UNIVERSITI PUTRA MALAYSIA

PROLINE ACCUMULATION IN OIL PALM POLYEMBRYOGENIC CULTURES UNDER VARIOUS CONDITIONS

AHHAD TARHIZI BIN HASHIM

FSAS 1992 3

PROLINE ACCOMDLATION IN OIL PALM

POLYEMBRYOGBNIC CULTURBS

UNDER VARIOUS CONDITIONS

By

AHHAD TARHIZI BIN HASHIM

Thesis Submitted in Fulfillment of the Requirements for the Degree of Master of Science. in Faculty of

Sc ience and Environmental Studies , Universiti Pertanian Malays ia

1 9 92

IN THE NAME OF ALLAH, MOST GRACIOUS, MOST MERCIFUL

Dedicated To :

My Wife Rabiah Abdul Wahid

My Sons Hasan Al-Baari

Muhammad Yazid

Ahmad 'Ulwan

Ahmad Abrar

Muhammad Hanif

My Parent. : Haabim and Rabiah

My Brothers and Sisters

My Teachers and Priends . • • . • . . •

Tidak suatu b eneane pun yang men!mp e di bum! dan ( tidak

pul a ) pada di rimu s endir i melainkan telah di tuli s di dalam

k i t ab (L a u h u l M a h f u z h ) s e b e l um kami m e neip t ak annya.

Sesungguhnya yang demi kian itu adalah mudah bagi ALLAH. Kami

je l a s kan yang demi ki an i tu supaya jangan kamu berdukac i t a

te rhadap a p a yang luput dari kamu dan supaya kamu jangan

terlalu gemb ira terhadap apa yang diberikanNya kepadamu. Dan

ALLAH tidak menyukai . e t i ap orang yang sombong lagi membangga

diri

(57 22 - 2 3 )

ACKNOWLEDGEMBNTS

All praise be to the Almighty ALLAH , the Merciful and the

Beni f i cient . Had it not been due to His wil l and favour , the

completion of this s tudy would not have been poss ible .

I wish to express my deep appreciat ion and gratitute t o

my supervisor , Associate Professor Dr . Marziah Mahmood for her

dedicated effort s , invaluable guidance , cons tant support and

encouragement throughout the course of this study .

I am grat e ful to a l l staf f members of the P lant S cience

and B i o t e chno l ogy Uni t , B iology Divis ion , POR I M , all s t a f f

members o f the Department o f Biochemistry and Microbiology , UPM

and friends who help me in one way or another .

My s incere appreciation is extended to Dr . Yusuf Basiron

(Di r e ct o r General o f PORIM ) , Dato ' Dr . Haj i Abdul Hal im Haji

Hassan ( former Director General of PORIM) , Professor Dr . Jalani

Sukaimi (Director o f Biology) , Dr . K. Paranj othy (Head o f PSB

Unit ) and Cik Rohani Haj i Othman (Senior Research Officer) for

their support and encouragement in this study .

Finally, I am greatly indebted to PORIM for the financial

support .

iv

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i v

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , xi

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , xxii

ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxv

CHAPTER

1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Growth Limitation Methods in Storage of In Vitro Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Low Temperature Incubat ion of Cultures . . . . 6

Minimal Growth Media . . . . . . . . . . . . . . . . . . . . . . 8

Modif ication of Gaseous Environment . . . . . . . 9

Biochemical and Phys iological changes in Plant Cell s and Tissues in Relation with Exposure to Low Temperature Regimes . . . . . . . , . . . . . . . . . . . . . 1 0

Changes in Proline Accumulation . . . . . . . . . . . 11

Other Amino Acids and Soluble Protein . . . . . 12

Enzyme Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Sugars and Related Compounds . . . . . . . . . . . . . . lS

v

Abscisic Acid (ABA) . . . . . . . . . . . . . . . . . . . . . . . 16

Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Plasma Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7

Inhibition of Photosynthesis . . . . . . . . . . . . . . 1 8

Changes i n Other Metabolites . . . . . . . . . . . . . . 1 9

Proline Accumulation under Other Stress Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

Moisture Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

Salinity Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2

Flooding Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2

Other Conditions that Induce Proline Accumulation . . . . . . . . . . . . . . . . . . . . . . 2 3

Proline Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4

Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 2 4

Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 S

Poss ible Roles for Prol ine Accumulation in Stres s Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9

Osmoregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9

Hydration Characteristic o f Biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9

The Conservation of Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

Sink for Soluble Nitrogen . . . . . . . . . . . . . . . . . 3 1

Other Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . , . 3 3

The Effect of Low Temperature on Growth and Multiplication of Oil Palm Polyembryogenic Cultures as a possible Means of In Vitro Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3

vi

Proline Acumulation in Oil Palm Polyembryogenic Cultures as Affected by Low Temperature Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5

The Effects of Other Growth- Limiting Treatments on Proline Content in Oil Palm polyembryogenic Cultures . . . . . . . . . . . . . . . . . . . . . . . 3 7

The influence o f High Concentration of Sucrose and Non-agitated MS Liquid Media on Proline Accumulation in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . . . . . . . 3 7

The Effects o f Various Concentrati ons of Sucrose on Proline Accumulation in Oil Palm Polyembryogenic Cultures . . . . . . . . . 3 8

The Application of Exogenous Sucros e in Prolonging the Storage Duration at Low Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 8

The Effect of Exogenous Proline on Physi cal Appearance of the Oil Palm Polyembryogenic Cultures Incubated at Low Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9

Proline Accumulation in Oil Palm Polyembryogeni c Cultures as Affected by Applications o f Specific Amino Acids . . . . . . . . . . . . 3 9

The Influence of Amino Acids on Proline Accumulation in Oil Palm polyembryogenic Cultures . . . . . . . . . . . . . . . . . . . 3 9

The Interaction of Relevant Amino Acids on Proline content in Oil Palm Polyembryogenic Cul tures . . . . . . . . . . . . . . . . . . 4 0

The Influence of Various Level s of pyrroline-S - carboxylic Acid (PSC) on Proline Accumulation in Oil Pal m polyembryogenic Cultures . . . . . . 4 1

The Effect o f Low Temperature on Proline Accumulation in Cultures Treated with Relevant Amino Acids and PSC . . . . . . . . . . . . . . 4 1

vii

Activities of Enzymes Related to Proline Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . , 4 2

Other Studies Related to Proline Content in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . 4 4

The influence o f Proline Analog and Enzyme Inhibitor on Proline Content in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . . . . . . . 4 4

The E fficiency o f Polyembryogenic Cultures in Utiliz ing The Exogenous Proline and Ornithine in Solid or Liquid Media . . . . 4 5

Duration Taken for Proline Accumul ated Cultures to Return to Normal Level After Transferring to the Normal Media . . . . . 4 5

4 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6

The Effect of Low Temperature on Growth and Mult iplication of Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6

The Influence o f Low Temperature S tress on Prol ine Accumulation Oil Palm polyembryogeni c Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1

The Effects o f Other Growth Limitation Treatments on Proline Content in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . 5 9

Proline Content in Oil Palm Polyembryogenic Cultures as Affected by High Concentration of Sucrose and Non-agitated Liquid Media at 2 7oC . . . . . . . . . . . . . . . . . . . . . . 5 9

The Influence of Various Concentrations of Sucrose on Proline Content in the Cultures . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . 6 3

Poss ibility o f Us ing Sucrose (prol ine inducer) and Exogenous Proline to Extend Low Temperature Tolerance of the Cultures at Low Temperature Incubation . . . . . . . . . . . . . . 6 7

viii

In Vit ro Studies in Understanding the Proline Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . 7 0

The Influence o f Relevant Amino Acids on Proline Content in Oil Palm Polyembryogenic Cultures at 2 7oC . . . . . . . . . . . 7 0

The Interaction o f Relevant Amino Acids on Proline Content in Polyembryogenic Cultures at 2 7oC . . . . . . . . . . . 7 3

The Influence o f P S C on Proline Content in Oil Palm Cultures at 2 7oC . . . . . . . . . . . . . . . 7 3

Proline Content i n the Cultures Supplemented with Various Concentrations of Amino Acids and PSC as Affected by Low Temperature Treatments . . . . . . . . . . . . . . . . . 7 9

Act ivities o f Specific Enzyme Relat ed t o Proline Biosynthesi s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 9

The Effect o f Low Temperature on the Activities of OT and PSC Reductase in Oil Palm PE Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3

The Influence o f Various Concentrations of Sucrose ( supplemented to media ) on the Activities of OT and PSC Reductase in Oil Palm PE Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

The Activities of OT and PSC Reductase in Oil Palm Polyembryogenic Cultures as Affected by Application of Relevant Amino Acids . . . . . . . . . . . . . . . . . . . . . . . 8 9

Some Studies Related to Proline Accumulation in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . 94

The Influence of Thioproline (proline analog) and Amino-oxyacetic acid ( enzyme inhibitor) on Proline Content in Oil Palm Polyembryogenic Cultures . . . . . . . . . . 94

The Efficiency of PE Cultures in Uti l i z ing the Exogenous Proline and Ornithine in Sol id and Liquid Media . . . . . . . . . . . . . . . . . . 94

ix

Duration Taken for Proline Accumulated Cultures to Return to Normal Level after Being Transferred to the Normal Growth Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 8

5 DISCUSS ION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 0

The Effects of Minimal Growth Treatments on Growth and Proline Accumulation in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . . . . 1 0 0

The Ef fects of Relevant Exogenous Subst rates on Prol ine Accumulation in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . . . . 1 1 0

Specific Activities o f Enzymes Related t o P roline Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Other Related Studies on Proline Accumulation in Oil Palm Polyembryogenic Cultures . . . . . . . . . . . . 1 2 0

6 SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 6

Implication and Suggestions . . . . . . . . . . . . . . . . . . . . . 1 2 9

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 2

APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1

APPENDIX A : PROLINE METABOLISM IN PLANTS . . . . . . . . . . . . . . . . . . . . 151

APPENDIX B : MS MEDIUM . . . . . . . . . . . . . . . . . 154

APPENDIX C : ADDITIONAL TABLES . . . . . . . . . 156

BIOGRAPHICAL S KETCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 8

x

Table

1

2

LIST OF TABLES

Shoot s Formation of the Cultures Incubated at 1 50C , 2 00C and 2 7oC ( control l . . . .

Regeneration Capacity of the Treated Cultures on Transferring to the Normal Condi tions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 The E f fect of Low Temperature on Growth o f Oil Palm Polyembryogenic Cultures (g f resh weight )

Page

52

5 3

( means of minimum three replicates ± S E ) . . . . . . 1 5 7

4

5

The E f fect of Low Temperature on Growth of Oil Palm Polyembryogenic Cultures ( g dry weight ) ( means of minimum three replicates ±SE) . . . . . .

The I nfluence of Low Temperature on Prol ine Accumulation in Oil Palm Polyembryogenic Cultures ( 3 0 days duration) ( �g/g fresh weight ) ( means of minimum three replicates ±SE) . . . . . .

6 The I nfluence of Low Temperature on Proline Accumulation in Oil Palm Polyembryogenic Cultures ( two days duration) ( �g/g fresh weight )

158

1 5 9

( means of minimum three replicates ±SE) . . . . . . 1 6 0

7 The Influence of Low Temperature on Proline Accumulation in Oil Palm Polyembryogenic Cultures ( 1 0 days duration) ( �g/g fresh weight ) ( means of minimum three replicates ±SE) . . . . . .

xi

161

8 Moisture Content in Oil Palm Polyembryogenic Cultures as Affected by Low Temperature Treatments ( means of minimum three repli cates ±SE ) . . . . . . 162

9 The Influence of Various Growth Limiting Treatments on Proline Accumulation in Oil Palm Polyembryogenic Cultures ( �g/g fresh weight ) (means of minimum three replicates ±SE ) . . . . . . 1 6 3

1 0

1 1

Moisture Content i n Oil Palm Polyembryogenic Cultures as Affected by Various Growth Limiting Treatments (means of minimum three replicates ±SE ) . . . . . .

The E f fects of Sucrose Treatments on P roline accumulation in Oil Palm Polyembryogenic Cultures ( �g/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . .

12 Moisture Content in Oil Palm Polyembryogenic Cultures as Affected Sucrose Treatments

164

1 6 5

(means of minimum three repl icates ±SE ) . . . . . . 166

13 The Influence of Exogenous Amino Acids (10 mM) on P roline Accumulation in Oil Palm Polyembryogenic Cultures (mg/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . 1 6 7

1 4 The Influence of Exogenous Amino Acids ( 10 0 �M) on P roline Accumulation in Oil Palm Polyembryogenic Cultures ( �g/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . 1 6 8

1 5 Moisture Content in Oil Palm Polyembryogenic Cultures as Affected by Relevant Amino Acids ( means of minimum three replicates ±SE ) . . . . . . 1 6 9

1 6 The Interaction of Relevant Exogenous Amino Acids on Proline Accumulation in Oil Palm Polyembryogenic Cultures ( mg/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . 170

xii

L7 The E ffects of Various Levels of PSC on Proline Accumulation in Oil Palm Polyembryogenic Cultures ( �g/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . L7L

L8 The Influence of Low Temperature on Prol ine Accumulation in Oil Palm Polyembryogenic Cultures Treated with Exogenous Amino Acids : Orn and Pro (mg/g fresh weight ) (means bf minimum three replicates ±SE ) . . . . . . L72

L9

2 0

The Influence of Low Temperature on Proline Accumulation in Oil Palm Polyembryogenic Cultures Treated with Exogenous Amino Acids : Arg ang Glu (mg/g fresh weight ) (means of minimum three replicates ±SE ) . . . . . .

The E ffect of Low Temperature on Proline Accumulation in oil Palm Polyembryogenic Cultures Treated with P5C ( LO O�M) ( �g/g fresh weight ) (means of minimum three repl icates ±SE ) . . . . . .

2 L Preliminary Study on Activities o f Specific Enzymes Related to Proline Synthesis in Normal Oil Palm Polyembryogenic Cultures

L73

L74

(means of minimum three replicates ±SE ) . . . . . . L75

2 2

2 3

The E ffect o f Low Temperature on the Activity of PSC Reductase in Oil Palm Polyembryogenic Cultures ( �moles NADH oxidized/ mini mg protein) ( means of minimum three replicates ±SE ) . . . . . .

The Influence of Low Temperature on the Activity of Ornithine Aminotransferase in oil Palm Polyembryogenic Cultures ( �moles NADH oxidized/ mini mg protein) (means of minimum three replicates ±SE ) . . . . . .

xiii

L76

L77

2 4

2 5

The E f fect o f Low Temperature on Soluble P rotein Content in Oil Palm Polyembryogenic Cultures ( mg/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . .

The Influence of Sucrose Treatments on the Activity of P5C Reductase in Oil Palm Polyembryogenic Cultures ( �moles NADH oxidi zed/ mini mg protein) ( means of minimum three replicates ±SE ) . . . . . .

2 6 The E ffects of Sucrose Treatments on the Activity of Ornithine Aminotransferase in Oil Palm Polyembryogenic Cultures ( �moles NADH oxidized/ mini mg protein)

178

179

( means of minimum three replicates ±SE ) . . . . . . 1 8 0

2 7 The Influence o f Sucrose Treatments on S oluble Protein in Oil Palm Polyembryogenic Cultures ( mg/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . 181

28 The Effects of Relevant Amino Acids on the Activity of P5C Reductase in Oil Palm Polyembryogenic Cultures ( �moles NADH oxidized/ mini mg protein) ( means of minimum three replicates ±SE ) . . . . . .

2 9 The Influence o f Relevant Amino Acids on the Activity of Ornithine Aminotransferase in Oil Palm Polyembryogenic Cultures ( �moles NADH oxidi zed/ mini mg protein)

182

( means of minimum three replicates ±SE ) . . . . . . 183

30 The Effects of Relevant Amino Acids on Soluble Protein in Oil Palm Polyembryogenic Cultures ( mg/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . 184

3 1 The Influence of Thioprol ine and Amino­oxyacetic Acid on Proline Accumulation in Oil Palm Polyembryogenic Cultures ( �g/g fresh weight ) ( means of minimum three replicates ±SE ) . . . . . . 1 8 5

xiv

3 2 Comparison Between Solid and Liquid Media on Proline Accumulation in Oil Palm Polyembryogenic Cultures (mg/g fresh weight) (means of minimum three replicates ±SE) . . . . . .

3 3 Reduct ion in Proline Content at the Normal Condit ions After 2 0 days Treatment (mg/g fresh weight)

1 8 6

( means o f minimum three replicates ±SE) . . . . . . 1 8 7

xv

Figure

1

2

3

LJ:ST OF FJ:GURES

S tages in Product i on of Plantlets from Callus Cultures ( from Lioret , 19 B 1 ) . . . . . .

The Growth of Oil Palm Embryoids Cultured at lSoC , 2 00C and 2 7oC ( control ) (g fres� weight/culture ) . . . . . . . . . . . . . . . . . . . . .

The Growth of Oil Palm Embryoids Cultured at lSoc, 2 00C and 2 7oC ( control ) (g dry weight / culture ) . . . . . . . . . . . . . . . . . . . . . . .

4 The Influence of Low Temperature on Proline Accumulation in the Embryoid

Page

5

s o

so

Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6

5 The Effect of Low Temperature(lsoC ) and Control ( 2 7°C ) on P roline Accumulation in Oil Palm polyembryogenic Cultures (hourly) . . .

6 The Effect of Low Temperature ( 15°C ) and Control ( 2 7°C) on Proline Accumulat ion in the Embryoid Tissues

57

( 2 - day Intervals ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 8

7 Moisture Content in the Embryoid Tissues as Affected by Low Temperature Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0

B The Influence of Various Growth Limiting Treatments on Prol ine Accumulation in the Embryoid Tis sues . . . . . . . . . . . . . . . . . . . . . . . . .

9 Moisture Content in the Embryoids Tissues as Affected by Various Growth Limiting

6 2

Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4

xvi

1 0 The Effects of Various Concentrations Sucrose on Proline Accumulation in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . 6 5

1 1 Moisture Content i n the Embryoid Tissues as Affected by Sucrose Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6

1 2 The Influence of Amino Acids ( 1 0 mM) on Proline Accumulation in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

13 The Influence of Amino Acids ( 1 0 0 �M) on Proline Accumulation in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . 72

14

15

Moisture Content in the Embryoid Tissues as Affected by Relevant Amino Acids . . . . . . . . . .

The Interaction o f Relevant Amino Acids on Prol ine Accumulation in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . .

16 The Influence of Various Levels of PSC on Proline Accumulation in

7 6

77

the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . 7 8

1 7

1 8

The Effect of Low Temperature on Prol ine Accumulation in the Embryoid Tissues Treated with Amino Acids . . . . . . . . . . . . . . . . . . . .

The Effect of Low Temperature on Prol ine Accumulation in the Embryoid Tissues Treated with PSC . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19 Prel iminary Study on Specific Activity of Three Enzymes Related to Proline Synthes is in the Normal Embryoid

8 0

8 1

Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2

2 0

2 1

The Influence of Low Temperatures on Specific Enzyme Activity of P5C Reductase in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . .

The Effects of Low Temperature Treatments on the Activity of Ornithine Aminotrans ferase in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . .

xvii

84

8 5

2 2 The Effects of Low Temperatures o n Soluble Protein Content in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 6

2 3 The Influence o f Sucrose Treatments on the Activity of P5C Reductase in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . .

24 The Effects of Sucrose Treatments on the Activity of Ornithine Aminotrans ferase in the Embryoid

8 7

Tissues- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8

2 5 The Effects o f Sucrose Treatments on Soluble Protein in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

2 6 The Influence o f Amino Acids on the Activity of P5C Reductase in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . .

2 7 The Effects of Amino Acids o n the Activity of Ornithine Aminotransferase in the Embryo id

91

Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . 92

2 8 The Effects o f Amino Acids on Soluble protein in the Embryoid Tissues . . . . . . . . . . . . . . 93

2 9

3 0

The Influence of Thioproline and Amino-oxyacetic Acid on Prol ine Accumulation in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Comparison Between Solid (SM ) and Liquid Media (LM) on Proline Accumulation in the Embryoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . .

3 1 Reduction in Proline Content a t the Normal Condit ions After 20 days

9 5

9 7

Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 9

xvii i

Plate

1

2

3

4

LiST OF PLATES

Oil Palm Polyembryogenic Cul tures . . . .

Growth of Embryoids of Oil Palm at Normal Conditions ( six month old) . . . .

Oil Palm Polyembryogeni c Cultures Stored at lSoC for Six Months . . . . . . . .

Oil Palm Polyembryogenic Cultures Stored at 2 00C for Six Months . . . . . . . .

5 The Cultures ( two month old) Regenerated Normally at Normal Condition

6

7

8

after Six Months Incubat�on . . . . . . . . . .

The Soggy Appearance of the Embryoids which Normally Observed after Six Months s torage at lSoC or 2 00C ( l.SX) . . . . . . . . . . . . . . .

Storage in Non-agitated Liquid Media ( 1 / 12 MS ) for Three Months ( O . 7SX) . . .

Physical E ffect of Various Concentrations of Sucrose on Oil Palm Polyembryogeni c Cultures at 2 70C after 3 0 Days . . . . . . . . . . . . . . . .

9 The Influence of Proline and Sucrose Treatments on Inducing Low Temperature Tolerance in Cultures ( 8 month old) at lSoC ( 1 . 5X ) . . . . . . . . . . . . . . . . . . . . . . .

1 0 The Influence o f Proline and Sucrose Treatments on Inducing Low Temperature Tolerance in Cultures ( 8 month old) at 2 00C ( 1 . 3X ) . . . . . . . . . . . . . . . . . . . . . . .

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Page

34

4 7

4 8

4 9

54

5 5

61

6 8

6 9

6 9

1 1 The Effects o f Amino Acids ( related to proline biosynthesis ) on Cultures after 3 0 Days ( 2X : A, B, C) . . . . . . . . .

1 2 The Effects of Amino Acids ( related to proline biosynthesis ) on Cultures

1 3

after 3 0 Days ( 2 X : D, E , F ) . . . . . . . . .

Thioproline at 10 mM Induced Proline Accumulation and Caused Toxicity to the Cultures ( one month old : 1 . 1X) . . . . . . . . . . . . . . . .

xx

74

7 5

96

text:

LIST OF ABBREVIATIONS

T h e f o l l ow i ng a bb r e v i a t i on s w e r e u s e d i n t he

ABA

AOA

ARG

GLN

GLU

GSA

IBA

MS

ORN

OT

PSC

PE

PRO

RNA

SEM

T4C

xxi

Absc i s ic acid

Amino- oxyacetic acid

Arginine

Glutamine

Glutamic acid

Glutamic - T - semialdehyde

Indolebutyric acid

Mura s h i g e ( 1962 )

Ornithine

a n d S ko o g

O rn i t h i n e ferase

a m i n o t r an s -

p y r r o l in e - S - c a rb o xYl i c acid

Polyembryogenic

Proline

Ribonu c leic Acid

S t an d a r d E r r o r o f t h e Mean

Thioproline

Abstract of thesi s submitted to the Senate of Universiti Pertanian Malays ia in f ul f i llment of the requirements for the degree of Master of Science .

PROLINE ACCUHOLATION IN OIL PALM

POLYEMBRYOGENIC CULTURES UNDER VARIOUS CONDITIONS

By

AHMAD TARMIZI BIN HASHIM

October 1 9 9 2

Supervisor As soc . Prof . Haj j ah Marziah Mahmood , Ph . D .

Faculty Science and Environmental Studies

Low temperature of l SoC to 2 00C was observed to suppress

t h e g r o w t h and mu l t i p l i c a t i o n c ap a c i t y o f o i l p a l m

polyembryogenic cul ture s . Thi s condition enabled the cul ture s

to be mainta ined for a t l e a s t s ix months durat ion wi thout

subcul ture and thus was suitable for minimal growth storage of

in vitro cul tures .

P r o l ine , a . univ e r s al s t r e s s indi c a t o r , was found t o

a c cumu l a t e in o i l palm cultures under low t empe rature and

moisture stresses ( sucrose treatment ) but not under anaerobic

s tres s (non- agitated liquid media treatment ) . Sucrose treatment

a t 0 . 5 M c au s e d mo i s t u r e r e du c t i on and indu c e d p r o l i n e

a c cu m u l a t i o n. I n a d d i t i o n , when c omb i n e d w i t h l o w

xxii

temperature s torage i t extended the low temperature tolerance

and prolonged the subcul ture durat ion for at least nine months .

The appl ication o f exogenous proline and ornithine at 1 0

roM could a l s o induce p rol ine ac cumula t i on in the cul ture s .

However, only low level of proline accumulated with application

of g l u t ami c a c i d, g l utamine and arginine Incons i s t ent

pattern was observed wi th pyrrol ine - S - carboxyl ic acid ( PS C ) .

P S C reductase ( EC 1 . 5 . 1.2 ) and ornithine aminotrans ferase ( EC

2 . 6 . 1 . 1 3 ) were f ound to involve d i r e c t ly/ ind i r e c t l y with

proline biosynthes is in polyembryogenic cultures . The specific

activi t i e s of the s e enzyme s and s oluble protein were highe r

under low temperature and moi s ture stre s s es . S ince exogenous

ornithine could induce proline accumulation and there was an

act ive involvement of orni thine aminotrans ferase, orni thine

c o u l d be o n e o f t h e p r e f e r r e d p r e cu r s o r s f o r p r o l i n e

biosynthes is in o i l palm cultures.

S im ila r l y, Thi o p ro l i n e ( p r o l i n e ana l o g ) i nc r e a s e d

prol ine ac cumul a t i on but p roduced s ome tox i c e f f ect which

eventually kil led the culture s . Polyembryogenic cultures were

found to ut i l i z e the exogenous proline and ornithine more

efficiently in the liquid media than in the solid media . The

p ro l i n e i n a c cumu l a t e d cul t ur e s wh i c h we r e t re a t e d w i t h

exogenous prol ine , orni thine and subj ected t o low temperature

xxii i

s tres s was observed to return to normal level on tranferring to

the n o rmal media and c ondi t ions . Thi s sugge s t ed that the

prol ine accumulated was readily uti l i zable when returned to

n o rm a l c on d i t i o n s a n d c o u l d b e c o n s i d e r e d as a l ab i l e

metabolite in oil palm polyembryogenic culture s .

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