UNIVERSITI PUTRA MALAYSIA

TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS

OF RAMBUTAN (NEPHELIUM LAPPACEUM L.)

HIEW YEE HOOI

FP 1991 9

TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVAT10N OF

EMBRYOS OF RAMBUTAN (NEPHEL1UM LAPPACEUM L.)

BY

llIEW YEE H001

Thesis Submitted in Fu lfilment of the Requirements for the Degree of Master of Science

in the Facu lty of Agricul ture Universiti Pertanian Ma laysia

MARCH 1 9 9 1

DEDICATED

to my parents

for their understanding and encouragement which have been a

constant source o f i nspiration for me throughout this s tudy

ACKNOWLEDGEMENTS

Gra t e f u l acknowl edgement i s extended t o Univers i t i

Pertan i a n Ma l ays i a f o r enab l i ng t h i s s tudy t o b e c a r r i ed

out .

The author a l s o w i s hes t o e xpre s s her s i ncere

appre c i a t i on t o :

Pro f e s s o r C h i n Hoong Fong and Dr . Hor Yue Luan ,

superv i s o r and c o - s uperv i s o r o f t h i s s tudy , f o r t h e i r

couns e l and encouragement .

Dr . Z a l i ha C h r i s t ine A l ang for her permi s s ion t o u s e the

P l ant T i s s u e Cu l ture Laboratory f ac i l i t i es f o r c ar ryi ng

out a ma j or por t i on of t h i s researc h .

Pro f e s s o r Yap Thoo Cha i and Dr . Ridzwan A . Ha l im f o r

s ugge s t i ons o n s t at i s t i ca l analys i s o f data .

Dr . Bas karan K r i s hnapi l l ay for h i s adv i c e and guidance

throughout t h i s s tudy .

Enc i k Abdu l Gha n i Has h i m and Mr . Teoh Peng Kee f o r t h e i r

pro f e s s i onal photographi c work .

i i i

TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS i i i

L I ST OF TABLES vi i i

L I ST OF F I GURES x i i

L I ST OF PLATES x i i i

L I ST OF ABBREVIATIONS x v i

ABSTRACT xv i i

ABSTRAK

CHAPTER

1

2

x x

INTRODUCTI ON 1

REVI EW OF LITERATURE 6

Characteri stics of Recalci trant Seeds 6

Moisture Content of Recalcitrant Seeds 9

Desiccation and Chi l l ing Sens itivity o f Recal c itrant Seeds 1 2

Storage Behaviour of Recalci trant Seeds of Tropical Tree Fru its 1 8

I n Vitro Storage o f Germplasm Conservation 2 1

Nature of Plant Material 2 3

Pre- freezing Treatments 25

Cryoprotection 27

Freezing 33

S l ow Coo l i ng Method 3 4

Rapid Coo l i ng Method 3 4

Pre - f r e e z i ng Method 35

i v

3

PAGE

Thawing 3 9

Recovery 4 0

Cryopreservation of Zygotic Embryos 4 1

MATERIALS AND METHODS 4 5

Study Layout 4 5

Seed Materials 4 8

Experimenta l Procedures 4 8

Exc i s ion o f Embryos 4 8

Regu l a t ion o f Embryo Mo i s tu re 5 0

Chemi c a l s 5 1

Preparat ion o f S tock So l u t ions and Cul ture Med i a 5 1

I ncubat ion o f Cu l tures 5 3

Preparat ion o f Cryoprotectants 5 3

Coo l i n g and Thaw i ng Procedures 5 4

Measurements and Observations 5 5

Mo i s tu re Content 5 5

Dry Matter Content and S hoot to Root Rat io o f I n V i t ro Grown Seed l i ngs 5 7

Germ i n a t ion 5 8

Experiments 5 9

S e l ec t ion o f Opt i ma l S tage o f Fru i t Ma turi ty for E xc i s ed Embryos 5 9

S e l ec t ion o f Embryos for Cryopres ervat ion S tud i es 6 1

v

4

5

E s tabl i s hment o f a S u i ta b l e Med i um f o r the Growth a nd Deve l opment o f Exc i s ed Rambu tan Embryos

Des i c c a t i o n E f fects on Who l e S eeds and Embryos in Re l a t i o n t o V i ab i l i ty a nd S t o rage

Use o f Cryoprotectants

Modi f i ca t i o n of C u l ture Med i um for I mproved Surv i va l and Growth o f Cryopres e rved Embryos

RESULTS

Selection of Optima l Stage of Fruit Maturity for Excised Embryos

Selection of Embryos for Cryopreservation Studies

Establ ishment of a Suitable Medium for the Growth and Development of Excised Embryos

Des iccation E f fects on Whole Seeds and Embryos in Relation to Viabi l i ty and Storage

Use of Cryoprotectants

Mod i f i cation of Cu lture Medium for Improved Survival and Growth of Cryopreserved Embryos

DI SCUSS ION

Selection of Optimal Stage of Frui t

PAGE

6 3

6 6

6 8

7 2

7 4

7 4

8 2

8 8

1 1 2

1 1 9

1 35

1 3 7

Maturi ty for Excised Embryos 137

Selection of Embryos for Cryopreservation Studies 1 4 3

Establ ishment of Suitable Medium for the Growth and Development of Exc ised Embryos 1 4 3

v i

6

Des iccation E f fects on Whol e Seeds and Embryos in Relation to Viabi l i ty and Storage

Use of Cryoprotectants

Mod i fi cation of Culture Medium for Improved Survival and Growth o f Cryopreserved Embryos

SUMMARY AND CONCLUS ION

BIBLIOGRAPHY

APPENDIX

A

B

C

VITA

Background of the Fruit ( Nephel ium lappaceum)

Murashige and Skoog (1962 ) Inorganic Salts and Vitamins

ANOVA Tables

v i i

PAGE

1 4 7

1 5 1

1 5 7

1 5 9

1 6 3

1 8 0

1 8 0

1 9 1

1 9 2

2 0 9

TABLE

1

2

3

4 a

4 b

LIST OF TABLES

Viabi l i ty Percentage o f Embryos Exc i s ed f rom t he Three S tages o f F ru i t Matu r i ty A f t e r F ive Weeks I n C u l ture

Categorizing E xc i s ed Embryos by S ize , f rom a Random Samp l e o f Seeds Ta ken f rom Mature Unr i pe F ru i ts

Fresh We i ght and Mo i s ture Content o f Exc i sed Embryos f rom the Var i ous S e l e c t i o n Methods

Mean Dry Matter Content o f P l an t l e t s Grown I n V i t ro on Medi a Conta i n i ng NAA / K i n e t i n and W i th or Wi thout Act i va ted Charcoa l

Mean S hoot t o Root Rat i o o f P l a n t l e t s Grown I n Vi tro on Med i a Conta i n i ng NAA/K i ne t i n and W i t h or W i thout Act i va ted Charcoa l

Sa Mea n Dry Mat t e r Content o f P l a n t l e t s Grown I n V i tro on Medi a Cont a i n i ng NAA/BAP and W i t h or Wi thout Act i va t ed Charco a l

Sb Mean S hoot to Root Rat i o o f P l an t l e t s Grown I n Vi tro on Med i a Conta i n i ng NAA / BAP a nd W i t h or Wi thout Act i va ted Charcoal

6a Mean Dry Matter Content of P l an t l e t s Grown I n Vi tro o n Med i a Cont a i n i ng NAA/ 2 i Pand W i t h or Wi thout Act i vated Charcoa l

6 b

7

Mean S hoot to Root Rat i o o f P l a n t l e t s Grown I n V i tro o n Med i a Cont a i n i ng NAA/ 2 i P and W i t h or Wi thout Ac t i va ted Charcoal

Cr i t i c a l Mo i s ture Content and Surv i v a l Percentage o f E xc i s ed Embryos W i t h a nd W i t hout S torage i n Liqu i d Ni trogen

vi i i

PAGE

7 8

84

8 6

8 9

9 0

9 9

1 0 0

1 05

1 0 6

1 2 0

TABLE

8

9 a

9 b

lOa

lOb

1 1

12

13a

13b

S u rv i va l Percentage o f Exc i s ed Embryos A f t e r C ryoprotect i ng i n Va r i ou s Comb i na t i o n o f C ryoprotectants f o r Va r i o u s P e r i od o f Time

Surv i va l Percentage o f E xc i s ed Embryos C ryoprotected for Three Hours F o l l owed by S torage Treatments and D i re c t P l acement i n Liqu i d N i trogen

Surv i v a l Percentage of Exc i s ed Embryos C ryoprotected for S i x Hours Fo l l owed by S torage Treatments and D i rect P l acemen t i n Liqu i d N i t rogen

Mo i s t u re Content in E xc i s ed Embryos A f t er Cryoprotec t i o n a nd Des i cc a t i o n for I , 2, 3, 4 and 5 Hours .

S u rv i va l Percentage o f C ryopres e rved Embryos A fter C ryoprotec t i o n and Des i c c a t i o n for I, 2, 3, 4 and 5 Hours

S u rv i v a l Percentage of E xc i s ed Embryos A f ter S i x Hours o f Cryoprotec t i on Fo l l owed by Part i a l Des i cc a t i on for 1 and 2 Hours and Two Methods o f Coo l i ng

ANOVA Tabl e o f V i ab i l i ty Percentage o f E xc i s ed Embryos f rom Three S tages o f Fru i t Mat u r i t y A f t e r F i ve Weeks I n C u l ture

Mean Square Va l u e s for the Regre s s i on o f Mo i s ture Content and Dryi ng T i me o f Exc i s ed Embryos f rom the Three S tages of Fru i t Matur i ty

Mean Square Va l u e s for the Regres s i o n o f S u rv i va l Percentage a nd Mo i s ture Content of E xc i s ed Embryos f rom the Three S tages of Fru i t Maturi ty

i x

PAGE

1 23

1 2 5

1 2 6

1 2 9

12 9

134

1 9 4

1 9 5

1 9 5

TABLE

1 4

l S a

lSb

1 6 a

1 6 b

1 7 a

1 7 b

1 8 a

Mean Square Va l u e s f o r Mea n , Coe f f i c ient o f Var i a t i o n o f Fre s h We i g h t and Mo i s ture Content o f Exc i s ed Embryos for t he Var i ou s S e l e c t i o n Methods

ANOVA Tabl e of Mean Dry Matter Content o f P l an t l e t s Grown I n V i t ro on Medi a Conta i ni ng NAA / K i ne t i n and W ith or W i t hout Act i vated Charcoal

ANOVA Tabl e of S hoot t o Root Rati o o f P l ant l et s Grown I n V i t ro o n Med i a Cont a i n i ng NAA/K i ne t i n and W i t h o r W i thout Act i vated Charcoal

ANOVA Tabl e of Mean Dry Matter Content o f P l ant l et s Grown I n Vi tro on Med i a Conta i n i ng NAA/ BAP and With o r W ithout Act i va ted Charco a l

ANOVA Tabl e o f Shoot t o Root Rat i o o f P l ant l e t s Grown I n Vi tro o n Med i a Conta i n i ng NAA / BAP and W i t h or W i thout Act i va ted Charc o a l

ANOVA Tab l e o f M e a n Dry M a t t e r Content o f P l an t l e t s Grown I n Vi tro on Medi a Conta i ni ng NAA / 2 i P and W i t h o r Wi thout Act ivated Charco a l

ANOVA Tab l e o f Shoot to Root Rat i o o f P l ant l ets Grown I n V i t ro o n Med i a Cont a i n i ng NAA/ 2 i P and W i t h or Without Ac t i va t ed Charc o a l

M e a n Square Va l u e s for the Regre s s i o n o f Mo i s ture Content and Dry i ng T i me o f Who l e Seeds and Embryos Exc i s ed from Des i cca ted Seeds

x

PAGE

1 9 6

1 9 7

1 9 8

1 9 9

2 0 0

2 0 1

2 0 2

2 03

TABLE

1 8b

1 9

2 0 a

2 0b

2 1

2 2

2 3

2 4

M e a n Square Va l u e s for the Regre s s i on o f Surv i v a l Percentage and Mo i s ture Content o f Who l e Seeds, Embryos E xc i s ed f rom Des i ccated S eeds and Exc i s ed Embryos

ANOVA Tabl e o f Moi s ture Content and S u rv i va l Percentage of E xc i s ed Embryos Des i cc a t ed Over a Mo i s ture Range o f 1 0 % t o 1 5%

ANOVA Tabl e o f Surv iv a l Percentage o f Exc i s ed Embryos A f t e r C ryoprotec t i ng i n Va r i ou s Comb i na t i on o f C ryoprotectants f o r Var i ous P e r i od of Time

ANOVA Tabl e o f Surv i v a l Percentage o f E xc i s ed Embryos A fter Cryoprotect i ng i n Var i o u s C omb i nat i ons o f Cryoprotectants f o r Var i ous P e r i od of Time

ANOVA Tabl e o f Surv i v a l Percentage o f E xc i s ed Embryos Cryoprotected f o r Three Hours Fo l l owed by S t orage Treatments and D i rect P l acement i n L i q u i d N i t rogen

ANOVA Tab l e of Surv iva l Percentage o f E xc i s ed Embryos Cryoprotected f o r S i x Hours Fol l owed by S torage Treatmen t s and D i rect P l a c ement i n L i qu i d Ni trogen

ANOVA Tabl e of Surv i v a l Percentage o f C ryopres erved Embryos A f t e r Cryoprotec t i on a n d Des i cc a t i on f o r 1 , 2, 3, 4 and 5 Hours

ANOVA Tab l e o f Survi v a l Percentage o f E xc i sed Embryos A f t e r S i x Hours o f Cryoprotect i o n Fo l l owed by Part i a l Des i cc a t i on for 1 and 2 Hours and Two Methods o f Coo l i ng

x i

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2 03

2 04

2 04

2 0 5

2 0 6

2 0 7

2 0 8

2 0 8

F I GURE

la

Ib

2 a

2 b

2 c

LIST OF FIGURES

Mo i s ture Content o f E xc i sed Embryos f rom Three S tages of Fru i t Ma tu r i ty Over a Des i cc a t i on Peri od o f 1 0 Hours

Des i cc a t i o n To l erence o f E xc i s ed Embryos f rom Three S tages o f F ru i t Mat ur i ty Over a Des i cc a t i on P e r i od o f 1 0 Hours

Mo i s ture Content of Who l e S eeds and Embryos E xc i s ed f rom Them Over a Des i cc a t i on Period o f 5 0 Hours

Des i cc a t i o n To l erence and Embryos E xc i s ed S e eds

of Who l e S eeds f rom Des i c c ated

Des i cc a t i on Toleren c e o f Embryos E xc i s ed P r i o r To and A f te r De s i cc a t i o n

x i i

PAGE

7 9

8 1

1 1 4

1 1 5

1 1 6

PLATE

1

2

3

4

5

6

7 a - b

7 c - d

7 e - f

7 g - h

8 a

LIST O F PLATES

Rambut a n Seeds ( Vari ety R7 )

Embryos E xc i s ed f rom Rambu tan S eeds

Four Maturi ty S tages o f Fru i t ( a ) I mma ture Young ( b ) Mature Unripe ( c ) Mature R ipe ( d ) Mature Fu l ly Ripe

Deve l opment of E xc i s ed Embryos f rom the Four S tages o f Fru i t Maturity A f ter F i ve Weeks In Cu l ture ( a ) I mmature Young ( b ) Mature Unr i pe ( c ) Mature Ripe ( d ) Ma ture Fu l ly Ripe

Var i ab i l i ty in Embryo S i z e E xc i sed f rom a Random Samp l e o f Seeds

S e l ec t ed Embryo Mea s u r i ng 3.02_4.0� i n S u r f ac e Area / S ide Used i n the S tudy

Growth a nd Dev e l opment o f E xc i sed Embryos in Med ium With and Wi thout Charcoa l ( Wi th the Pres ence of ( a ) O, ( b ) 0 . 1 , ( c ) 1 . 0 and ( d ) 10 . Omg / 1 NAA )

Growth and Deve l opment o f E xc i s ed Embryos i n Medium W i t h a nd Wi thout Charc o a l ( Wi t h the Presence o f ( a ) O, ( b ) O . l, ( c ) l . O and ( d ) l O . Omg/ l NAA i n Comb i na t i o n W i t h O . lmg / l K i net i n )

Growth and Deve l opment o f E xc i s ed Embryos i n Medium W i t h and Wi thout Charc o a l ( Wi t h the Presence o f (a ) O, (b ) O . l, (c ) l . O and (d ) l O . Omg / l NAA i n Comb i na t i on W i t h 1 . Omg / l K i n e t i n )

Growth and Devel opment o f E xc i s ed Embryos i n Med i um W i t h and W i t ho u t Charc o a l ( Wi t h the Pre s ence o f (a ) 0, (b ) O . l, ( c ) l . O and ( d ) l O . Omg / l NAA i n Comb i na t i on W i t h l O . Omg / l K i ne t i n )

P l a nt l et Devel opment i n Med i um Conta i n i ng O . lmg / l Each o f NAA and K i net i n i n the Abs ence o f Charcoa l

x i i i

PAGE

49

49

7 5

7 6

8 5

8 5

92

93

94

95

96

PLATE

8b

9 a

9 b

l O a

l Ob

1 1

1 2

13a-b

13c

1 4 a

1 4 b

P l an t l e t Devel opment i n Med i um Cont a i ni ng 1.0mg / l Each of NAA and K i ne t i n in the Pre s ence of Charc o a l

P l ant l e t Dev e l opment i n Cont a i n i ng O . lmg / l Each of BAP i n the Abs ence of Charcoal

P l a n t l et Deve l opment i n Cont a i n i ng 1.0mg / l Each o f BAP i n the Presence o f Charc o a l

P l ant l e t Devel opment i n Cont a i n i ng O . lmg / l E a c h of 2 i P i n the Abs ence o f Charc o a l

Med i um NAA and

Med i um NAA and

Med i um NAA and

P l ant let Devel opment in Medi um Conta i n i ng 1 . 0mg / l Each of NAA and 2 i P in the Presence of Charc o a l

C a l l u s Growth i n Med i um Conta i n i ng 1 0.0mg / l Each o f NAA and BAP i n the Abs ence of Charcoa l

Dead E xc i s ed Embryos w i t h C r i t i ca l Mo i s ture Content Aft e r Expo s u re t o L i qu i d N i t rogen

Embryos S howi ng Root Deve l o pment A f te r Cryoprotect i on Fo l l owed by Rapi d Free z i ng

Embryos Showing S hoot Deve l opment A f ter Cryoprotect ion Fo l l owed by Rap i d Free z i ng

Devel opment o f Embryos i n Enri ched Weeks I n Culture

Non-Cryopres e rved Medi um After 5

Devel opmen t o f Cryopreserved Embryos i n Enri c hed Medi um A f ter 5 Weeks I n Cu l tu re

x i v

PAGE

9 7

1 0 2

1 03

1 0 7

1 0 8

1 1 1

1 2 1

1 3 1

132

1 3 6

136

PLATE

1 5

1 6

1 7

1 8

1 9

2 0

2 1

2 2 a

2 2 b

The Rambutan Fru i ts

D i f ferent Shape a nd S i z e o f Rambutan Seeds ( a ) : Rambutan Seeds of Va r i ety R7

Embryon i c Ax i s o f Rambu tan That E nc l os ed W i t h i n the Cotyledons

The D i f ferent Attachment Pos i t i ons o f Rambutan Embryos Between the Two Unev e n l y S i z e Cotyl edons

Budded Rambutan Trees ( Va r i ety R7 )

Lea f s and Young S hoots o f Rambutan ( Va r i e ty R7 )

An I n f l orescence Hav i ng S t ructura l ly Hermaphrod i te F l owers ( Va r i ety R7 )

Young and I mmature Rambutan Fru i t s ( Va r i e ty R7 )

Ma tured R i pe Rambutan F ru i t s ( Va r i ety R7 )

xv

PAGE

1 8 2

1 8 2

1 8 3

1 8 3

1 8 9

1 8 9

1 9 0

1 9 1

1 9 1

LIST OF ABBREVIATIONS

The f o l l ow i ng abbrevi a t i ons were u s ed i n the text:

NAA �-Naptha l ene a c e t i c a c i d

SAP

2 i P

LSD

DNMRT

RH

E tOH

w/w

v / v

m i n

°c

mg

g

1

u l

gil

mg / l

MS

%

DMSO

rpm

GA )

Senzyl ami nopuri ne

2 - i s opentyl aden i ne

Leas t S i gni f i cant D i f f erence

Duncan ' s New Mu l t ip l e Range Tes t

Re l a t i ve Hum i d i ty

E t hyl Al coho l

we i ght by wei ght

vol ume by vol ume

m i nu t e s

Degree c e nt i grade

m i l i gram

gram

l i t re

m i c ro l i tre

grams per l i tre

m i l i grams per l i tre

Murashige and S koog Medi um formu l a t i on

percentage

Dimethyl s u l f o x ide

revo l u t i o n per m i nute

G i bbere l l i c acid

xv i

Abs t ract o f the thes i s pres ented to the S enate o f Un ivers i t i Perta n i an Ma l ays i a a s f u l f i l ment o f the requ i rements for the degree of Mas ter of S c i ence

TOWARDS THE DEVELOPMENT O F A PROTOCOL FOR CRYOPRESERVATION OF

EMBRYOS OF RAMBUTAN ( Nephel ium lappaceum L.)

by

HIEW YEE HOOl

MARCH , 1 9 9 1

Supervi s o r Pro f e s s o r C h i n Hoong Fong

Facu l ty Agr i c u l ture

Key Words Nephe l i um l appaceum , excis ed embryos ,

c ryopres ervation .

Long t erm s t orage o f rec a l c i trant s eeds by

conve n t i ona l s t orage methods thus f a r have been

unsucces s f u l . In t h i s s tudy , the exc i s ed embryos f rom

s eeds o f Rambu tan ( Nephe l i um l appac eum ) , a reca l citrant

t ropi c a l f ru i t spec i es has been a ttempted f o r l ong t erm

s torage through c ryopr"e serva t i on.

xv i i

S tage o f f ru i t matu r i ty was found to be one o f the

important factor for the surv i val of embryos in v i tro .

The embryos exc i s ed f rom mature unripe f ru i t s were

s elected a s the mos t appropr i a te for u s e in t h i s s tudy .

Mi n i m i z i ng variability i n f re s h we i ght and moi s ture

content was found to be es sent i al i n t h i s s tudy .

S ele c t i o n o f u n i form s i z ed embryos, mea s u r i ng 3 . 0 2 - 4 . 0 2mm

i n s u r f a c e area per s ide o f the cubi c al bloc k s o f

cotyledonary t i s sue that enclos ed the embryos ; ens ured

un iform f r e s h we i ght and mo i s ture content .

A modi f i ed Mura s h i ge and Skoog medi um conta i n i ng

1 . 0 mg ll each o f NAA and K i ne t i n, 2 gil o f a c t i va ted

charcoal and 1 7 0 mgll of NaH -,,0 4 . H .p was f ound to be

s u i table medi um f o r the development and growth of the

exc i s ed embryos o f rambutan .

Cri t i cal mo i s ture l i m i t o f rambutan embryos was

found to be around 1 0 - 1 5% . Attempts a t d i re c t s torage o f

exc i s ed embryos i n l i qu i d n i t rogen a f ter des i cc a t i o n t o

near c r i t i cal mo i s ture l i m i t wa s found t o be

unsucces s f ul .

xvi i i

Embryos c ryoprotected w i t h l O% DMSO+ I O% Glycerol f o r 6

hours f o l l owed by part i a l des i cca t i on to mo i s ture

contents o f 2 9 - 3 3% and s l ow

appro x i ma t e l y lOC / m i n be f o re

free z i ng

p l u n g i ng i nto l iq u i d

n i trogen s urv i ved c ryopres erva t i o n a n d gave appro x i mate ly

40% v i abi l i ty . However, the s u rv i v i ng embryos were

abnorma l in growth with e i ther root or s hoot deve l opment

only .

I mprovement i n growth and dev e l opment o f

cryopreserved embryos was attempted by suppl ement i ng the

i n i t i a l ly e s t a b l i s hed med i um with add i t i ona l growth

promo t i ng subs tances namel y,

aspara g i ne and aden i ne

GA 3' g l utami ne, arg i n i ne,

s u l phate . However, t he

deve lopment o f the s u rv i v i ng embryos i n the e n r i c hed

med ium was not d i f ferent f rom the i n i t i a l medi um in wh i ch

abnorma l growth o f p lant l e t s was obs erved .

x i x

Abs t rak tes i s yang d i kemukakan kepada Senat Uni vers i t i Pert a n i a n Ma l ays i a bagi memenuh i syara t - syarat untuk mempero l e h i I ja z ah Mas t e r S a i n s .

TERHADAP PERKEMBANGAN SATU PROTOKOL BAGl PENYIMPANAN KRIO EMBRIO RAMBUTAN

( Nephel ium lappaceum L.)

o l eh

HIEW YEE HOOl

MAC, 1 9 9 1

Penye l i a Pro f e s s o r C h i n Hoong Fong

Faku l t i Pert a n i a n

Setakat i n i, penyimpanan b i j i ben i h tempoh panjang

me 1 a l u i kaedah penyimpanan b i a s a, be l um menemu i kejaya a n .

Da l am kaj i a n i n i , embr i o yang t e l ah d i a s i ngkan dari b i j i

ben i h rambutan (Nephe l i um l appaceum ) , s eje n i s s pe s i s

reka l s i tran, te l ah d i kaji s ebaga i bahag i a n tumbuhan u n t u k

peny i mpa nan tempoh panjang me l a l u i kaedah kri ogen i k .

xx

Peri ngkat kematangan buah d idapa ti s ebagai s a t u

f a k t o r pen t i ng demi pengeka l a n keadaan terus hidup embryo

s ecara in v i tro . Embrio dari biji ben i h d a l am buah matang

tetapi b e l um mas ak, d i dapa t i s esua i s e ka l i d i guna d a l am

kaji an i n i .

Pengurangan va r i ab i l i t i bagi berat bas a h dan

kandunga n kel embapan embrio d idapa t i amat penting da l am

kaj i a n i n i . Pemi l i han embr i o yang s a i z nya s e ragam,

berukuran l u a s permukaannya 3 . 0 2 - 4 . 0 2mm tiap s e be l ah

ketu l a n k i u b t i s u kot i l edon yang mengandungi embrio

ada l a h amat p e r l u demi memas t i kan berat basah dan

kandunga n k e l embapan embrio yang s e ragam d i perol e hi .

Med ium Mura s h i ge dan S koog diubahsu ai , yang

menga ndu ngi 1 . 0 mg/ l NAA dan Kinetin masing-masing , 2 gil

arang d i ak t i fkan dan 1 7 0 mg / l NaH2 P04 . H2 0 d i dapati s e s uai

untuk perkembangan dan tumbes aran embri o rambutan .

Untuk embri o rambutan, had kritik a l k e lembapan

d idapa t i l e b i h kurang 1 0 - 1 5% . Percubaan untuk menyimpan

embrio s ecara t erus d a l am n i trogen c ec air s e l epas

d i ke r i ngkan s e h i ngga hampir had kritikal, didapa ti tidak

berjaya .

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Emb r i o yang d i rawat dengan DMSO 1 0 % + G lycero l 1 0 %

s e l ama 6 j am d i i ku t i o l e h s eparuh kekeri ngan s e h i ngga 2 9 -

3 3 % k e l embapan dan d i bekukan dengan perl ahan pada kadar

1°C/mi n s e h i ngga - 4 0°C s ebelum d i l e ta kkaan keda l am

ni trogen ceca i r , d i dapat i terus h i dup dan menu n j ukkan

v i abi l iti l ebih kurang 4 0 % . Wa l a ubaga i mana pun , ha nya

pe rtumbu han embr i o yang abnorma l dengan perkemba nga n

baha g i a n akar a tau pucuk s a haja d idapat i .

Untu k memperol e h i tumbesaran norma l bagi emb r i o

s e l epa s k r i o awetan , penambahan bahan pengga l akan

tumbe s aran , i a i tu GA 3' g l utami ne , argi n i ne , aspara g i n e

d a n ade n i ne s u lphate kepada medi um ku l tur yang d i t entukan

dahu l u , telah d i cuba . Wa l aubaga imana pun , pertumbuhan

embr i o s e l epas krioawetan d a l am medi a dengan tambahan

bahan pengg a l a kan ini t idak berbeza dari yang terdahu l u

d i mana pertumbuhan p l a n t l et abnorma l mas i h d i pe rhat i ka n .

x x i i

CHAPTER 1

INTRODUCTION

Seeds i s one o f the most important i nputs i n crop

p r oduct i on ; i t s qu a l i t y g re a t l y i n f l u e n c e s s u b s e qu e n t

performance a nd y i e l d . Thus , the ma intenance o f seed

viab i l ity in storage i s o f part i cu l a r importance . Roberts

( 1 9 7 3 ) introduced the terms ' orthodox ' and ' reca l c i trant'

to descr ibe the storage behaviour o f seeds .

O rthodox s e e d s a re tho s e that c a n t o l e r a t e

d e s i c c a t i o n a n d f r e e z i n g temp e ra t u r e s . They c a n b e

genera l ly dried to moisture contents i n the range o f 1 - 5 %

without damage . I n dehydrated state , orthodox seeds can

be s t or e d for l on g p e r i od s w i th no s e r i ou s l o s s o f

viab i l ity . Reca lc itrant seeds on the other hand , are

those seeds that are norma l ly shed in a moist condit ion

and do not undergo dry ing during maturation . Attempts to

dry these seeds below some relat ively high critical value

of mo isture content ( 12 - 3 1 % ) resul t in des i ccation inj ury

and d e a th . Futhermore , reca l c i t r a n t s e e d s a re a I s o

sens it ive to free z ing inj ury at l ow temperatures .

1

2

Many o f the maj or pl antat ion crops , fru it trees and

t i mb e r s p e c i e s p rodu c e r e c a l c i t r a n t s e e d s . L i t t l e

attent ion has been given to the storage o f the seeds o f

trop ica l tree fru it spec ies ( Hanson , 1 9 8 4 ) and there are

m a n y s to ra g e p r ob l em s y e t t o b e s o l ved . Be f o r e

cons idering the various storage methods ava i labl e , it i s

n e c e s s a ry t o i d e n t i fy tho s e s e e d s s p e c i e s t o wh ich

reca l c itrant behaviour has been attributed ( Roberts and

King , 1 9 8 2 ) .

A number o f l ist o f reca l c itrant seed species have

b e e n produced ( Ch i n , 1 9 7 8 ; Ch i n a nd Rob e rt s , 1 9 8 0 ;

Harri ngton , 1 9 7 2 ; Roberts , 1 9 7 5 ; Roberts and King , 1 9 8 2 ) .

Un fortunate ly , comprehens ive comp i l at ion i s hampered by

incomp l ete i n format ion for many spec ies . Furthermore , in

some cases seed may have been reported to have been

k i l l ed by drying , a nd there fore might be c l a ss i f ied a s

reca lc itrant , when it i s pos s i b l e that the dry ing method

wa s a t f a u l t a n d that t he s e e d i s r ea l l y o rthodox .

I nevitably , any cata l ogue o f reca l c itrant seed species i s

l ikely to be subj ected t o con s iderabl e mod i f icat ion a s

m o r e r e c a l c i t r a n t s p e c i e s a re i d e nt i f i e d a n d c e rt a i n

s p e c i e s provi s i o na l l y recorded a s r e c a l c i t ra nt a r e

c l a s s i f i ed a s o rthodox ( K i n g a nd Rob e rt s , 1 9 8 0 ) . I n

re a l i z i n g the impo r t a n c e o f i d e n t i f i ca t i on o f

reca l c itrant seeds , Chi n et a l . ( 1 9 8 4 ) proposed a method