Thesis Submitted in Partial Fulfilment of the. Requirements for the Degree of Doctor of Philosophy in the Faculty of Agriculture. Universiti Pertanian Malaysia.
UNIVERSITI PUTRA MALAYSIA
TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.)
BASKARAN KRISHNAPILLAY
FP 1989 8
TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.)
By
BASKARAN KRISHN APILLA Y
Thesis Submitted in Partial Fulfilment of the Requirements for the D egree of Doctor of Philosophy in the Faculty of Agriculture Universiti Pertanian Malaysia
July 1 98 9
DEDICATED
to the memory of my late
Father
VARGHESE KRISHNAPILLAY
and
to my Mother whose patience and understanding has been a constant source of inspiration for me throughout this study
ACKNOWLEDGEMENT
It pleases me to have this opportunity of expressing my sincere gratitude to Universiti Pertanian Malaysia and in particular the Faculty of Agriculture for enabling this study to be carried out. My sincere thanks are also due to the International Board for Plant Genetic Resources (IBPGR/FAO), Rome for financial support through their grant No. 8 5 /1 1 4 (a) without which this study would not have been possible.
I am extremely grateful and greatly indebted to my supervisor P rofessor Chin Hoong Fong for his dedicated efforts, stimulating discussions, active participation and constant encouragement throughout the planning and execution of this research.
My sincere thanks also goes to my co-supervisor Dr Hor Yue Luan for
his
advice,
constructive
criticisms,
constant
encouragement
and
guidance throughout this study.
I take this opportunity also to thank Dr Zaliha Christine Alang of the Biotechnology Department for allowing m e the use of the Plant Tissue Culture Laboratory facilities for carrying out a major portion of this research and also for her constant assistance provided throughout this study.
Thanks are also due to Dr Gan Yik Yuan for her help and
guidance during protein separation studies using electrophoresis.
iii
My sincere appreciation is extended to M r Ho Oi Kuan for his technical assistance in the preparation of the electron m icrographs; Mr How
Peng
Guan
for technical assistance in
the preparation of
the
histological slides; Capt. Yap Y en Kiew for the construction of the starch gel electrophoresis kit;
Encik Abdul Ghani Hashim (Fakulti Sains dan
Pengajian Alam Sekitar) for his professional photographic work; Encik Abdul Ghani Hashim (Unit Hydroponik) for the loan of his printer during the preparation of this manuscript; the Faculty of Forestry, U.P.M. for use of their m isting and glasshouse facilities and M r Ong Choon Hoe for his valuable friendship and assistance in the laboratory during the course of the study.
Thanks are also due to the Director, Dr M M Guha of Agricultural Research and Advisory Bureau for allowing the use of his office facilities during the entire course of this study.
Sincere thanks are due to Mr and M rs Ramachandran for help provided for proof reading of the original drafts and in the preparation of the final manuscript.
Lastly but not the least, I wish to thank all my family members especially Rajan, Richard, Sara and George K rishnapillay for being very understanding and for their constant encouragement and support throughout the course of this study.
iv
TABLE OF CONTENTS PAGE iii
ACKNOWLEDGEMENT LIST OF TABLES
ix
LIST OF PLATES
xiv xviii
LIST OF FIGURES
xix
LIST OF ABBREVIATIONS
xx
ABSTRACT
xxiii
ABSTRAK CHAPTERS
1
INTRODUCTION
1
1.1
Need for the Study
1
1.2
Selection of Crop for the Study
2
1.3
Objectives of the Study
3
2
REVIEW OF LITERATURE
6
2.1
Present State of Recalcitrant Seed Storage
6
2.2
Characteristics of Recalcitrant Seeds
7
2.3
Moisture Content of Recalcitrant Seeds
9
2.4
Desiccation Sensitivity of Recalcitrant Seeds
11
2.5
Chilling Sensitivity in Recalcitrant Seeds
16
2.6
Potential of In Vitro Storage for Germplasm Conservation of Difficult Materials
19
2.7
Cryopreservation of Embryos
21
2.8
Chemical Dehydration and Toxicity
24
2.9
Freezing Injury
25
v
PAGE 2.10
Mechanism Injury
2.11
Cryoprotective Agents
2.12
Use of Cryoprotective Mixtures
2.13
Thawing
34
2.14
Recovery!Recovery Medium
35
2.15
Summary
36
3
MATERIALS AND M ETHODS
37
3.1
Study Layout
37
3.2
Seed Materials and Sampling
40
3.3
Experimental Procedures
42
Packaging and Storage
42
Excision of Embryos
42
Regulation of Embryo Moisture
42
Glassware Cleaning for In Vitro Studies
43
Chemicals
44
Preparation of Stock Solutions and Culture Media
44
Incubation of Cultures
45
Preparation of Cryoprotectants
46
Cooling and Thawing Procedures
47
of
Preparation of Separation of Electrophoresis
Natural
Tolerance
to
Freezing
27 29
Compo\Ulds
and
Starch Gel and Sample Starch Proteins by
Their
for Gel
33
47
Preparation of Sections for Light Microscopy
50
Preparation o f Sections for Transmission Electron Microscopy
51
Photography
53
vi
PAGE 3.4
3.5
Measurements and Observations
53
Moisture Content
53
Dry Matter Content and Shoot to Root Ratio of In Vitro Grown Seedlings
54
Conductivity of Embryo Leachate
57
Uptake o f Oxygen During Respiration
57
Comparison o f Some Metabolic Enzymes After Electrophoretic Separation
58
Experiments
58
Selection o f Optimal Stage of Fruit Maturity for Excised Embryos (Experiments la - c)
58
Selection of Embryos for C ryopreservation Studies (Experiments 2 a - b)
61
Establishment of a Suitable Medium for the Growth and Developm ent of Excised Jackfruit Embryos ( Experim ents 3a - c)
62
Dehydration Effects on Embryo Storage (Experim ents 4 a - e )
65
Viability
and
Use o f C ryoprotectants (Experiments 5a - g)
69
Modification o f Culture Medium for Improved Growth and Planting Out of C ryopreserved Seedlings (Experiments 6a - b)
74
3.6
Statistical Analysis
76
4
RESULTS
77
4.1
Selection of Optimum Excised Embryos
4.2
Selection of Embryos for Cryopreservation Studies
84
4.3
Establishment of a Suitable Medium for the Growth and Development of Excised Jackfruit Embryos
88
4.4
Dehydration Storage
Embryo Viability and
103
Effects on
vii
Stage of
Maturity for
77
PAGE 4.5
Use of Cryoprotectants
1 26
4.6
Modification of Culture Medium for Improved Growth and Planting Out of the Cryopreserved Embryos
147
5
DISCUSSION
1 53
6
SUMMARY AND CONCLUSION
179
BIBLIOGRAPHY
182
APPENDIX
196
A
Background of the Fruit Artocarpus heterophyllus
196
B
Murashige and Skoog (1962) Inorganic Salts and Vitamiru;
204
C
Preparation of Chemicals
205
D
Anova Tables
211 227
VITAE
viii
LIST OF TABLES PAGE
TA BLE 1
Viability Percentage of Embryos Excised from the Three Stages of Fruit Maturity, After Six W eeks in Culture
79
2
Moisture Content and Viability Percentage of Excised Embryos from the Three Stages of Frui t Maturity After Desiccation Over a Period of Six Hours
79
3
Categorizing Excised Embryos by Size, from a Random Sample of Seeds Taken from Mature Unripe F ruits
86
4
Fresh W eight, Moisture Content and Coefficient of Variation of Moisture Content of Excised Embryos from the Various Selection Methods
86
5
Mean Dry Matter Content of Plantlets Grown In Vitro on M edia Containing N A A /Kinetin and With or Without Activated Charcoal
90
6
Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N AA/Kinetin and With or Without Activated Charcoal
91
7
Mean Dry Matter Content of Plantlets Grown In Vitro on M edia Containing N A A/BAP and With or Without A ctivated Charcoal
94
8
Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N A A /BAP and With or Without A ctivated Charcoal
95
9
Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing N A A /2iP and With or Without A ctivated Charcoal
98
10
Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N AA/2iP and With or Without Activated Charcoal
98
11
M oisture Content of Excised Embryos Obtained for the Various M ethods of Drying
1 04
12
In Vitro Survival Percentage of Excised Embryos After Various M ethods and Periods of Drying
1 04
ix
PAG E
TABLE 13
Moisture Content and Survival Percentage of Excised Embryos Desiccated Over a Moisture Range of 1 0-1 5 %
109
14
P hysiological and Biochemical Changes i n Excised Embryos Desiccated Over a Period of One to Six Hours
111
15
Survival Percentage of Stored Excised Embryos (Desiccated to 1 4-1 5 % and 1 2-1 3 % Moisture Content Respectively) Cultured Weekly Over a Period of Four Weeks
123
16
Survival P ercentage of Excised Embryos Dried to N ear C ri tical Moisture Level and Stored in Liquid Nitrogen
123
17
Survival Percentage of Excised Embryos After Cryoprotecting in Various Combination of Cryoprotectants for Various Period of Tim e
127
18
Survival Percentage of Excised Embryos Cryoprotected for Six Hours Followed by Storage Treatments and Direct Placem ent in Liquid N itrogen
129
19
Survival Percentage of Excised Embryos Cryoprotected for Twelve Hours Followed by Storage Treatments and Direct Placem ent in Liquid Nitrogen
130
20
Moisture Content in Excised Embryos After Cryoprotection and Desiccation for One, Two and Three Hours
132
21
Survival Percentage of Cryopreserved Embryos A fter Cryoprotection and D esiccation for One, Two and Three Hours
133
22
Survival Twelve Partial and Two
Percentage of Excised Embryos After Hours of Cryoprotection Followed by Desiccation for One and Two Hours M ethods of Cooling
133
23
Survival Percentage of Cryoprotected Embryos Partially Desiccated and Subjected to Slow Freezing Protocol
137
x
PAGE
TA B L E 24
Assessment of Viability of Cryopreserved Embryos Stored in Liquid Nitrogen Over a Period of One Month Using the Slow Freezing Protocol
137
25
Mean Dry Matter Content and Shoot to Root Ratio of Control and Cryopreserved Embryos Cultured on Two Different Media
1 48
26
Survival Percentage of Control and Cryopreserved Seedlings in Polybags After Eight Weeks of Planting in the Nursery
1 48
27
AN OVA Table of Viability Percentage of Excised Embryos from Three Stages of Frui t Maturity After Six Weeks in Culture
211
28
Mean Square Values for Mini mum, Maximum, Mean and Range of Fresh Weights of Excised Embryos from the Various Selection M ethods
212
29
Mean Square Values for Minimum , Maximum, Mean, Range and Coefficient of Variation in Moisture Content of Excised Embryos from the Various Selection Methods
212
30
AN OV A Table of M ean Dry Matter C ontent of Plantlets Grown In Vitro on Media Containing NAA/Kinetin and W ith or Without Activated Charcoal
213
31
AN OVA Table of Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N AA/Kinetin and With or Without Activated Charcoal
214
32
A N OVA Table of Mean Dry Matter Content of Plantlets Grown In Vitro on M edia Containing NAA/BAP and With or Without Activated Charcoal
215
33
A N OVA Table of Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing NAA/BAP and W ith or Without Activated Charcoal
216
34
AN OVA Table of Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing NAA/2 i P and With or Wi thout Activated Charcoal
217
35
AN OV A Table of Shoot to Root Ratio of Plantlets Grown In Vitro on M edia Containing N AA/2iP and With or Without Activated Charcoal
218
xi
PAGE
TABLE 36
A N OVA Table of Moisture Content and Survival Percentage of Excised Embryos Desiccated Over a Moisture Range of 10-1 5 %
219
37
Mean Square Values of Moisture Content, In Vitro Survival Percentage, Leachate Conductivity, Tetrazolium Test and Respiration Rate of Excised Embryos Desiccated for a Period of One to Six Hours
219
38
Mean Square Values of Survival Percentage of Stored Excised Embryos (Desiccated to 141 5% Moisture C ontent), Cultured Weekly Over a Period of Four W eeks
220
39
Mean Square Values of Survival Percentage of Stored Excised Embryos (Desiccated to 1 21 3 % Moisture C ontent), Cultured Weekly Over a Period of Four Weeks
220
40
AN OV A Table of Survival Percentage of Excised Embryos D ried to N ear Cri tical Moisture Level and Stored in Liquid Nitrogen
221
41
AN OVA Table of Survival Percentage of Excised Various Embryos After Cryoprotecting in Combination of Cryoprotectants for Various Period of Tim e
221
42
A N OVA Table of Survival Percentage of Excised Embryos Cryoprotected for Six Hours F ollowed by Storage Treatments and Direct Placement in Liquid Nitrogen
222
43
AN OVA Table of Survival Percentage of Excised E mbryos Cryoprotected for Twelve Hours Followed by Storage Treatments and Direct Placement in Liquid Nitrogen
223
44
A N OVA Table of Survival Percentage of C ryopreserved E mbryos After C ryoprotection and Desiccation for One, Two and Three Hours
224
45
AN OVA Table of Survival Percentage of Excised Embryos After Twelve Hours of Cryoprotection Followed by Partial Desiccation for One and Two Hours and Two Methods of Cooling
224
xii
TAB L E
PAGE
46
AN OVA Table of Survival Percentage of Cryoprotected E mbryos Partially Desiccated and Subjected to Slow F reezing Protocol
225
47
AN OV A Table of Assessment of Cryopreserved Embryos Stored in Liquid Nitrogen Over a Period of One Month U sing the Slow Freezing Protocol
225
48
Mean Square Values for Mean Dry Matter Content and Shoot to Root Ratio of Control and Cryopreserved Embryos Cultured on Two Different Media
226
49
AN OV A Table of Survival Percentage of Control and Cryopreserved Seedlings in Polybags After Eight Weeks of Planting in the Nursery
226
xiii
LIST OF PLATES PLATE
PAGE
1
A Jackfruit of Variety NS. 1
41
2
Excision of the Sharp Scalpel
Embryo of Jackfruit Using a
41
3
The Selected Three Stages of Fruit Along With Their Seeds (a) Fully Ripe (b) Ripe Fruit (c) Mature U nripe F ruit
78
4
D eveloping Plantlets from Embryos Excised from Fruits at the Three Stages of Maturity After Six W eeks in Culture (a) F ully Ripe Fruit (b) Ripe Fruit (c) Mature Unripe Fruit (Culture tube diam . 2 5 m m)
80
5
Comparison of Four Metabolic Enzymes in the Embryos from F ruits at the Three Stages of Maturity (a) Fully Ripe Fruits (b) Ripe Fruits (c) Mature U nripe Fruits
83
6
Variability in Embryo Size Excised from a Random Sample of Seeds
85
7
Close up of Selected Embryo Measuring 4.05.0 m m U sed in the Study
85
8
Plantlet Development in Medium Containing 0.1 m g/l each of N A A and Kinetin in the Absence of Charcoal After Eight W eeks of Growth (Culture tube diam. 25 m m )
92
9
Plantlet Development in M edium Containing 1 . 0 m g/l each of N A A and K inetin in the Presence of Charcoal, A fter Eight Weeks of Growth (Culture tube dia m . 25 m m )
92
10
Plantlet Development i n Medium Containing 0 . 1 m g/l each of N A A and BAP in the Absence of Charcoal, After Eight W eeks of Growth (Culture tube dia m . 25 m m )
97
11
Plantlet D evelopm ent in Medium Containing 1 . 0 m g/l each of N AA and BAP in the Presence of Charcoal, After Eight Weeks of Growth (Culture tube dia m . 25 m m)
97
xiv
P LATE
PAGE
12
Plantlet Development in Medium Containing 0.1 m g/! each of NAA and 2iP in the Absence of Charcoal, After Eight Weeks of Growth (Culture tube diam. 25 m m )
100
13
Plantlet Development i n Medium Containing 1 . 0 m g/! each of N AA and 2iP in the Presence of Charcoal, After Eight Weeks of Growth (Culture tube dia m . 25 m m )
100
14
Comparison of Root Development With and Without Charcoal
M edia
101
15
Viability of Embryos Dried Over a Period of Six Hours by A m bi ent Drying Method (Culture tube diam . 20 m m )
105
16
Viability of Embryos Dried Over a Period of Six Hours by Air-Condition Drying Method (Culture tube diam. 20 m m )
105
17
Viability of Em bryos Dried Over a Period of Six Hours by Laminar Air Flow Drying M ethod (Culture tube diam. 20 m m )
1 06
18
Viability of Embryos Dried Over a Period of Six Hours by Silica-Gel Drying M ethod (Culture tube diam . 20 m m )
106
19
Stunted Growth of Embryos Dried to Moisture Content of 9-1 0 %
108
20
Embryo Growth and Development After Drying to Six Moisture Levels in a Lam inar Air Flow Cabinet (a) 14-1 5% (b) 1 3-14% (c) 1 2-1 3 % (d) 1 1- 1 2 % (e) 1 0-1 1 % (f) 9-1 0 %
1 08
21
C ellular Structure o f Radicle Tip and Cortex from Excised Embryos of Fresh Jackfruit Seeds (0 Hours of Drying) (a) Radicle Tip (x 2 0 0 ) (b) Cortex Tissue o f Radicle ( x 400)
114
22
C ellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to One Hour of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400)
116
23
Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Two Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400)
1 17
xv
in
PLATE
PAGE
24
Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Three Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400)
118
25
C ellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Four Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400)
119
26
Cellular Structure of Radicle Tip and Cortex from Excised E mbryos Subjected to Five Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400)
120
27
Cellular Structure of Radicle Tip and Cortex from Excised E mbryos Subjected to Six Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400)
121
28
Embryos at 1 4-1 5 % Moisture Showing Survival After Storage in Liquid Nitrogen for Twelve Hours (Culture tube diam. 25 m m )
125
29
Embryos at 1 3-14% Moisture Showing Survival A fter Storage in Liquid Nitrogen for Twelve Hours (Culture tube dia m . 2 5 m m )
125
30
Embryos at 1 2-1 3 % Moisture Showing Survival A fter Storage in Liquid Nitrogen for Twelve Hours (Culture tube diam . 25 m m )
1 25
31
Embryos Showing Either Shoot or Root Development After Cryoprotection Followed by Rapid Freezing After Four Weeks in Culture
135
32
Embryos Showing Normal Development of Shoot and Root A fter Cryoprotection Followed by Slow Freezing After Four Weeks in Culture
135
33
Ultrastructure of Jackfruit Embryo
Fresh
140
34
Ultrastructure of Radicle C ells from Jackfruit Embryos Cryopreserved After Desiccating to 1 4-1 5% Moisture Content
141
Radicle
xvi
Cells
from
P LATE
PAGE
35
Ultrastructure of Radicle C ells from Jackfruit Embryos C ryopreserved After Desiccating to 1 2- 1 3 % Moisture Content
142
36
Ultrastructure of Radicle C ells from Jackfruit Embryos Cryopreserved After Desiccating to 9-1 0 % Moisture Content
144
37
Ultrastructure of Radicle Cells from Jackfruit Embryos C ryopreserved Directly in Liquid Nitrogen After Twelve Hours of C ryoprotection and Partial Desiccation to 2 9-3 3 % Moisture Content
145
38
Ultrastructure of Radicle C ells from Jackfruit Embryos Cryopreserved After Twelve Hours of C ryoprotection Followed by Partial Desiccation to 2 9-3 3 % Moisture Content and Slow Cooling to -40 ° C
1 46
39
Control Embryos Developing on Initially Established and Enriched Media (a) Initially Established Medium (b) Enriched Medium
1 50
40
Cryopreserved Embryos Developing on Initially Established and Enriched Media (a) Initially Established Medium (b) Enriched Medium
151
41
Plantlets in Glasshouse the Misting Chamber
from
152
42
Comparison Between Control and C ryopreserved Plantlets After F ive W eeks in a Misting Chamber (a) Control Plantlet (b) C ryopreserved Plantlet
152
43
Fresh Seeds and Seeds Stored for Three Days (a) Fresh Seeds (b) Seeds Stored for Three Days Showing Signs of Germination
1 54
44
Jackfruit Trees in a Small Scale Plantation
198
45
Close up of Jackfruits Developing on the Tree
198
46
A Section Through a Jackfruit Fruit (f), Seed (s) and Embryo (e)
After
xvii
Removal
Showing
the
200
LIST OF FIGURES PAGE
FIGURE 1
Graph Showing Rate of Cooling for the Alcohol Bath (Julabo - Model F40-HC )
xviii
48
LIST OF ABBREVIATIONS
The following abbreviations w ere used in the text :
NAA
olJ-Napthalene acetic acid
BAP
Benzylam inopurine
2iP
2-isopentyl adenine
LSD
Least Significant Difference
DN M RT
Duncan's N ew Multiple Range Test
RH
Relative Humidity
EtOH
Ethyl alcohol
TBA
Tertiary Butyl Alcohol
w/w
weight by weight
v/v
volume by volume
min
minutes
°C
Degree centigrade
mg
m illigram
g
gram
I
litre
J.Ll
microlitre
Kg/cm 2
K ilogram per square centim etre
).18
Microsemens
gIl
grams per litre
m g/l
m iligram per litre
MS
Murashige and Skoog M edium formulation
%
percentage
D MSO
Dimethysulf oxide
Kg
Kilogram
rpm
revolution per minute
G A3
Gibberellic acid
xix
ABSTRACT
Abstract of the thesis submitted to the Senate of Universiti Pertanian Malaysia in partial fulfilm ent of the requirements for the Degree of Doctor of Philosophy.
TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVA110N OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.)
by
BASKARAN KRISHNAPILLAY
July, 1989
Supervisor
Professor C hin Hoong Fong
C o-Supervisor
Dr Hor Yue Luan
F aculty
Agriculture
Long term storage of recalcitrant seeds by conventional storage m ethods have by far been unsuccessful. In this study the excised embryos of seeds of jackfruit (Artocarpus heterophyllus). a recalcitrant species has been attempted at for long term preservation using cryogenic m eans.
Stage of fruit maturity was critical in the study where embryos excised from mature unripe fruits w ere found to be the m ost appropriate.
xx
Selection of uniform sized e mbryos (4.0 - 5.0 m m in length) minimised variability in e mbryo fresh weight and moisture content to a considerable extent.
A modified Murashige and Skoog medium containing 1 . 0 mgll each of NAA and BAP, 2 gil of activated charcoal and 1 7 0 mgll of NaH 2 P 04 . H2 0 was found to be a suitable medium for development and growth of the excised jackfruit e mbryos.
The safe critical moisture limit for excised jackfruit e mbryos was found to be around 1 4-1 5%.
Storage of e mbryos dried to this moisture
limit by conventional storage methods w ith a view to long term storage was unsuccessful. month.
The maximum storage period achieved was only one
Attempts at direct storage in liquid nitrogen after desiccation
to near critical moisture lim i t was also found to be unsuccessful.
Embryos cryoprotected with 1 0 % D MSO
+
0 . 5 % proline for 1 2 hours
followed by a partial desiccation to moisture contents of 2 9-33 % and slowly frozen to -40 ° C at approximately 1 ° C/min before plunging into liquid nitrogen survived cryopreservation and gave approximately 6 0 % normal recovery.
No significant decline in the viability percentage was
observed in those e mbryos stored by this method for a period of one month.
For normal recovery growth of e mbryos after cryopreservation, addition of other growth promoting substances namely GA 3 ' glutamine, argenine, asparagine and adenine sulphate to the initially established medium was found to be beneficial. xxi
Only 5 0 % of the w ell developed plantlets transferred to polythene bags w ere able to survive the transplanting shock and resume normal growth in the glasshouse. The plantlets required a hardening period of five w eeks in a high humidity chamber before they were able to withstand the ambient conditions in the glasshouse.
xxii
ABSTRAK
Abstrak tesis yang dikem ukakan kepada Senat U niversiti P ertanian Malaysia bagi m e m enuhi sebahagian daripada syarat-syarat untuk memperolehi Ijazah Doktor Falsafah.
TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.)
oleh
BASKARAN KRISHN APILLAY
July, 1 989
Penyelia
Professor C hin Hoong �ong
Penolong P enyelia
Dr Hor Yue Luan
Fakulti
Pertanian
P enyimpanan biji benih tempoh panjang m elalui kaedah penyimpanan biasa, belum berjaya setakat ini.
Dalam kajian ini, embrio-embrio yang
telah diasingkan dari biji benih nangka (Artocarpus heterophyllus), sejenis spesies
rekalsitran,
telah
dikaji
sebagai
bahagian
penyimpanan tempoh panjang m elalui kaedah kriogenik.
xxiii
tumbuhan
untuk
Peringkat kematangan buah didapati sangat penting.
Embrio dari
biji benih dalam buah yang matang tetapi belum masak, didapati yang sesuai sekali. panjangnya)
Pemilihan e m brio yang saiznya seragam (4.0 - 5 . 0 m m
didapati
mengurangkan variabiliti dalam
berat segar dan
kandungan kelembapan e mbrio.
Medium
Murashige dan Skoog diubahsuai, yang mengandungi 1.0
mgll N A A dan BAP masing-masing, 2 gil arang diaktifkan dan 1 7 0 mgll N a H2 P 04 . H2 0 didapati sesuai untuk perkembangan dan tumbesaran e mbrio nangka.
U ntuk embrio nangka, had kritikal kelem bapan didapati lebih kurang 1 4-1 5%.
Ujian
untuk
m enyimpan
e mbrio
yang
dikeringkan
kepada
kelembapan ini, melalui kaedah biasa tidak berjaya. Tempoh penyimpanan maksimum yang dicapai hanyalah selama sebulan sahaja. Percubaan untuk menyim pan e mbrio secara terus dalam nitrogen cecair selepas dikeringkan sehingga hampir had kritikal, juga didapati tidak sesuai.
Embrio yang dirawat dengan D MSO 1 0 %
+
0.5% prolin selama 1 2
jam diikuti oleh separuh kekeringan sehingga 2 9-33 % kelembapan dan dibekukan dengan perlahan pada kadar l ° C/min sehingga -40 ° C sebelum diletakkan kedalam nitrogen cecair, didapati terus hidup dan menunjukkan kepulihan lebih kurang 6 0 %.
Tiada penurunan yang bererti dalam peratus
viabiliti bagi e mbrio yang disimpan melalui kaedah ini selama sebulan.
U ntuk krioawetan,
m e mperolehi penambahan
tumbesaran bahan
normal
penggalakan xxiv
bagi
em brio
tumbesaran
(iaitu
selepas GA 3 ,
