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UNIVERSITI PUTRA MALAYSIA
GENETIC STUDIES OF STEVIOSIDE CONTENT AND OTHER AGRONOMIC CHARACTERS AND THEIR RESPONSES TO
CLONING IN STEVIA REBAUDIANA BERT.
WONG LOCK JAM
FP 1991 7
GENETIC STUDIES OF STEVIOSIDE CONTENT AND OTHER AGRONOMIC CHARACTERS AND THEIR RESPONSES TO
CLONING IN STEVIA REBAUDIANA BERT.
By
WONG LOCK JAM
Thesis Submitted in Fulfilment of the Requirements for the Degree of Doctor of Philosophy in the
Faculty of Agriculture Universiti Pertanian Malaysia
September 1991
I would l ike t o dedicate
this the s i s t o Maggi e f o r giving me
the best yea r s of her l i fe
ACKNOWLEDGEMENTS
I am grate ful to the D i r e c tor -Genera l of MARD I ,
Data' Dr . Hj . Yus o f b Hashim , for award ing me the grant
to purs ue t h i s s tudy . Spe c i a l thanks to the Deputy
D irector-Gen€ra l ( Re s ea rc h ) , Dr . S ahar an b H j . Anang , f or
h i s encouragement and k ind s uppor t .
I acknowledge w i t h grat itude the t e c hn i c a l guidance
and profe s s ion a l advice of my l earned s upervi s or, P rof .
Dr . Yap Thoo Cha i . I am a l s o thankful for h i s pat ienc e ,
under s tanding and he lpful s ugge s t ion s in the preparat ion
of t his thes i s .
I would l i ke to thank Tuan H j . Ahmad b . Abdul Wahab
and t he s t a f f of Centra l Ana lyt ic a l S e rv i c e s for showing
me how to extract glucosides from Srevia leaf s amples and
for a s s i s t ing me in identifying and quantifying the maj or
g lucos ide compounds pres ent in the extrac t s . I would a l s o
l ike t o thank En . Anthony Tan for h i s unres erved
cooperation , and En . Moh E s a b . S a iman for he lp ing to
analyze chem i c a lly the f in a l s et of Stevia l e a f s amp l e s .
i i i
My warm e s t thanks to Mr . L im Boon Thong for prov i ding the
pure s te v i o s ide s amp l e s . I am indebted to En . Mohd
Za i lani b . Mohd J a i s and En . Muth uve lu Ch inna for the ir
invaluabl e a s s i s tanc e .
Las t but by no means the lea s t , I am thank f u l t o my
f am i ly for the ir under s t anding and pat ienc e . However , it
wa s the i r s ubt l e e nc ouragement that kept me going .
iv
ACKt;OWLEDGEMENTS
L I ST OF TABLES
L I ST OF F I GURES
L I ST OF PLATES
AB STRACT
ABSTRAK
CHAPTER
I INTRODUCTION
TABLE OF CONTENTS
I I L I TERATURE REVI EW
S t e vi a reba udi a na B e r t on i . - A New Crop
Taxonomy o f S t e vi a re ba u di ana
S tevi o s ide and Other Gluc o s ide
Page
i i i
v i i i
xi
x i i i
xiv
xvi i
1
4
4
6
C ompounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
D i a l l e l Mat ing S y s tems
Hayman ' s Methods
Gr i f f ing ' s Methods
Oakes' Methods
I I I MATERIALS AND METHODS
Evaluat i on of St e vi a C o l l ec t i on
1 4
15
21
25
28
( Exper iment 1) • . • • • • • • . • • • . • . • • • • . . • • . • . . • 28
v
Studie s on S t e vi a C lona l Mat e r i a l s ( Exper iment I I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0
S tudi es o n 7 x 7 D i a l l e l C r o s s ( Exper iment I I I ) . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3
C o l l e c t ion o f Data
Stat i s t ic a l Ana lys i s
I V RESULTS
Eva luat ion o f S t e vi a C o l le c t ion
3 5
42
48
( Exper iment I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Mean Va lues 48
Corre lat ion S tudie s 58
Studi e s on S t e vi a Clona l Mat e r i a ls ( Experiment I I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Ana lys i s o f V a r i ance 64
Mean Values 67
Corr e l a t i o n s 6 8
Studi e s on 7 x 7 D i a l l e l Cro s s ( Exper iment I I I ) .. . .......... .... . ........ 71
Ana lys i s o f V a r i ance 71
Combin ing Abi l ity Ana lys e s 76
Corre l a t i on s 79
Heter o s i s 80
Variance {Vr ) - Cova r iance (Wr ) Ana lys i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3
Stevios ide Content 85
Stevios ide We ight 92
P lant Height 92
v i
Dry Leaf We ight 94
Dry Stem We ight 95
V D I SCUS S ION AND CONC LU S I ON 96
Stevios ide Content 96
Stevios ide We i ght 102
P l ant Height , Dry Lea f Wei ght and Dry S t em We i ght . . . . . . . . . . . . . . . . . . . . . . . 104
BIBLIOGRAPHY 108
APPENDI X TABLES 114
VITA 118
vii
LIST OF TABLES
Tab le
1
2
3
4
5
6
7
8
9
10
1 1
1 2
1 3
Stev io s ide Content o f 6-month-o ld Stevia P l an t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Main G luco s ide Contents ( % ) o f S. rebaudi ana . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .
Corre lat ions ( r ) between the Ma i n Gluc o s ides i n S . reba udiana . . . . . . . . . . . . . . . .
Degree s o f Freedom for Hayman ' s Ana lys is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Expec t ed Mean Squares for B iomet r i c a l Genet i c a l Mode l
Expected Mean S quar e s for GCA and S CA Ana lys es f o r Gri f f ing's Method 2 <mode l I ) . . . . . . . . . . . . . . . . . . . . . . . .
P l ant ing Mater i a l s Us ed i n the Study . . . • . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . .
Expected Mean S quare s and Degrees of Freedom for the Ana lys i s o f Var iance . . . . . . . . . . . . . . . . . . . . . . .
The Degr e e s o f Freedom for the Ana lys i s of Var i ance . . . . . . . . . . . . . . . . . . . . . .
Degrees o f Freedom for GCA and S CA Analy s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corre lat i ons ( r value s ) betwee n Selected Chara c t e r s in JPK ( l ) Popu lat i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Co�r e l at ions ( r va lue s ) between S e l ected Charac t e r s in OKA Pop u l a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corre lat i ons (r va lues ) between Se lected Charac ters in J PK(2 ) Popu l at ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v i i i
Page
1 0
1 0
1 1
1 7
1 8
2 5
2 9
4 3
45
4 6
6 0
6 1
6 2
1 4
1 5
1 6
1 7
1 8
1 9
20
21
22
23
S t ev i o s ide Contents o f S e l ected Stevia L ine s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mean Squares o f P l ant He i ght , Branch Number , L e a f Number , Leaf We ight , S tem We i ght , S tevios ide Content and Stevios ide We i ght of 18 S t e vi a Lines . . . . . . . . . . . . . . . . .
Genotyp i c Vafl i ance s <02), Error Var i ances (0 I and Her i�abi l it i e s o f S even Tra i t s o f 1 8 S t e vi a l ines
Summar i zed Res u l t s of S even Tra i t s o f 1 8 S t e vi a Line s . . . . . . . . . . . . . . . . . . • . . . . .
Phenotypic and Genotyp ic C o r re l a t i ons among 1 8 S t e vi a L i ne s
Mean Va lues o f S ix Trait s , E s t imated f r om PI Data o f a 7 x 7 D ia l le l C r o s s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ana l ys e s o f Var iance and Comb ining Ab i l ity o f a 7 x 7 D ia11e l C ro s s - Mean Square s , GCA and SCA Values S ix Tra i t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Est imat e s o f �enera l C ombi ning Abi l ity <under l ined leading d i agon a l f i gure s ) and Spec i fic Comb ining Ab i l ity E f fe c t s o f ( 1 ) Stevios ide C ontent , (2) Stev i o s ide We i ght , (3) P l ant He ight , ( 4 ) Dry Leaf Weight , and ( 5 ) Dry S t em We ight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Phenotyp i c and Genotypi c C o r re lat ions among Five Tra it s , E s t imated from FI Data o f a 7 x 7 D i a l l e l Cros s . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Performanc e o f FI Cros s e s Expres s ed as %MP ( per c ent o f mid-parent ) and a s %BP ( per c ent o f better parent ) . . . . . . . . . . . . . . . . . . . . . . . . .
ix
63
6 5
6 6
6 9
7 0
72
73
7 7
8 0
8 2
2 4 E s t imates o f C ovari anc e /Variance Regres s ion ( W
r/V
I) , Degree o f
Dominance ( z =n/d ) , Product and Average Frequenc i e s of Homozygous ( u ) and Hetero zygou s ( w ) Genotypes of Five Tra i t s E s t imated from the Fl Data of a 7 x 7 D i a l l e l Cro s s . . . . . . . . . 8 4
2 5 Average Stev i o s ide Content s o f Fl Cro s s e s and Parent s . . . . . . . . . . . . . . . . . . . . . . . 1 0 2
2 6 Mean Va lues o f J P K ( l ) P opu l at i o n 1 1 5
2 7 Mean Va lues o f OKA Popu l at i on 1 1 6
2 8 Mean V a lues o f J P K ( 2 ) P opula t ion 1 1 7
x
F igure
1
2
3
4
5
6
7
LIST OF FIGURES
The B a s i c S tructure o f S t evi a Sweeteners
A Fl ow Chart of Glucos ides Extra c t ion and Determinat i o n by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D i s t r ibut i on o f ( a ) P l ant he igh t , ( b ) M a i n B ranch Number , ( c ) S e condary Branch Number, ( d ) Tota l Branch Number , ( e ) Leaf Area , ( f ) LAI , ( g ) Dry Lea f
We ight , ( h ) Dry F lowe r / Stem We ight , ( i ) S tevi o s ide Content , and { j } Rebaud i o s i de A Content o f JPK ( I ) Popu lat ion
D i s t r ibut ion o f ( a ) P lant He ight , ( b ) Main B ranch Number , ( c ) S e c o ndary Branch Number, (d ) Tot a l B r a n c h Number, ( e ) Lea f Are a , ( f ) LAI , ( g ) Dry Lea f
Weight , ( h ) Dry Flowe r / S tem We i ght , ( i ) S tevi o s ide Content , and ( j ) Rebaud i o s ide A Content o f OKA Popu l a t i on
D istribution o f ( a ) P lant He i ght , ( b ) M a i n B r an c h Number , ( c ) S e co ndary Branch Number, ( d ) Tot a l B r a n c h Number , ( e ) Dry Lea f We ight , ( f ) Dry F l ower / St em
We ight , ( g ) Stevios ide Content , and ( h ) Rebaudi o s ide A Content o f J PK ( 2 ) Popu l a t i on . . . . . . . . . . . . . . . . . . . . • . . . . . .
Wr-Vr Graph o f Stevi o s i de Con t ent o f a Seve n -parent D i a l l e l o f S t e vi a reba udi a na ( A ) Al l Arr ays, and ( B ) Omi t t ing Array O KA 4 3 . . . . . . . .
Wr- Vr Graph o f Stevio s ide We i ght o f a Seve n -parent D i a l l e l o f Ste vi a reba udi a na ... . . ...... . . .... .. .
xi
Page
1 3
40
52
54
56
8 7
8 8
8
9
1 0
Wr-Vr Graph o f Plant Hei ght o f a Seven-pa rent D i a l lel o f S t e via reba udiana ( A ) Al l Arrays , and ( B ) Omi t t ing Array JPK 4 7
Wr-Vr Graph o f D r y Lea f Wei ght o f a Seven-parent D ia l lel o f S t e via reba udiana ( A ) Al l Arrays , and ( B ) Omitt ing Array JPK 42 . . . . . . . • . . . .
Wr-Vr Graph o f Dry S tem Weight o f a Seven-parent Oia l le l of Stevia reba udiana . . . . . . . . . . . . . . . . . . . .
x i i
8 9
9 0
91
LIST OF PLATES
P late
1 S t e via reba udi a n a B e r t on i . ( A ) P lants Two Weeks be fore
Harve s t ing ( B ) P lants Ready for
Page
Harve s t ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 S oxh let Apparatus Used f o r Extract ing Gluc o s ides f r om S t e via L eaves . . . . . . . . . . . . . 39
xi i i
Abs tract o f the s i s s ubm itted to the Senate o f Un ivers i t i Pertanian Ma l ays i a in f u l f i lment o f the requirements for the degree o f Doc t o r o f Ph i lo s ophy .
GENETIC STUDI E S OF STEVIOSIDE CONTENT AND OTHER AGRONOMIC CHARACTERS AND THEIR RESPONSES TO
CLONING IN STEVIA REBAUDIANA BERT.
By
WONG LOCK JAM
Sep tember 19 9 1
Supervi sor: Pro f . Dr . Yap Thoo Cha i
Facul t y : Agricu lture
S tevia rebaudiana Bert . produce s the sweetener
s tevi os ide , wh ich is 3 0 0 t imes swe e t e r than c ane sugar .
However, the mean s tevios ide content o f c ommerc i a l
cult ivars w a s genera l ly l ow and among the better
c u l t ivars the average stevios ide c ontent was in the
region of s even per c ent . Stud i e s were set up t o
inves tigate t h e genetic sys tem o f stevios ide c ontent and
other t r a i t s , the e f fe c t s o f c loning and the r e l a t ionsh ip
of s tevios ide content to the other t r a i t s . Three
s egregat ing popu l a t i ons were s tud i ed , whereupon 18 lines
were s e l ec t ed for s t udying the effe c t s of c l oning .
xiv
The mean s tevi o s ide conten t s o f J P K ( l ) , JPK ( 2 ) and
aKA popu lat ions were 5 . 0% , 4 . 7% and 5 . 8% respec t ive ly .
P lant height s for the three respect ive popu lat ions were
4 5 . 1 cm , 4 1 . 9 cm and 4 2 . 0 cm . The mean LA! for J PK ( l ) and
aKA were 1 . 7 and 1 . 0 respect ively . Dry lea f and dry s tem
weight were 6 . 3 9 and 1 7 . 6 9 per p l ant for J PK ( l ) , 7 . 2 g
and 1 2 . 6 g for JPK ( 2 } , and 4 . 9 g and 1 6 . 1 g f o r aKA .
The c lonal s tudies revealed h ighly s ign i f icant
d i f ferences between l ines for p l an t height , branch
number , lea f area , l ea f weight , s tem we ight , s tevi o s ide
content and s tevioside weight . However , c lo ning h a s n o
ef fec t on t h e above t r a i t s .
I n the 7 x 7 d i a l lel ana lys i s , ef fec t s due t o
genotypes were h ighly s ign i f icant for s tevi o s ide content,
s tevi o s ide wei ght, p l ant height, leaf and s tem we�ght s .
Heterobe l t ios i s w a s a l s o detec ted i n the s ame t r a i t s , but
was more prom inent f o r both s tevio s i de c on tent and
s t evio s ide weigh t . For the f ormer , 1 9 . 1% o f the c r o s s es
showed heterobelt i o s is , whereas f o r t he latter i t was
9 . 5% . Analyses tor GCA and SCA ef fec t s s howed addit ive
genes were more impo rtant than n o n - a dd i t ive genes for
s tev i o s ide c ontent and stevios ide weight . Non-addi t ive
genet ic ef fec t s were important for p l ant he ight and lea f
we ight . For s tem weight , both add it ive and non- addit ive
xv
e f fec ts were important . JPK 24, J P K 4 8 and aKA 1 0 had
the h i ghes t GCA est imates for both s tevi o s ide c ontent and
s tevios ide weight . The s ame l ines a l s o had very h igh
s tevios ide c ontents , 1 1 . 5% , 9 . 8% and 8 . 6% , respec t i vely ,
and s tevios ide weights , with 6 9 , 4 4 and 5 4 mg per p l ant ,
respec t ively . The Wr/VI analys i s s howed gene act i o n for
s tevi o s ide c ontent among the s ix paren t s , but exc l uding
aKA 43 , was part i a l dom inance . aKA 4 3 exhib ited non
a l le l i c interact ion for this tra i t i n its c r o s ses . For
s tevi o s ide wei ght the gene act i on was a l s o p a r t i a l
dom inance . The non- s tevios ide t r a i t s o f p lant he ight ,
leaf and s tem weights had gene act ion varying from
c omp lete dom inance t o over -do m inance .
I t was concluded that further improvemen t f o r both
s tevios ide c ontent and stevio s ide we i ght c o u l d be
s imultaneous ly achi eved , s ince both t r a i t s were h i ghly
c orrel ated , by inter c ro s s ing among JPK 2 4 , JPK 48 and OKA
1 0 f o l lowed by selec t ion among the progen ies and the
genot ype s o f s elect ed li nes can then be fi xed by clo n ing.
xvi
Abs trak tes i s d ikemukakan kepada Senat Un iver s i t i Pertanian Ma lays ia bagi memenuh i syarat i j azah Doktor F a l s afah
KAJIAN GENETIK KANDUNGAN STEVIOSIDE DAN CIRI-CIRI AGRONOMI LAI N DAN TINDAKBALAS KEPADA KLONI NG
D I DALAM STEVIA REBAUDIANA BERT.
01eh
WONG LOCK JAM
September 1 9 9 1
Penye l i a P ro f . Dr . Yap Thoo Chai
Fakult i Pertanian
Stevi a reba udi a na B ert . mengh a s i l ka n s atu pema n i s ,
s tevios ide , yang 3 0 0 ganda lebi h m a n i s j i ka dibandingkan
dengan gul a t ebu . Wa l a u baga imanapun , purata kandungan
s tevios ide bagi ku l t ivar komer s i a l pada keseluruhannya
ada l ah rendah , dan b a g i kult iva r yang ba ik ada l ah da lam
l ingkungan t u j uh per atus . Ka j ian telah d i j a l ankan untuk
menyiasat s i s tem genet i k yang mempengaruhi kandungan
stev i o s ide , kes an p embiakan k lon , dan perhubungan a n t a ra
kadar s tev i o s ide dengan c ir i - c i r i l a in . Tiga popu l a s i
pengas ingan telah d i k a j i , di mana 1 8 t i t i s an terp i l ih
untuk pengkajian kes an pemb iakan k lon .
xv i i
Min kandungan stevios ide bagi popu l a s i JPK ( l ) ,
JPK ( 2 ) dan OKA ada l ah 5 . % , 4 . 7% dan 5 . 8 % . Ket inggian
pokok bagi ket iga -ket iga popu l a s i t e r s ebut ada l a h 4 5 . 1
em , 4 1 . 9 em dan 4 2 . 0 em dan b i l angan rant ing mas ing
m a s ing ada lah 3 4 . 3 , 1 8 . 9 dan 3 9 . 8 s epokok . Min LAI
( indeks luas permukaan daun ) bagi JPK dan OKA ada lah 1 . 7
dan 1 . 0 . Berat daun ker ing dan berat dahan bagi sepokok
ada l a h 6 . 3 9 dan 1 7 . 6 9 bagi p opu l a s i J PK ( 1 ) , 7 . 2 9 dan
12 . 6 9 bagi popu l a s i J PK ( 2 ) , 4 . 9 9 dan 1 6 . 1 9 bagi
popu l a s i OKA . Perka i t a n kadar s tevios ide ada l ah ket ara
dengan ket inggian poko k , b i l angan rant i ng , luas permukaan
d aun dan berat daun bagi popu l a s i J PK ( l ) , t e t ap i untuk
popu l as i OKA perka itan kadar s tevi o s ide h anya dengan luas
permukaan daun . Manaka l a bagi popu l a s i J PK ( 2 ) , perka i tan
yang ketara hanya dengan berat dahan s ah a j a .
Pemb iakan k l on t idak mempengaruh i ket inggian pokok ,
b i l angan rant ing , luas permukaan daun , berat daun , berat
dahan , kandungan s tevios ide dan berat stevios ide .
Perbezaan antara t i t i s an ada l ah ket a r a bagi s etiap ciri
e ir i yang dieat i t kan .
Bagi ana l i s i s kaeukan s eparuh d i a l e 1 7 x 7 ,
perb e zaan antara genot ip ada lah berk e s an bagi kandungan
s t e v i o s ide, berat s tevios ide, ket inggian pokok , berat
daun dan be rat dahan . Heterobe l t i o s i s j uga ada lah ket ara
xv i i i
tentang c i r i-c i r i t e r s ebut , khus us nya bagi kandungan
stevios ide dan berat s tevio s i de . Ana l i s i s untuk ke s an GCA
( general combin ing a b i l ity ) dan SCA ( spec i f ic c ombin ing
abi l i ty ) terhadap tuj uh t i t i s an menun j ukkan bahawa gen
adi t i f ada lah lebih berke s an j ika d iband ingkan dengan gen
bukan adit i f bagi kandungan s te v i o s ide dan berat
s tev i o s ide . Gen bukan adi t i f ada l a h b e rke s a n bagi
ket inggian pokok dan berat daun . Kedua - dua gen adi t i f dan
bukan adi t i f ada lah berke s an bagi berat dahan . Ket iga
t iga klon induk JPK 2 4 , JPK 4 8 dan OKA 1 0 mempunyai GCA
yang p a l ing t i nggi bagi kandungan s tevios ide dan be rat
stevios ide , j uga mempuny a i min kandungan s tevios ide yang
p a l i ng t inggi , i a i t u 1 1 . 5% , 9 . 8% dan 8 . 6% , dan m in berat
s tevios ide yang p a l i n g t inggi , i a i tu 6 9 , 4 4 dan 5 4 mg
s e t i ap s atu . Ana l i s i s Wr/Vr menun j ukkan pergerakan gen
separuh domi n a n bagi kandungan s te v i o s ide antara enam
d a r ipada s e j um lah t u j uh klon i nduk . OKA 4 3 me l a lu i
t it i s an kacukan mempamerkan interaks i bukan a Ie l . Jen i s
pergerakan g e n untuk b e r a t s t evi o s ide ada lah s eparuh
dom in a n . Untuk
ke t ingg i an pokok ,
c i ri -c i r i bukan
berat daun dan
stevios ide s ep e r t i
b e r a t dahan , j en i s
perge rakan gen ada l a h dar i dom inan penuh ke
dom i nance'
ove r -
Kes impu l annya , kemajuan l a n j ut an b a g i kedua-dua
kandungan stevios ide dan berat st evios ide boleh d i c ap a i
xix
dengan k a c ukan anta r a JPK 24, JPK 48 dan OKA 1 0 d i iku t i
dengan pem i l ihan dan pembiakan t i t i s an t e rp i l i h di l akukan
dengan m e l a l u i cara pemb i a kan k lon .
xx
CHAPTER I
INTRODUCTION
I n the exp lo it a t ion o f plants for the produc t i o n o f
natur a l sweetener s , Ste vi a rebaudi a na Bert . s tands out a s
a n important c r op because o f i t s intr ins ic value a s a
producer o f the s we etener s t evio s ide. The latter i s about
300 t imes swe e t e r t h a n c a ne sugar , i . e . 3 . 5 g of s tevi o
s ide would have t h e e quivalent s we e t ne s s o f I kg o f c an e
sugar . S ince s tevi o s i de h a s negl ig i b l e carbohydrate value
and the amount t o be c on sumed e ac h t ime is very s mall , it
is aptly dubbe d 's ugarle s s sugar I . Although it t a s te s
l ike sugar , s t ev i o s ide is d i f ferent f r om s ugar i n t h a t i t
does not contribute t o health p r ob l em s s uch a s diabet i c s ,
tooth decay , obes ity and heart r e l a t e d d i s e a s e s.
stevi a i s a n i n tr oduce d crop i n Ma l ays ia and t here
are at least three reas ons f o r intere s t i n its c u l t iva
t ion in this c ountry. F i r s t ly , t h e t ropi ca l environment
o f t h i s country , c h aracter i z e d by h igh annual r a infall
with hardly any d i s t inct agr icultur a l drought and
perpetual sun s h in e t hroughout the year , is c apabl e o f
sustain ing c on t inuous cropp ing all the year round .
Secondly , t h i s crop was f ir s t brought into Malays ia by
I
2
Japanese entrepreneur s w i th the main purpose o f produc ing
S t e vi a l eaves che a p l y f o r the s tevi o s ide factor i e s in
Japan a s it i s not economica l t o do s o in the ir own
country . Th irdly , S t e vi a cult ivat ion has potent i a l in our
country becaus e o f i t s ready market for sweet eners .
Ma l ays ia import s more than one m i l l ion tonnes o f sugar
annua l ly , incurring a l o s s in fore ign exchange exc e eding
6 0 0 m i l lion r in gg i t per a nnum ( Kementer i an Pertan i an ,
1 9 8 4 ; 1 9 8 5 ; and 1 9 8 6 ) . Loca l produc t ion o f cane s ugar
c ould o n ly meet 2 0 per c ent of the t o t a l demand .
Furthermore , i t i s not econom ic a l ly feas ible for any
expans ion in s uga r - c ane hectareage s ince agroc l imat ical ly
sui tabl e l and i s not read i l y ava i l ab l e .
Unfortunate ly , the intere s t i n S t e vi a f a i led to
sus t a in i t s cult ivat ion on a c omme rc ia l s c a le . The m a in
r e a s on for the f a i lure was the unava i labi l ity o f widely
acc ept abl e S t e vi a cult ivars with good adaptat i on and h i gh
s tevios ide c ontent . S te vi a leaves produced by l o c a l
f armers c o u l d n o t b e marketed . Wh en t h e J a panes e
entrepreneurs f ir s t introduced S t e vi a cUlt ivat ion in
Malays ia , each c ompany b rought thei r own cult ivars from
J apan . The main r e a s on for do i ng so was the h igh
var i ab i l ity in s t ev i o s ide contents among the cult ivar s .
The stevios ide contents o f the i r cult ivars were later
found t o vary from 4% t o 7% ( Wong and Lim , 1 9 8 0 ) .
B e l ieving the irs were super i o r , S t e vi a c u l t ivars were
3
regarded a s c ompany secret . As s uc h , one c ompany would
not buy Stevi a leave s produce d by another . I n s t e a d , each
company wou l d s end S t evi a l e aves back t o their parent
compan ies i n Japan .
I n order t o a s s i s t S t e vi a c u l t ivat ion in t h i s
c ountry , t h e deve l opment o f l oc a l ly adapted genotypes
w i t h h i gh s tevios ide content i s e s s en t i a l . Henc e , s tudies
have been s e t up t o inve s tigate the genet ic system
a f fe c t ing the t r a i t on stevios i de c ontent ; the e f fect o f
s elect i on and c l on ing o n i t ; and i t s r e l a t ions h ip to
s elected characters of the p lant. Hope f u l ly , informat ion
and c u l t i va r s gene r a t ed f rom the s tudies c onducted would
be u s e f u l for p lanni ng our S t e vi a breeding programme in
Malays i a .
CHAPTER I I
LITERATURE REVI EW
The review o f l it e rature is organ is ed under two main
s e c t ions , the f ir s t sect ion is on the deve l opment and
c ult ivat ion o f Stevi a reba udi ana as i t i s a new c rop
( re fe r P late 1), and the second is o n the dial l e l mat ing
s ys tems becau s e of its r e levance t o the res earc h herein
conducted.
Stevia rebaudiana Bertoni. - A New Crop
S tevi a rebaudi ana Berton i . is a s ma l l shrub f ound mainly
i n the Arnambai Highlands and the h igh northern p l at eau o f
Paraguay ( Crosby and Wingard , 1979; Gos l ing , 19Q1).
Henc e , the p lant i s nat ive t o Paraguay <Shoc k , 1982;
Soe j ar t o e t a l . , 1983). It has been used in Paraguay and
Braz i l as a s weetener for a long t im e and i s we l l known
tor s weetening the Paraguayan mate' tea (Inglett, 1981;
Cramme r and Ikan, 1986).
4
