IX




211 Distribution 3





Growth

231 Seed Maturity 8




IV

r

II I

I

t

31

32

33

34

35

36

37

38

CHAPTER IV

41

42

43

44

45

CHAPTER V

REFERENCES

APPENDICES

Plant Material 12











Germination





Seedling Growth


36

40

v


MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI



Coconut water

Banana homogenate

Potato homogenate




Sodium hydroxide

Hydrochloric acid





milligram per liter


VI

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

DECLARATION








1i~ Date 12062015

Ling Wan Ting





III

ibull


TABLE OF CONTENTS

ACKNOWLEDGMENTS II

DECLARATION III



LIST OF TABLES VII


ABSTRACTABSTRAK IX




211 Distribution 3





Growth

231 Seed Maturity 8




IV

r

II I

I

t

31

32

33

34

35

36

37

38

CHAPTER IV

41

42

43

44

45

CHAPTER V

REFERENCES

APPENDICES

Plant Material 12











Germination





Seedling Growth


36

40

v


MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI



Coconut water

Banana homogenate

Potato homogenate




Sodium hydroxide

Hydrochloric acid





milligram per liter


VI

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~


TABLE OF CONTENTS

ACKNOWLEDGMENTS II

DECLARATION III



LIST OF TABLES VII


ABSTRACTABSTRAK IX




211 Distribution 3





Growth

231 Seed Maturity 8




IV

r

II I

I

t

31

32

33

34

35

36

37

38

CHAPTER IV

41

42

43

44

45

CHAPTER V

REFERENCES

APPENDICES

Plant Material 12











Germination





Seedling Growth


36

40

v


MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI



Coconut water

Banana homogenate

Potato homogenate




Sodium hydroxide

Hydrochloric acid





milligram per liter


VI

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

II I

I

t

31

32

33

34

35

36

37

38

CHAPTER IV

41

42

43

44

45

CHAPTER V

REFERENCES

APPENDICES

Plant Material 12











Germination





Seedling Growth


36

40

v


MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI



Coconut water

Banana homogenate

Potato homogenate




Sodium hydroxide

Hydrochloric acid





milligram per liter


VI

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~


MS medium

B5 medium

CW

BH

PH

PGRs

SFC

Rpm

NaOH

HCI

Psi

CRD

ANOVA

DMRT

mgIL

DAI



Coconut water

Banana homogenate

Potato homogenate




Sodium hydroxide

Hydrochloric acid





milligram per liter


VI

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

6

LIST OF TABLES

Table Page Number


platform










0

VII

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

LIST OF FIGURES

Figure Page Number







after inoculation)









VIII


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~


Ling Wan Ting



ABSTRACT



ABSTRAK



IX

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

CHAPTER I

INTRODUCTION




















1























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~























2

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

CHAPTER II

LITERATURE REVIEW













211 Distribution






3

~bull



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~



















4



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~



5





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~





length

6









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~









Hybrids 2014)








Hybrids 2014)

7












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~












231 Seed Maturity











8
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~
















2014)







9





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~





Parmar 2010)


















10




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~




11

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~

CHAPTER III


31 Plant Material


Malaysia













12












13
















germination





14

~












13
















germination





14

~
















germination





14

~

effects of different basal media and … of different basal...declaration i declare that this thesis...

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