oil palm (elaeis guineensis pro top lasts_ isolation, culture and micro call us formation

8/3/2019 Oil Palm (Elaeis Guineensis Pro Top Lasts_ Isolation, Culture and Micro Call Us Formation

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Plant Cell, llssue and Organ Culture 46: 35-4 1, 1996. 35 1996KluwerAcademic Publishers. Printed n the Netherlands.

O i l p a l m (Elaeis guineensis) p r o t o p l a s t s: i s o l a ti o n , c u l t u r e a n d m i c r o c a l l u sf o r m a t i o nRa v ig a de v i Sa mba ntha mur th i , S i t i Ha s na h Pa r ma n & M o hd . Ro s la n M d. No o rPlant Science and Biotechnology Unit, Palm Oil Research Institute of Malaysia, P.O. Box 10620, 50720 KualaLumpur, MalaysiaReceived 31 October 1995; accepted n revisedform 10 Jun e 1996

Key words." Elaeis guineensis, oil palm, protoplas ts

A b s t r a c t

Procedures a re desc r ibed fo r the e f f i c ien t i so la t ion o f p ro top las ts f rom a var ie ty o f o i l pa lm (Elaeis guineensisJacq . ) t i s sues . Var ious f ac to r s inc lud ing donor sou rce , com pos i t ion o f enzym e m ix tu re and cu l tu re m ed ium a f fec tedthe y ie ld an d v iab i l i ty o f the p ro top las t s Po lyem bryo gen ic cu l tu res o f o i l pa lm w ere the m os t su i t ab le s t a r tingm ate r ia l in t e rm s o f y ie ld , v iab i l ity and m etabo l ic com petence . Pec to lyase Y-23 in as soc ia t ion w i th ce l lu lase andhem ice l lu lase was r equ i r ed fo r the e f f i c ien t r e lease o f p ro top last s f rom the o i l pa lm t i ssues . L im i ted ce l l d iv i s ion tofo rm m icroca l lus was observed a t ve ry low f r eque ncy (


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3 6- e m bryo n i c a xe s o f ge rmi na t i ng s ee ds , a nd- you ng i n f l o re sc e nc e s .

Surface s te r i l i za t ion was ca rr ied out on a l l t i ssuese xc e p t po l y e m bry oge n i c c u l t u re s a s fo l lows : soa k i ngi n a so l u t i on c on t a i n i ng 8% Cl o rox (v / v ) a nd 0 .1%(v / v ) Twe e n 20 f o r 5 mi n fo l l ow e d by r i n s i ng i n th re echan ges o f s te r i le d i s t i l l ed wate r . T he t i ssues weret he n t r e a t e d wi t h 70% e t ha no l fo r 1 mi n a nd r i n se dthree t imes w i th s te r i le d i s t i l led wate r . Th e t i ssues w eres l i c e d f i ne l y a nd p l a smol yse d (30 mi n ) i n me d i um A(Ta b l e 1 ). Th i s me d i um wa s a mod i f i c a t ion o f t hoseo f G a m b o r g e t a l. ( 1 9 6 8 ) a n d K a o ( 1 9 8 2 ). M e d i u m Aw a s r e m o v e d a n d r e p l a c e d w i t h e n z y m e s o lu t io n c o n -t a i n i ng va r i ous c ombi na t i ons o f 2% (v / v ) Ce l l uc l a s t( N o v o I n d u s t r i A / S N o v o A l l ~ D K - 2 8 8 0 B a g s v a e r d ,D e n m a r k ) , 1 % ( v / v ) P e c t i n e x 3 X L ( N o v o F e r m e n tL t d . Voge se ns t ra s se s 132 , CH-4013 Ba se l S wi t z e r -l a nd ) , 0 .25 % (w/ v ) P e c t o l ya se Y-23 (S e ish i n P ha r -ma c e u t i c a l , 4 -13 , Koa m i c ho , Ni homb a sh i , Tokyo ,Ja pa n ) , 1% (w/ v ) M a c e ro z ym e R- 10 (Ya ku l t P ha rma -c e u t i c a l L t d . , t -1 -19 , Hi ga sh i S h i nba sh i , Mi na t o -ku ,T o k y o ) a n d 0 . 5 % ( w / v ) h e m i c e ll u l a se ( A m a n o P h a r -ma c e u t i c a l s L t d . , Na goya , J a pa n ) a nd 0 .01% (w/ v )t ryps i n i nh i b i t o r (S i gma , U S A) . Tryps i n i nh i b i t o r wa si nc l ude d i n a l l t he e nz ym e mi x t u re s t o a r re s t pos si b l ep ro t e a se a c t i on by c on t a m i na n t s p re se n t i n t he e nz y m esolut ions .

T h e e n z y m e m i x t u r e w a s a d j u st e d to p H 5 .7 .S uc rose wa s a dde d t o ob t a i n a n osmol a l i t y o f 800m O s m ( k g H 2 0 ) - 1 . O s m o l a l i ty m e a s u r e m e n t s w e r ec a r r i e d o u t w i t h a W e s c o r o s m o m e t e r . T h e e n z y m eso l u t i on wa s f i lt e r s te r i li z e d . Di ge s t i on wa s c a r r i e d ou tfo r 5 h i n t he da rk a nd t he mi x t u re f i l t e re d t h rougha 1 2 0 # m f o l l o w e d b y 6 9 # m p o r e s iz e n y l o n m e s ht o r e move c e l l c l umps a nd va sc u l a r t i s sue . The f i l -t r a te wa s c e n t r i fuge d (100 0 ; 5 mi n ) . Un de r t he sec ond i t i ons , p ro t op l a st s f rom po l ye m bryo ge n i c c u l t u resf o r m e d a f l oa t in g b a n d w h i c h w a s r e m o v e d a n d p u r i -f i ed b y r e s u sp e n s i o n in m e d i u m A ( 8 0 0 m O s m ) a n dc e n t r i fuga t i on (100 0 ; 5 m i n ) . Th e f l oa t ing p ro t op l a s tl a ye r wa s p i pe t t e d o f f . P ro t op l a s t s f rom t he o t he r t is -s u e s f o r m e d a p e l l e t w h i c h w a s w a s h e d i n m e d i u m Aa nd t he n l a ye re d on t o 25% (w/ v ) suc rose so l u ti on . Thef l oa t i ng l a ye r , wh i c h c on t a i ne d t he p ro t op l a s t s , wa swa she d i n me d i um A. P ro t op l a s t s i so l a t e d f rom t heve ge t a t i ve a p i c e s o f c lona l r a me t s we re ve ry de nse a ndd i d no t f l oa t e ve n i n 25% (w/ v ) suc rose so t he pe l l e twa s w a she d i n me d i u m A a nd f i lt e re d t h rough a 30 #mp o r e s i z e n y l o n m e s h .

Determination o f protoplast yield, v iability an dmetabolic integrityP ro t op l a s t y i e l d wa s e s t i ma t e d wi t h a ha e moc y t om e t e r .

Eva ns B l ue , w h i c h i s a s ta i n t ha t is e xc l ud e d byv i a b l e c e l l s wa s u se d t o me a su re p ro t op l a s t v i a b i l i t y(Na ga t a a nd Ta ke be , 1970) . P ro t op l as t s we re ke p t i nme d i um A be fo re v i a b i li t y de t e rmi na t i ons .

The m e t a bo l i c in t e g r i t y o f the o i l pa l m p ro t op l a s tp re pa ra t i ons wa s ga uge d by t he i r a b i l i t y t o r e sp i re14CO2 and sy nthes ize [14C] l ip ids wh en in cub a ted w i th[ 1 -14C] a c e t at e . Th e de t e rm i na t i ons w e re c a r r i e d ou t onf re sh l y i so la t e d p ro t op l as t s . Two m l o f p ro t op l a s t su s -pe ns i on (104 - 105 c e ll s in me d i um A ) w e re i nc uba t e dwi t h 15 #C i o f [1-14C] a c e t i c ac i d (263 nm ol , Am e r -sha m In t e rna ti ona l , Eng l a nd) fo r 5 h w i t hou t sha k i ng ,i n a c on i c a l f l ask f it t ed wi t h a K on t e s rub be r s t opp e ra nd a p l a s ti c c e n t re we l l . Th e w e l l c on t a i ne d f l u t e d f i l-t e r p a p e r m o i s te n e d w i t h 0 .2 m l o f 0 .2 M K O H t o tr a p14CO2. The i nc uba t i on wa s s t oppe d wi t h 0 .2 ml 4MH2 S 04 . The m oi s t f i lt e r pa pe r wa s t r a ns fe r re d d i re c t l yi n t o a s c i n t i ll a t ion v i a l fo r c oun t i ng . Th e suspe ns i onwa s e x t ra c te d wi t h 3 x 15 ml c h l o ro fo r m/ m e t ha no l(2 : 1 ; v /v ) . The e x t ra c t wa s w a she d w i t h 0 .2 vo l 0 .8%(w/ v ) NaC1 so l u t ion . Th e c h l o r o fo rm l a ye r wa s c a re -fu l l y r e mov e d a nd a n a l i quo t t r a ns fe r re d to a s c i n t il -l a t i on v i a l a nd e va pora t e d t o d ryne s s unde r a s t r e a mof n i t roge n . Te n ml o f s c i n t i ll a t ion f l u i d we re a dde d t oe a c h s a mpl e a nd c oun t i ng c a r ri e d ou t on a LK B W a l la cRackbe ta 1217 l iquid sc in t i l l a t ion counte r .

The fa t t y a c i d c ompos i t i on o f t he r a d i oa c t i vel ip i d s wa s a na l yse d by ra d i o ga s c h roma t og ra p hy asde sc r i be d by S a m ba n t ha m ur t h i e t al . (1987) .Protoplast cultureDe ns i t y w a s a d j us t e d t o 1 x 105 p ro t op l a s ts / ml i n m e d i -u m A w i th 1 .2 # M N A A ( 8 0 0 m O s m ) . A l i q u o ts ( 2. 5ml ) we re d i spe nse d i n 60 x 15 m m p l a s t i c c u l t u rep l a te s . Asp i r in (10 -50 m g 1 - l ) s i l ve r n i t r a te (20 mg 1-1 2 ,4 -D (0 .5 mg 1 -1 ) a nd z e a t i n (1 .5 #M ) w e re a l soa dde d t o t he c u l t u re me d i a e i t he r i nd i v i dua l l y o r i nva r i ous c ombi na t i ons i n some e xpe r i me n t s . The c u l -t u re p l a t e s we re s e a l e d wi t h P a ra f i l m a nd i nc uba t e d(da rk ) a t 25 C . D ur i ng c u l t u re , t he o smol a l i t y o f t hem e d i u m w a s p r o g r e s s i v e ly r e d u c e d b y t h e a d d i t io n o f0 . 2 5 m l m e d i u m A c o n t a i n i n g 1 0 0 m M s u c r o s e e v e r y5 da ys un t i l da y 25 . F o r so l i d c u l t u re , 1 .2% S e a P l a quea ga rose (F MC Bi o P roduc t s , Roc k l a nd , U .S .A. ) wa sa dde d . To p re ve n t t he p l a t es f ro m d ry i ng up a nd n u t r i-e n t s f rom be i ng de p l e t e d , 0 .5 m l so l u t i on A c on t a i n i ng


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Table 1. Compositionof M edium A.Compound Concentration Com pou nd Concentrationa. Mineral salts mM b. Vitamins mMK2HPO4 7.5 Thiam ine HC1 30.0(NH4)2SO4 7.5 Py rid ox ine C1 5.0NH4-acetate 1 0 .0 O- Ca lciu mantothenate 1.0KH2PO4 7.3 p-Am ino benzo ic acid 0.1K2SO4 10.0 Biotin 0.2

/LM Vitamin D3 0.1KI 4.5 Folic acid 0.5H3BO3 5 0 .0 Choline-chloride 7.2MnSO4.H20 6 0 .0 My oinositol 555.0ZnSOn .7H20 7.0 Nicotinic acid 5.0Na2Mo04.2H20 1.0CuSOa.5H20 0.1Compound Concentration Com poun d Concentrationc. Sugars and organic acids /zM d. Others mMSucrose 400.0 MES 5.0Ribose 2.0 DT T 1.0Ascorbate (Ascorb ic acid) 1.0 PVP 0.1(%)Citrate (Citric acid) 2.0Malate (Maleic acid) 1.0 pH adjusted to 5.7 with KO H

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1 0 0 m M s u c r o s e w a s a d d e d t o t h e s o l id m e d i u m e v e r y7 d a y s f r o m d a y 1 4 o n w a r d s .C e l l w a l l r e g e n e r a t i o n w a s d e t e r m i n e d e v e r y d a yf r o m d a y 1 o n w a r d s u s i n g 0 . 0 1 % C a l c o fl u o r W h i t e S Ta c c o r d i n g t o t h e m e t h o d o f N a g a t a a n d T a k e b e ( 19 7 0 ) .A s s e s s m e n t s w e r e c a r r i e d o u t i n t r i p l i c a t e f o r e v e r ye x p e r i m e n t .

R e s u l ts a n d d i s c u s s io n

C o m p o s i t io n o f p r o t o p l a s t i s o la t io n m i x t u r eA c om bina t ion o f c e l l u l a se ( C e l luc l a s t ) , pe c t ina se( P e c t ine x I I I /P e c to lya s e 3 (- 23 ) a nd he m ic e l lu l a s e( H e m i c e l l u l a s e A m a n o 1 0 ) w a s r e q u i r e d f o r e ff i ci e n tm a c e r a t i o n o f t h e o i l p a l m t i ss u e s ( T a b l e 2 ) . T h e s u b -s t i t u ti on o f P e c t ine x I I I w i th P e c to lya s e Y - 23 inc r e a se dm a c e r a t i o n a n d w a s e s s e n t ia l f o r p o l y e m b r y o g e n i c c u l-tu r e s . A c om b ina t io n o f C e l luc l a s t , P e c to lya s e Y - 23 ,P e c t i n e x I I I a n d H e m i c e l l u l a s e A m a n o 1 0 g a v e t h eh i g h e s t p r o t o p l a s t y i e l d f o r a l l d o n o r s o u r c e s a n d t h ise n z y m e m i x t u r e w a s u s e d f o r s u b s e q u e n t i s o la t i o n p r o -c e du r e s .

V i a b i li t y a n d m e t a b o l i c i n t e g r i t yT h e v i a b i li t y o f p r o to p l a s t s o b t a i n e d f r o m t h e v a r i -ous don o r t i s sue s is g ive n in Ta b le 3 . P r o top la s t s f r omp o l y e m b r y o g e n i c c u l t u re s w e r e h i g h l y v i a b l e ( > 9 9 % ) .T h e m e t a b o l i c c o m p e t e n c e o f t h e p r o t o p l a s t s i s a l s oi n d i c at e d i n T a b le 3 . P r o t o p l a st s f r o m p o l y e m b r y o -g e n i c c u l t u re s a n d v e g e t a t i v e a p ic e s w e r e m e t a b o l i c a l -l y t h e m o s t a c t i v e ( in t e r m s o f l i p id s y n t h e s i s a n d C O 2pr oduc t ion . ) . A na lys i s o f t he r a d ioa c t ive l i p id s syn -t h e s i s e d b y p r o t o p l a s t s ( b o t h f r o m p o l y e m b r y o g e n i cc u l tu r e s a nd ve g e ta t i ve a p i c e s ) i nd i c a t e d tha t t he l i p idm e t a b o l i s m h a d b e e n a l te r ed . T h e p r o t o p l a s t s s y n t h e -s i s ed h i g h l e v e l s o f p a l m i t o l e i c a c id ( u p t o 2 7 % ) . T h i sf a t ty a c i d is n o r m a l l y p r o d u c e d o n l y i n t r a c e a m o u n t sb y o i l p a l m t i s s u e s ( < 0 .1 % ) . T h e a l t e r e d m e t a b o l i s mw a s p r o b a b l y i n d u c e d a s a r e s u lt o f o s m o t i c s t r es s .P r o t o p l a s t s o u r c eTis sue s t ha t a r e t he m se lve s e a s i l y r e ge ne r a b le i . e .a m e n a b l e t o t i s s u e c u l t u r e a r e m o r e l i k e l y t o b ea m e n a b l e t o p r o t o p l a s t c u l t u re a n d r e g e n e r a t io n . T h eva r ious t i s sue s u se d f o r p r o top la s t i so l a t i on in t h i ss t u d y h a v e p r e v i o u s l y b e e n s u c c e s s f u l ly u s e d f o r t is s u e


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Table 2. Protoplast yield with different enzymatic mixtures.Protoplast yield (g f.wt. )-1

Enz ym e Embryogenic Vegetative Seed Inflorescence Embryonicmixture culture apex embryo axisCelluc last < 102 < 103 < 102 < 103 < 103Ma cer ozy me - < 102 < 103 < 102 < 102 < 103R1 0Celluclast,Macerozyme -R1 0Celluclast,Pectinex,HemicellulaseCelluclast,Pectolyase Y-23,HemicellulaseCelluclast,Pectolyase Y-23,Pectinex 3XL,Hemicellulase

< 105 14- 0.2 x1~ < 103 24 -0 .5x 1~ < 103

< 102 24 -0. 3x 1~ 64-0.3x105 14-O.ltimes105 14-0 .2x1~

54-0.4x105 24-0.3x105 54-0.6x 1~ 54-0.8x105 74 -1 .2 x1 ~

1.54-0.3x 106 1.0 4-0 .1 106 0. 5x 106 1.04-0.2x 106 54- 0.3 x 105

Values represent the mean of 5 independent experiments. Each experiment was carded out in triplicate.

Table 3. Viability and metabolic integrity of protoplasts isolated from various oil palmtissues.

Protoplast Viabilitysource

Freshly 24h 48h 72hisolated

Incorporation of [1-14C]into labelled productsper 105 freshly isolatedprotoplasts (counts/rain 10 -7 )

CO2 Lipid

Embryogenic >99 >99 9 5 4 - 3 954-2 8.24 -1.1 4.54-0.8cultureVegetative >99 724-8 654-4 50 4- 5 7.8+0 .8 3.24-0.9apexSeed embryo 424-10 1 0 4 -8 14-0.5 0 0.14-0.03 0.024-0.001Inflorescence 834-9 6 5 4 - 7 524-6 454-7 1.04-0 .1 0.1+0.04Embryonic 704-12 624-6 4 5 4 - 8 334-6 2.04-0.3 0.54-0.03axis

Values represent the mean of 3-5 independent experimeuts. Each experiment was cardedout in triplicate.


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cul ture of o i l palm (Paranjothy, 1982; Brac kpoo l e t a l . ,1986) an d w ere thus iden t i f i ed as goo d dono r sou rcesfo r p ro top las t i so la t ion and cu l tu re . O f the v ar ious t is -sues used , p ro top las ts f ro m po lye m bry ogen ic cu ltu resand veg e ta t ive ap ices appeared to be the m o s t p rom is-ing m ate r ia l in t e rm s o f y ie ld and m etabo l ic com pe-tence . However , p ro top las t s f rom vege ta t ive ap iceswere fu l l o f s t a r ch g ranu les and o f ten bu r s t, r e leas -ing the s tarch granules . Th ey were th us d if f icul t towork w i th . Fu r therm o re , the v iab i l i ty o f these p ro to -p las ts qu ick ly d ec l ined du r ing incuba t ion . P ro top las t sf rom po lye m b ryoge n ic cu l tu res were thus the m ate r ia lo f cho ice . T he p ro top las t p repara t ions f rom po lyem -bryogen ic cu ltu res were very c lean . Con tam ina t ion byce l l deb r is and u nd iges ted ce l l s cou ld no t be de tec tedm icroscop ica l ly . Fu r therm ore , Ca lco f luo r W hi te s t ain -ing conf i rm ed the absence o f und iges ted ce l l wa l ls inthe p ro top las t p repara t ions . A d rawba ck o f the po lyem -bryogen ic cu l tu res used in ou r s tudy however , was tha tm a ny o f the ce l ls we re vacuo la te . I t i s genera l ly know ntha t dense cy top lasm ic ce ll s undergo ce l l d iv i s ion be t-t e r than v acuo la te ce l l s.

Reproduc ib le m ethods o f p lan t r egenera t ion havebeen r epor ted fo r p ro top las t s f rom r ice (Abdu l lah e tal ., 1986; To r iyam a et a l . , 1988, ma ize (R hode s et al .,1989; Don n et a l. , 1990), b ar ley (Wan g and Lo rz, 1994;Zh ang et a l ., 1995) and whe at (L i e t a l. , 1992; Ahm adand Sagi , 1993) . These successes were accomplishedus ing suspens ion cul tures as the protoplas t source. Inour exper im en ts how ever , o i l pa lm suspens ion cu l tu reswere no t ava i l ab le . T he po lye m bry ogen ic cu l tu res usedwere f rom so l id m ed ium . T h is m ay be the m ain r ea -son fo r ou r l im i ted succes s as em b ryogen ic suspens ioncu l tu res wou ld be a be t t e r sou rce o f to t ipo ten t cel l s.Ano ther r eason fo r the l im i ted succes s in cu l tu r ing thep ro top las ts m ay be the f ac t tha t the m etabo l i sm o f thep ro top las ts ha d been a l t e red . T he a l t e red m etabo l i smm a y have in som e w ay a l t e r ed m i to t i c com petence .

Co-cu l tu r ing o f r eca lc i tr an t p ro top las ts espec ia l lythose f rom m onoco ty ledons , w i th nu r se cu l tu res hasbeen r epo r ted to indu ce ce l l d iv i s ion and inc rease p la t-ing ef f ic iencies (Jain et a l ., 1995; G hosh B iswas et a l .,1994) . Nurse cul tures general ly ass is t in maintaininga cr i t ical dens i ty of act ive cel ls , producing suff ic ientl eve l s o f "g row th p rom ot ing f ac to r s " fo r sus ta ined ce l ld iv i s ion . Such f eeder ce l l s have been used succes s fu l lyto p rom ote ce l l d iv i s ion and ca l lu s fo rm at ion in p ro -t o p la s ts f r o m m o n o c o t y l e d o n s su c h a s b a n a n a ( M e g i aet al. , 1992), r ice (Jain et al. , 1995) and maize (Shilli-to et a l. , 1989). B ass and H ugh es (1984) repor ted theregenera t ion o f o i l pa lm p ro top last s u s ing nu r se cu l tu re

39which com pr i s ed o i l pa lm ce l l suspens ion as the f eederlayer and oi l palm protoplas ts on a f i l ter paper d isc . Inthe absence o f suspens ion cu l tu res in ou r l abora to ry ,a t t em pts were m ade to use po lye m b ryogen ic cu l tu resf rom so l id m ed ium as a f eeder l ayer . However , theexper im en ts were n o t succes s fu l.Culture medium

In add i t ion to m ed ium A , a t t em pts were m ad e to i so-la te and cu l tu re p ro top las ts in o ther basa l m ed ia sucha s M u r a s h ig e a n d S k o o g ( 1 96 2 ) m e d i u m , K M m e d i u m(Kao and Michay luk , 1975) and W oody P lan t m ed i -um (W PM ) repor ted by Russe l l and Mc Co wn (1986) .However , m ed ium A cons i s ten t ly per fo rm ed be tt er .G lu ta th ione and ca ta lase were im por tan t com po nen tso f the cu l tu re m ed ium . Var ious l eve ls o f g lu ta th ioneand catalase were tes ted (Table 4) . A t the t ime o f iso-lat ion and 24h later , a l l the protoplas ts had m ore tha n99% v iab il i ty . In the absence o f these two com ponen ts ,v iabi l i ty decrea sed s ignif icant ly af ter 1 week (Table 4) .T he bes t l eve l to m ain ta in ce l l v iab i l ity was 1000 un i t sca ta lase in com bina t ion w i th 2 m M g lu ta th ione andthese were the re fo re inc luded rou t ine ly in the cu l tu rem ed ia . T he m arked im p rovem en t in p ro top las t v iab i l i-ty in the p resence o f bo th these com pon en ts sugges tedt h a t o x y g e n d a m a g e b y l i p id p e r o x id e s m a y b e o n eof the f ac to r s con t r ibu t ing to the poor r esponse o f o i lpalm p rotoplas ts to cul ture . S imila r resul ts were repor t-ed by I sh i i (1989) who found tha t in r i ce ce l l s , theenzy m e t r ea tm en t used fo r p ro top las t i so la t ion causedthe ce l l s to genera te the superox ide r ad ica l . Add i t ionof ca ta lase o r superox ide d i sm utase to the m ed iumresu l ted in s ign i fi can t im prov em e n t in p ro top las t v ia -bi l i ty . Aspir in (O-acety l-sal icyl ic acid) , s i lver n i t rate ,2 ,4 -D and zea t in d id n o t have an e f f ec t on v iab i l ity o rce l l d iv i sion a t the concen t r a t ions used . Repor t s ha veshow n the benef icial ef fec t of aspir in (Carsw ell e t a l . ,1989) and silver salts (Perl et al. , 1988) on protoplastd iv i s ion and co lony fo rm at ion . T hese com poun ds a rebe l i eved to exer t the i r e f f ec t by inh ib i t ion o f e thy -lene produ ct ion (Per l e t al . , 1988 an d Carsw ell e t a l . ,1989) . However , inclus ion of these chemicals in thecu l tu re m ed ium ind iv idua l ly and in com bina t ion d idno t im prove v iab i l ity o r d iv i s ion o f o i l pa lm p ro top las -t s .

In l iqu id m ed ium , Ca lco f luo r s t a in ing showed tha tce l l wa l l fo rm at ion occur red a f te r two days . H owev-er , protoplas t d iv is ion was not observed and the cel lsaggrega ted a f te r two w eeks in cu l tu re m ed ium . On so l-id m ed ium , however , bo th ce l l wa l l r egenera t ion and


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40Table 4. Effec t o f ca ta lase and g lu ta th ione on v iab i l i ty o f pm-toplasts isolated from e mbry ogen ic cultures of oi l palm.

Com pound Viabi li ty (%)Catalase Glu ta th ione 72h 1 W k 2 W kuni t s /ml mM

0 0 954-2 454-6 54-30 1 954-3 754-4 104-50 2 974-1 804-8 274-50 3 964-2 734-7 284-8

500 0 964-1 784-8 554-3500 1 974-1 704-3 534-5500 2 964-3 754-6 604-6500 3 954-2 804-10 604-101000 0 964-2 834-5 674-51000 1 964-3 894-3 694-61000 2 994-1 994-1 754-61000 3 984-1 854-8 684-71500 0 954-1 794-6 604-51500 1 964-2 804-2 594-31500 2 974-1 804-6 574-51500 3 964-1 754-8 534-5

Value represen t the m ean o f 3 -5 independent exper iments. Eachexpe rimen t was carried ou t in tripl icate.

l i m i t e d c e l l d i v i s i on w e re ob se rve d . F i r s t c e ll d i v i s i onwa s o bse rv e d a f t e r t h re e da ys o f c u l tu re a nd c o l on i e so f up t o 12 c e l l s c ou l d be obse rve d a f t e r 10 da ys .F u r t he r d i v i s i on t o fo rm mi c roc a l l u se s (300-500 c e l l s )o c c u r r e d a t a v e r y l o w f r e q u e n c y ( < 0 .0 1 % o f p r o to -p l a s ts p l a t e d ) a f t e r th re e t o fou r we e ks . F u r t he r g ro wt hof t he mi c roc a l l u se s c ou l d n o t be sus t a i ne d on t he s a mem e d i u m .

Ef fo r t s a re i n p rog re s s t o i m prov e t he c u l tu re sys -t e m i n o rde r t o ob t a i n sus t a i ne d c e l l d i v i si on . Re ge n-e ra b l e e mbryoge n i c suspe ns i on c u l t u re s ha ve be e nre por t e d fo r t he o i l pa l m i n o t he r l a bo ra t o r i e s (Touc he te t a l. , 1991; T e ixe i ra e t al . , 1 995) . Effo r t s a re a lso inp rog re s s t o ob t a i n e mb ryog e n i c suspe ns i on c u lt u re s asa sou rc e ma t e r i a l fo r p ro t op l a s t s .

Acknow l e d g emen tT h e a u t h o rs t h a n k t h e D i r e c to r - G e n e r a l o f P O R I M , D r .Yuso f Ba s i ron fo r pe rm i s s i on t o pub l i sh t he se r e su l ts .

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oil palm (elaeis guineensis pro top lasts_ isolation, culture and micro call us formation

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