Citrus Tissue Culture

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Plant Physiol. (1978) 6 2 , 885-888 Citrus Tissue Culture STIMULATION OF FRUIT EXPLANT CULTURES WITH ORANGE JUICE Received f o r publication June 1 2, 1978 a n d i n revised form August 1 , 1978 JoHN W . EiNsFr Department o f Plant Sciences, University o f Cal!fornia, Riverside, California 92521 ABSTRACT hi ntro growth o f explant (juice vesicle oralbedo tissues) cultures from citron (Citrus medics), lemon ( C . limos), grapefruit ( C . paradisj), sweet orange ( C . sinesis), a n d mandarin ( C . redculata) fruits w a s stimulated b y addition o f orange juice (10% v / v optimum) t o a basal medium containing Murashige a n d Skoog salts, 5 0 grams p e r liter sucrose, 1 0 0 milligams p e r liter myo-lnositol, 5 m i a m s p e r liter thiamine-HCI, 2 milligrams p e r liter 2,4-dichlorophenoxyacetic acid a n d 0 . 5 millirams p e r liter kinetin. I n analyzing this effect o f orange juice o n citron explant cultures, w e failed t o obtain increased yields b y addition of appropriate concentrations of citric acid t o t h e basal medium butobtained growth stimulation when t h e medium w a s supplemented with juice from an "acidless" orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid i s involved. Addition o f t h e inorganic a s h corresponding t o 1 0 % (v/v) orange juice t o th e basal medium h ad no effect o n yields. Similarly, t h e stimulatory effect of orange juice could n o t b e explained based o n its content o f sucrose o r o f organic growth factors already present i n thebasal medium. T h e u s e o f tissue culture methods f o r Citrus crop species h as already h a d practical benefits. Most notable among these a re techniques f o r obtaining virus-free a n d mycoplasma-free using i n vitro grafting o f apical meristems from infected plants onto decapitated seedlings (7). As with other woody species which ar e difficult t o culture i n vitro, future advances with Citrus tissue culture will depend on a better understanding o f nutritional factors a n d development o f methods f o r controlling cellular differentia- tion a n d organogenesis (4, 9). To improve techniques f o r Citrustissue culture, w e a r e investi- gating conditions that enhance callus culture growth. It h a s been observed that vigorous growth o f tissue cultures derived from fruit o f many Citrus species requires addition o f orange juice t o a basal medium containing Murashige a n d Skoog salts, sucrose, vitamins, a n d a number o f other organic growth factors (6). Erner e t a l . (3), using callus tissues recultured from orange albedo f o r bioassays, obtained a partial purification o f an active component that co- chromatographed with citric acid. O n the basis o f this evidence a nd t h e activity o f citric acid i n promoting recultured orange albedo, they that the stimulating effect o f orange juice i s a result o f i t s citric acid content. I n experiments t o extend these findings t o fruit explants we have observed orange juice-stimulated growth f o r a number o f Citrus cultivars. We have failed t o observe a n effect o f citric acid when added a s a supplement to t h e basal medium i n the appro- priate concentrations. This paper reports results indicating that the stimulation b y orange juice o f Citrus fruit explant cultures i s a consequence o f some organic factor(s) other than citric acid. MATERIALS AND METHODS Chemicals, Composition of Media. Sucrose a n d t h e organic growth factors myo-inositol, thiamine HCl, 2,4-D, an d kinetin were al l obtained from Sigma Chemical Company. Murashige a nd Skoog salt mixture ( 5 ) a n d Phytagar were obtained from Grand Island Biological Company. The basal medium f o r Citrus explant cultures contained t h e recommended concentration o f Murashige a n d Skoog salts, 5 0 g / l sucrose, 100 mg/l myo-inositol, 5 mg/l thiamine.HC1, 2 mg/l a n d 0 . 5 mg/l kinetin, a n d 1% Phytagar (v/v) ( p H 5.6). Orange juice, obtained locally an d partially clarified b y centrifugation a t 10,000g f or 3 min, was added a s a supplement prior t o autoclaving. Juice from t h e "acidless" orange variety, Lima, was obtained b y hand-squeezing c u t fruit. T h e juice from Lima fruit is p H 6. 8 a nd h as titratable acidity o f 1 1 meq/l compared with t h e juice from Valencia oranges which i s p H 3 .9 a n d titratable acidity o f 1 1 5 meq/l (8). Plant Materials a n d Tissue Culture. Young (100 days postpol- lination) citron, c v . Citron o f Commerce, fruit were used f o r most o f t h e experiments because o f t h e availability o f fruit throughout t he year. These were obtained from t h e University o f California-South Coast Field Station (Orange County, Santa Ana, Calif.). Lima variety orange fruit was from theUniversity of California-Citrus Experiment Station (Riverside, Calif.). Fruit o f other Citrus species were purchased locally. T h e fruit f o r tissue cultures were taken t o t h e laboratory where they were washed with dishware detergent a n d then rinsed with cold water. Subsequent sterilization steps involved soaking i n 0.5% (w/v) sodium hypochlorite f o r 1 0 m i n followed by a brief di p i n 9 5 % (v/v) ethanol a n d flaming. Tissue pieces (20 m g ) o f juice vesicle or albedo were then c ut a n d planted singly in tubes ( 2 5 x 1 5 0 m m ) containing 2 0 m l o f medium. Cultures were incubated i n the dark a t 2 7 C f o r 2 8 days after which time t h e tissues were harvested a n d fresh weights determined. Preparation o f Orange Juice Ash. To hydrolyze pectic sub- stances, a 0.2-ml volume o f HNO3-HCI (1:3) wa s added t o 4 0 m l o f partially clarified orange juice i n a quartz crucible. T h e mixture w a s evaporated t o dryness over a h o t plate a n d then transferred t o a n ashing oven f o r 7 h r a 5 5 0 C ; i e . sufficient time t o produce a white ash. RESULTS Effects o f Orange Juice a n d Citric Acid o n Explant Cultures. Explant cultures o f juice vesicles o r albedo tissues from Citrus fruits proliferate i n vitro b y cell division a n d expansion f o r 4 weeks. T h e final fresh weight yield c a n b e increased substantially b y addition o f 10%1o (v/v) orange juice t o t h e basal medium. Increases i n yield have been observed with juice vesicle explant cultures of citron ( C . medica), lemon ( C . limon), grapefruit (C. paradisi), a n d sweet orange (C . sinensis), a n d with albedo explant cultures o f citron, lemon, mandarin ( C . reticulata) cultivars. Figure 1 shows representative dose/reponse values f o r three Citrus spe- cies. T h e optimum concentration o f approximately 10%'o (v/v) juice caused increases in yield a s great a s 6-fold compared t o controls cultured o n basal medium. To determine whether t h e effect o f orange juice was due t o i t s 8 8 5

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P l a n t P h y s i o l . ( 1 9 7 8 ) 6 2 , 8 8 5 - 8 8 8

C i t r u s T i s s u e C u l t u r e

STIMULATION OF FRUIT EXPLANT CULTURES WITH ORANGE JUICE

R e c e i v e d f o r p u b l i c a t i o n J u n e 1 2 , 1 9 7 8 a n d i n r e v i s e d f o r m A u g u s t 1 , 1 9 7 8

JoHN W. E i N s F r

D e p a r t m e n t o f P l a n t S c i e n c e s , U n i v e r s i t y o f C a l ! f o r n i a , R i v e r s i d e , C a l i f o r n i a 9 2 5 2 1

ABSTRACT

h i n t r o g r o w t h o f e x p l a n t ( j u i c e v e s i c l e o r a l b e d o t i s s u e s ) c u l t u r e s f r o m

c i t r o n ( C i t r u s m e d i c s ) , l e m o n ( C . l i m o s ) , g r a p e f r u i t ( C . p a r a d i s j ) , s w e e t

o r a n g e ( C . s i n e s i s ) , a n d m a n d a r i n ( C . r e d c u l a t a ) f r u i t s w a s s t i m u l a t e d b y

a d d i t i o n o f o r a n g e j u i c e ( 1 0 % v / v o p t i m u m ) t o a b a s a l m e d i u m c o n t a i n i n gM u r a s h i g e a n d S k o o g s a l t s , 5 0 g r a m s p e r l i t e r s u c r o s e , 1 0 0 m i l l i g a m s p e r

l i t e r m y o - l n o s i t o l , 5 m i a m s p e r l i t e r t h i a m i n e - H C I , 2 m i l l i g r a m s p e r

l i t e r 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d a n d 0 . 5 m i l l i r a m s p e r l i t e r k i n e t i n . I na n a l y z i n g t h i s e f f e c t o f o r a n g e j u i c e o n c i t r o n e x p l a n t c u l t u r e s , we f a i l e d t o

o b t a i n i n c r e a s e d y i e l d s b y a d d i t i o n o f a p p r o p r i a t e c o n c e n t r a t i o n s o f c i t r i ca c i d t o t h e b a s a l m e d i u m b u t o b t a in e d g r o w t h s t i m u l a t i o n w h e n t h e m e d i u m

w a s s u p p l e m e n t e d w i t h j u i c e f r o m a n " a c i d l e s s " o r a n g e v a r i e t y ( c v . L i m a ) .T h e s e f a c t s s u g g e s t t h a t s o m e c o m p o n e n t ( s ) o t h e r t h a n c i t r i c a c i d i s

i n v o l v e d . A d d i t i o n o f t h e i n o r g a n i c a s h c o r r e s p o n d i n g t o 1 0 % ( v / v ) o r a n g e

j u i c e t o t h e b a s a l m e d i u m h a d n o e f f e c t o n y i e l d s . S i m i l a r l y , t h e s t i m u l a t o r ye f f e c t o f o r a n g e j u i c e c o u l d n o t b e e x p l a i n e d b a s e d o n i t s c o n t e n t o f s u c r o s e

o r o f o r g a n i c g r o w t h f a c t o r s a l r e a d y p r e s e n t i n t h e b a s a l m e d i u m .

T h e u s e o f t i s s u e c u l t u r e m e t h o d s f o r C i t r u s c r o p s p e c i e s h a s

a l r e a d y h a d p r a c t i c a l b e n e f i t s . M o s t n o t a b l e among t h e s e a r et e c h n i q u e s f o r o b t a i n i n g v i r u s - f r e e a n d m y c o p l a s m a - f r e e s t o c k su s i n g i n v i t r o g r a f t i n g o f a p i c a l m e r i s t e m s f r o m i n f e c t e d p l a n t so n t o d e c a p i t a t e d s e e d l i n g s ( 7 ) . As w i t h o t h e r w o o d y s p e c i e s w h i c ha r e d i f f i c u l t t o c u l t u r e i n v i t r o , f u t u r e a d v a n c e s w i t h C i t r u s t i s s u ec u l t u r e w i l l d e p e n d on a b e t t e r u n d e r s t a n d i n g o f n u t r i t i o n a l f a c t o r sa n d d e v e l o p m e n t o f m e t h o d s f o r c o n t r o l l i n g c e l l u l a r d i f f e r e n t i a -t i o n a n d o r g a n o g e n e s i s ( 4 , 9 ) .To i m p r o v e t e c h n i q u e s f o r C i t r u s t i s s u e c u l t u r e , we a r e i n v e s t i -

g a t i n g c o n d i t i o n s t h a t e n h a n c e c a l l u s c u l t u r e g r o w t h . I t h a s b e e no b s e r v e d t h a t v i g o r o u s g r o w t h o f t i s s u e c u l t u r e s d e r i v e d f r o m f r u i to f m a ny C i t r u s s p e c i e s r e q u i r e s a d d i t i o n o f o r a n g e j u i c e t o a b a s a lm e d i u m c o n t a i n i n g M u r a s h i g e a n d S k o o g s a l t s , s u c r o s e , v i t a m i n s ,a n d a n u m b e r o f o t h e r o r g a n i c g r o w t h f a c t o r s ( 6 ) . E r n e r e t a l . ( 3 ) ,u s i n g c a l l u s t i s s u e s r e c u l t u r e d f r o m o r a n g e a l b e d o f o r b i o a s s a y s ,o b t a i n e d a p a r t i a l p u r i f i c a t i o n o f an a c t i v e c o m p o n e n t t h a t c o -

c h r o m a t o g r a p h e d w i t h c i t r i c a c i d . On t h e b a s i s o f t h i s e v i d e n c ea n d t h e a c t i v i t y o f c i t r i c a c i d i n p r o m o t i n g r e c u l t u r e d o r a n g ea l b e d o , t h e y c o n c l u d e d t h a t t h e s t i m u l a t i n g e f f e c t o f o r a n g e j u i c ei s a r e s u l t o f i t s c i t r i c a c i d c o n t e n t .

I n e x p e r i m e n t s t o e x t e n d t h e s e f i n d i n g s t o f r u i t e x p l a n t s w e

h a v e o b s e r v e d o r a n g e j u i c e - s t i m u l a t e d g r o w t h f o r a n u m b e r o fC i t r u s c u l t i v a r s . We h a v e f a i l e d t o o b s e r v e a n e f f e c t o f c i t r i c a c i dw h e n a d d e d a s a s u p p l e m e n t t o t h e b a s a l medium i n t h e a p p r o -p r i a t e c o n c e n t r a t i o n s . T h i s p a p e r r e p o r t s r e s u l t s i n d i c a t i n g t h a tt h e s t i m u l a t i o n b y o r a n g e j u i c e o f C i t r u s f r u i t e x p l a n t c u l t u r e s i sa c o n s e q u e n c e o f some o r g a n i c f a c t o r ( s ) o t h e r t h a n c i t r i c a c i d .

MATERIALS AND METHODS

C h e m i c a l s , C o m p o s i t i o n o f M e d i a . S u c r o s e a n d t h e o r g a n i c

g r o w t h f a c t o r s m y o - i n o s i t o l , t h i a m i n e H C l , 2 , 4 - D , a n d k i n e t i nw e r e a l l o b t a i n e d f r o m S i g m a C h e m i c a l C o m p a n y . M u r a s h i g ea n d S k o o g s a l t m i x t u r e ( 5 ) a n d P h y t a g a r w e r e o b t a i n e d f r o mG r a n d I s l a n d B i o l o g i c a l C o m p a n y . T h e b a s a l medium f o r C i t r u se x p l a n t c u l t u r e s c o n t a i n e d t h e r e c o m m e n d e d c o n c e n t r a t i o n o fM u r a s h i g e a n d S k o o g s a l t s , 5 0 g / l s u c r o s e , 1 0 0 m g / l m y o - i n o s i t o l ,5 m g / l t h i a m i n e . H C 1 , 2 m g / l 2 , 4 - D , a n d 0 . 5 m g / l k i n e t i n , a n d1 % P h y t a g a r ( v / v ) ( p H 5 . 6 ) . O r a n g e j u i c e , o b t a i n e d l o c a l l y a n dp a r t i a l l y c l a r i f i e d b y c e n t r i f u g a t i o n a t 1 0 , 0 0 0 g f o r 3 0 m i n , w a s

a d d e d a s a s u p p l e m e n t p r i o r t o a u t o c l a v i n g . J u i c e f r o m t h e" a c i d l e s s " o r a n g e v a r i e t y , L i m a , w a s o b t ai n e d b y h a n d - s q u e e z i n gc u t f r u i t . T h e j u i c e f r o m L i m a f r u i t i s pH 6 . 8 a n d h a s t i t r a t a b l ea c i d i t y o f 1 1 m e q / l c o m p a r e d w i t h t h e j u i c e f r o m V a l e n c i a o r a n g e sw h i c h i s pH 3 . 9 a n d t i t r a t a b l e a c i d i t y o f 1 1 5 m e q / l ( 8 ) .

P l a nt M a t e r i al s a n d T i s s u e C u l t u r e . Y o u n g ( 1 0 0 d a y s p o s t p o l -l i n a t i o n ) c i t r o n , c v . C i t r o n o f C o m m e r c e , f r u i t w e r e u s e d f o r m o s to f t h e e x p e r i m e n t s b e c a u s e o f t h e a v a i l a b i l i t y o f f r u i t t h r o u g h o u tt h e y e a r . T h e s e w e r e o b t a i n e d f r o m t h e U n i v e r s i t y o f

C a l i f o r n i a - S o u t h C o a s t F i e l d S t a t i o n ( O r a n g e C o u n t y , S a n t a

A n a , C a l i f . ) . L i m a v a r i e t y o r a n g e f r u i t w a s f r o m t h e U n i v e r s i t y o fC a l i f o r n i a - C i t r u s E x p e r i m e n t S t a t i o n ( R i v e r s i d e , C a l i f . ) . F r u i to f o t h e r C i t r u s s p e c i e s w e r e p u r c h a s e d l o c a l l y .

T h e f r u i t f o r t i s s u e c u l t u r e s w e r e t a k e n t o t h e l a b o r a t o r y w h e r et h e y w e r e w a s h e d w i t h d i s h w a r e d e t e r g e n t a n d t h e n r i n s e d w i t h

c o l d w a t e r . S u b s e q u e n t s t e r i l i z a t i o n s t e p s i n v o l v e d s o a k i n g i n 0 . 5 %( w / v ) s o d i u m h y p o c h l o r i t e f o r 1 0 m i n f o l l o w e d b y a b r i e f d i p i n95% ( v / v ) e t h a n o l a n d f l a m i n g . T i s s u e p i e c e s ( 2 0 m g ) o f j u i c ev e s i c l e o r a l b e d o w e r e t h e n c u t a n d p l a n t e d s i n g l y i n c u l t u r e t u b e s

( 2 5 x 1 5 0 m m ) c o n t a i n i n g 2 0 m l o f m e d i u m . C u l t u r e s w e r ei n c u b a t e d i n t h e d a r k a t 2 7 C f o r 2 8 d a y s a f t e r w h i c h t i m e t h et i s s u e s w e r e h a r v e s t e d a n d f r e s h w e i g h t s d e t e r m i n e d .

P r e p a r a t i o n o f O r a n g e J u i c e A s h . To h y d r o l y z e p e c t i c s u b -s t a n c e s , a 0 . 2 - m l v o l u m e o f HNO3-HCI ( 1 : 3 ) w a s a d d e d t o 4 0 m lo f p a r t i a l l y c l a r i f i e d o r a n g e j u i c e i n a q u a r t z c r u c i b l e . T h e m i x t u r ew a s e v a p o r a t e d t o d r y n e s s o v e r a h o t p l a t e a n d t h e n t r a n s f e r r e dt o a n a s h i n g o v e n f o r 7 h r a t 5 5 0 C ; i e . s u f f i c i e n t t i m e t o p r o d u c ea w h i t e a s h .

RESULTS

E f f e c t s o f O r a n g e J u i c e a n d C i t r i c A c i d o n E x p l a n t C u l t u r e s .E x p l a n t c u l t u r e s o f j u i c e v e s i c l e s o r a l b e d o t i s s u e s f r o m C i t r u sf r u i t s p r o l i f e r a t e i n v i t r o b y c e l l d i v i s i o n a n d e x p a n s i o n f o r 4w e e k s . T h e f i n a l f r e s h w e i g h t y i e l d c a n b e i n c r e a s e d s u b s t a n t i a l l yb y a d d i t i o n o f 1 0 % 1 o ( v / v ) o r a n g e j u i c e t o t h e b a s a l m e d i u m .I n c r e a s e s i n y i e l d h a v e b e e n o b s e r v e d w i t h j u i c e v e s i c l e e x p l a n tc u l t u r e s o f c i t r o n ( C . m e d i c a ) , l e m o n ( C . l i m o n ) , g r a p e f r u i t ( C .p a r a d i s i ) , a n d s w e e t o r a n g e ( C . s i n e n s i s ) , a n d w i t h a l b e d o e x p l a n tc u l t u r e s o f c i t r o n , l e m o n , m a n d a r i n ( C . r e t i c u l a t a ) c u l t i v a r s . F i g u r e1 s h o w s r e p r e s e n t a t i v e d o s e / r e p o n s e v a l u e s f o r t h r e e C i t r u s s p e -c i e s . T h e o p t i m u m c o n c e n t r a t i o n o f a p p r o x i m a t e l y 1 0 % ' o ( v / v )o r a n g e j u i c e c a u s e d i n c r e a s e s i n y i e l d a s g r e a t a s 6 - f o l d c o m p a r e dt o c o n t r o l s c u l t u r e d o n b a s a l m e d i u m .

To d e t e r m i n e w h e t h e r t h e e f f e c t o f o r a n g e j u i c e w a s d u e t o i t s

8 8 5

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P l a n t P h y s i o l . V o l . 6 2 , 1 9

1 , 3 , 3 00 _ D

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CONC. OF J U I C E , %

F I G . 1 . E f f e c t o f c o n c e n t r a t i o n o f orange j u i c e a d d e d as a s u p p l e m e n tt o b a s a l medium on y i e l d s o f C i t r u s e x p l a n t c u l t u r e s . A : j u i c e o f c i t r o n ; B :a l b e d o t i s s u e o f c i t r o n ; C : a l b e d o t i s s u e o f l e m o n ; D : a l b e d o t i s s u e o f

orange. G r o w t h p e r i o d w a s 2 8 d a y s .

w i ~ ._.--

F I G . 2 . C o m p a r i s o n o f y i e l d s o f c i t r o n j u i c e v e s i c l e a n d a l b e d o e x p l a n tc u l t u r e s grown on m e d i a s u p p l e m e n t e d w i t h V a l e n c i a or L i m a ( i . e . " a c i d -l e s s " ) v a r i e t y orange j u i c e . C u l t u r e f l a s k s e a c h c o n t a i n e d 5 0 m l o f m e d i u m ;

g r o w t h p e r i o d w a s 2 8 d a y s .

c i t r i c a c i d c o n t e n t ( 2 ) , we a d d e d c i t r i c a c i d i n c o n c e n t r a t i oe q u i v a l e n t t o 0 . 5 , 1 , 5 , a n d 1 0 % ( v / v ) orange j u i c e ( i . e . 0 . 2 6 , 0 . 52 . 5 , a n d 5 . 2 mm c i t r i c a c i d , r e s p e c t i v e l y ) . R e s u l t s o f t h e s e e x p e rments w i t h c i t r o n j u i c e v e s i c l e a n d a l b e d o e x p l a n t c u l t u r e s h a v

b e e n c o n s i s t e n t l y n e g a t i v e . C i t r i c a c i d w a s f o u n d t o b e w i t h o

e f f e c t i n r e p e a t e d t e s t s r e p r e s e n t i n g over 5 0 0 c u l t u r e s s u g g e s t i

t h a t s o m e o t h e r c o m p o n e n t ( s ) o f orange j u i c e w a s r e s p o n s i b l e ft h e s t i m u l a t i o n o f g r o w t h .

J u i c e f r o m t h e L i m a c u l t i v a r o f orange, a n " a c i d l e s s " v a r i e tc o n t a i n s a p p r o x i m a t e l y 1 / 1 0 0 t h e c i t r i c a c i d c o n c e n t r a t i o n o f t

j u i c e f r o m t h e c o m m e r c i a l V a l e n c i a c u l t i v a r . F i g u r e 2 s h o w s te f f e c t s o f t h e two j u i c e s i n s t i m u l a t i n g y i e l d s o f c i t r o n j u i c e v e s i ca n d a l b e d o c u l t u r e s . I t ca n b e seen t h a t j u i c e f r o m t h e " a c i d l e sorange h a s d e f i n i t e s t i m u l a t o r y a c t i v i t y i n t h e s a m e c o n c e n t r a t i

range a s t h e n o r m a l j u i c e w h i c h , a g a i n , s u g g e s t s t h a t s o m e co m

p o n e n t ( s ) o f t h e j u i c e s o t h e r t h a n c i t r i c a c i d i s i n v o l v e d .Inorganic C o n s t i t u e n t s . T h e p o s s i b i l i t y t h a t t h e e n h a n c e

g r o w t h o f c i t r o n e x p l a n t c u l t u r e s i s a r e s u l t o f i n o r g a n i c c o n s t i te n t s i n orange j u i c e w a s t e s t e d b y co m pa r in g y i e l d s o b t a i n e d o

t h e b a s a l medium w i t h y i e l d s on m e d i a s u p p l e m e n t e d w i t h 1 0

( v / v ) ora n g e j u i c e or w i t h t h e i n o r g a n i c n u t r i e n t s i n a s h corr

s p o n d i n g t o t h i s s a m e c o n c e n t r a t i o n o f ora n g e j u i c e . T h e r e s u lo f t h e s e e x p e r i m e n t s i n T a b l e I s h o w n o e f f e c t o f t h e a s h c o m p a r

t o s t i m u l a t i o n b y w h o l e j u i c e w h i c h i n d i c a t e s t h a t i n o r g a n

c o n s t i t u e n t s i n ora n g e j u i c e d o n o t cause t h e i n c r e a s e d y i e l d s . T hr e c o m m e n d e d s i n g l e s t r e n g t h c o n c e n t r a t i o n o f M u r a s h i g e a n

S k o o g s a l t s i s o p t i m a l f o r c i t r o n e x p l a n t c u l t u r e s ( F i g . 3 ) a s i t

f o r r e c u l t u r e d C i t r u s c a l l u s ( 6 ) .O r g a n i c C o n s t i t u e n t s . Th e e f f e c t o f orange j u i c e on y i e l d s

c i t r o n e x p l a n t c u l t u r e s i n t h e p r e s e n c e o f d i f f e r e n t sucrose conce

t r a t i o n s w a s i n v e s t i g a t e d t o d e t e r m i n e w h e t h e r orange j u i c e f un i s h e s c a r b o h y d r a t e t h a t i s p r e s e n t i n g r o w t h - l i m i t i n g c o n c e n t r

t i o n s i n t h e b a s a l m e d i u m . I t i s known t h a t 1 0 9 % ( v / v ) orange j u is u p p l e m e n t a t i o n w o u l d i n c r e a s e t h e s u c r o s e a n d t o t a l s ug ar co

c e n t r a t i o n s o f t h e m e d i a b y a p p r o x i m a t e l y 5 g / l a n d 1 0 g /r e s p e c t i v e l y ( 1 ) . As e x p e c t e d , orange j u i c e p a r t i a l l y r e s t o r e s g r o w

o f e x p l a n t c u l t u r e s on m e d i a l a c k i n g sucrose ( F i g . 4 ) . T h e y i e lo f c a l l u s are s t i l l very l o w a n d t h e magnitude o f t h e i n c r e a s e d

t o orange j u i c e i s much l e s s t h a n t h a t o b t a i n e d on t h e o p t i m

s u c r o s e c o n c e n t r a t i o n s i n d i c a t i n g t h a t sucrose c o n t a i n e d i n oran

j u i c e i s n o t t h e reason f o r i t s a c t i v i t y .T a b l e I I s h o w s t h e e f f e c t o f o m i s s i o n o f m i n o r o r g a n i c c o n s t i

u e n t s o f t h e b a s a l m e d i u m on y i e l d s o f c i t r o n j u i c e v e s i c l e c u l t u r eTh e l a c k o f e f f e c t r e s u l t i n g f r o m t h e o m i s s i o n o f e i t h e r m y

i n o s i t o l ( t r e a t m e n t c ) or k i n e t i n ( t r e a t m e n t d ) i n d i c a t e s t h a t t h eg r o w t h f a c t o r s a r e n o t e s s e n t i a l . R e m o v a l o f 2 , 4 - D f r o m t h e b a sm e d i u m s u b s t a n t i a l l y d e c r e a s e s y i e l d ( t r e a t m e n t e ) b u t a d d i t io f orange j u i c e t o t h e s e c u l t u r e s d o e s n o t s i g n i f i c a n t l y i m p r o

t h e y i e l d ( t r e a t m e n t f ) . T h i s s h o w s t h a t ora n g e j u i c e s u p p l i e s l i t t

i f a n y , m a t e r i a l s u b s t i t u t i n g f o r t h e 2 , 4 - D r e q u i r e m e n t . O m i s s i

o f t h i a m i n e H C I h a s t h e m o s t d r a m a t i c e f f e c t on y i e l d ( t r e a t m eg ) . T h i s s u g g e s t s t h a t t h e stimulation o f g r o w t h o f t i s s u e smedium l a c k i n g g r o w t h f a c t o r s b u t c o n t a i n i n g ora n g e j u i c e ( t r e

Table I . Effects o f a s h a n d whole orange juice o n y i e l d s o f explant

cultures derived f r o m citron j u i c e vesicle a n d albedo tissues. Values

are m e a n s o f 5 r e p l i c a t e f l a s k s + standard error. Growth period was 2 8

d a y s .

Yield ( m g f r e s h w t )Additions t o Basal Medium

J u i c e V e s i c l e A l b e d o

None 4 5 0 + 2 7 7 5 + 4

As h o f 1 0 % ( v / v ) Orange Juice 4 0 2 + 3 5 7 8 + 6

1 0 % ( v / v ) Orange Juice 6 2 5 + 8 0 1 4 2 + 1 9

8 8 6 EINSET

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P l a n t P h y s i o l . V o l . 6 2 , 1 9 7 8

E

3

I

n

Lo

CITRUS EXPLANT CULTURES

E

100

50

O

0 IX 2 X 3 X

C O N C . OF MS SALTS

F I G . 3 . E f f e c t o f i n c r e a s e d i n o r g a n i c n u t r i e n t c o n c e n t r a t i o n s o n r e -

sponse o f c i t r o n e x p l a n t c u l t u r e s t o orange j u i c e ; I x MS s a l t s ar e t h ec o n c e n t r a t i o n s o f i n o r g a n i c s s p e c i f i e d b y M u r a s h i g e a n d S k o o g . A : j u i c ev e s i c l e s o f c i t r o n ; B : a l b e d o t i s s u e o f c i t r o n ; ( 0 ) : u n s u p p l e m e n t e d m e d i a ;( 0 ) : s u p p l e m e n t e d w i t h 10% ( v / v ) orange j u i c e . E a c h p o i n t r e p r e s e n t s f i v er e p l i c a t e c u l t u r e s . G r o w t h p e r i o d w a s 2 8 d a y s .

E

( n

L U .

E

3 .

n)

U i .

8 8 7

C O N C . O F S U C R O S E , g / 1

F I G . 4 . E f f e c t o f sucrose c o n c e n t r a t i o n on response o f c i t r o n e x p l a n tc u l t u r e s t o orange j u i c e . A : j u i c e v e s i c l e s o f c i t r o n ; B : a l b e d o t i s s u e o f

c i t r o n ; ( 0 ) : u n s u p p l e m e n t e d m e d i a ; ( 0 ) : s u p p l e m e n t e d w i t h 1 0 % ( v / v )orange j u i c e . E a c h p o i n t r e p r e s e n t s f i v e r e p l i c a t e c u l t u r e s . G r o w t h p e r i o dw a s 2 8 d a y s .

Table I I . Y i e l d s o f j u i c e vesicle e x p l a n t c u l t u r e s i n t h e a b s e n c e o f

growth f a c t o r s . T h e + indicates presence o f a growth f a c t o r , - a b s e n c e .Concentrations o f growth f a c t o r s are as i n d i c a t e d i n t h e Materials and

M e t h o d s . Orange j u i c e was a d d e d to 1 0 % ( v / v ) . Values are m e a n s o f 5r e p l i c a t e f l a s k s + s t a n d a r d error. Growth p e r i o d w a s 2 8 d a y s .

Growth FactorsTreatment Yield ( m g )

i-Inositol Kinetin 2 , 4 - D T h i a m i n e . H C l Orange Juice

a + + + + + 7 2 1 + 5 3b + + + + - 5 1 8 + 3 4c - + + + - 5 3 2 + 9 3d + - + + - 5 0 9 + 7 3e + + - + - 3 0 0 + 5 5f + + - + + 3 9 0 + 6 1g + + + - - 1 4 7 +19h - - - - - 90+ 6

i - - - - + 3 4 8 + 4 8

m e n t i v e r s u s t r e a t m e n t h ) may b e a t l e a s t p a r t i a l l y a r e s u l t o f t h e v e r s u s t r e a t m e n t b ) i s n o t a c o n s e q u e n c e o f t h i a m i n e , h o w e v e r ,t h i a m i n e

c o n t e n t o f t h e j u i c e ( 1 ) . E n h a n c e d g r o w t h o f c u l t u r e s o n b e c a u s e t h e b a s a l m e d i u m c o n t a i n s t h e o p t i m u m t h i a m i n e . H C 1t h e b a s a l m e d i u m s u p p l e m e n t e d w i t h o r a n g e j u i c e ( t r e a t m e n t a c o n c e n t r a t i o n ( 6 ) .

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8 8 8 EINSET P l a n t P h y s i o l . V o l . 6 2 , 1 9 7

DISCUSSION

T h e s t i m u l a t i o n o f C i t r u s t i s s u e c u l t u r e s b y o r a n g e j u i c e s u p p l e -m e n t s h a s now b e e n e x t e n d e d t o e x p l a n t c u l t u r e s d e r i v e d f r o m an u m b e r o f f r u i t t y p e s . U n l i k e t h e s t i m u l a t i o n o f r e c u l t u r e d c a l l u so f o r a n g e a l b e d o , t h e e f f e c t o f o r a n g e j u i c e o n c i t r o n e x p l a n tc u l t u r e s c a n n o t b e a t t r i b u t e d t o c i t r i c a c i d b a s e d o n t h e l a c k o fe f f e c t o f c i t r i c a c i d a d d e d t o t h e b a s a l m e d i u m a n d t h e d e m o n -s t r a t e d s t i m u l a t i o n o f g r o w t h c a u s e d b y j u i c e f r o m t h e " a c i d l e s s , "

v a r i e t y o f o r a n g e .E x p l a n t c u l t u r e s d e r i v e d f r o m j u i c e v e s i c l e t i s s u e s o f c i t r o n f r u i t

h a v e s h o w n t h e h i g h e s t d e g r e e o f g r o w t h s t i m u l a t i o n i n r e s p o n s et o o r a n g e j u i c e s u p p l e m e n t s . O u r e v i d e n c e w i t h t h e s e c u l t u r e ss u g g e s t s t h a t n e i t h e r i n o r g a n i c c o n s t i t u e n t s n o r t h e c a r b o n s o u r c es u c r o s e i s t h e c o m p o n e n t o f o r a n g e j u i c e c a u s i n g i n c r e a s e d y i e l d s .I t s e e m s u n l i k e l y , a l s o , t h a t t h e o r a n g e j u i c e f u r n i s h e s s t i m u l a t o r yq u a n t i t i e s o f t h o s e o r g a n i c g r o w t h f a c t o r s a l r e a d y p r e s e n t i n t h eb a s a l m e d i u m .

W h e t h e r t h e a c t i v e o r g a n i c c o m p o n e n t ( s ) i n o r a n g e j u i c e i s a

k n o w n p l a n t g r o w t h s u b s t a n c e r e m a i n s t o b e d e t e r m i n e d . U n a n -s w e r e d a l s o i s t h e q u e s t i o n o f t h e r e l a t i o n s h i p , i f a n y , o f t h e i n

v i t r o r e s p o n s e t o p o t e n t i a l f a c t o r s r e g u l a t i n g f r u i t g r o w t h a nd e v e l o p m e n t i n C i t r u s .

A c k n o w l e d g m e n t s - I t h a n k c W. P . B i t t e r s f o r a d v i c e a n d f o r f u r n i s h i n g f r u i t a n d C . K . L a b a

a u s k a s f o r h e l p i n t h e p r e p a r a t i o n o f t h e a s h e d o r a n g e j u i c e s a m p l e . T h e t e c h n i c a l a a s i s t a n c e o f

C h e n g i s g r a t e f u l l y a c k n o w l e d g e d .

LITERATURE CITED

1 . B I R D S A L L , J J , PH D E R S E , L J T E P L Y 1 9 6 1 N u t r i e n t s i n C a l i f o r n i a l e m o n s a n d o r a n g e s .V i t a m i n , m i n e r a l a n d p r o x i m a t e c o m p o s i t i o n . J Am D i e t A s s o c 3 8 : 5 5 5 - 5 5 9

2 . C L E M E N T S RL 1 9 6 4 O r g a n i c a c i d s i n c i t r u s f r u i t s . I . V a r i e t a l d i f f e r e n c e s . J F o o d S c i 2 9 : 2 7 6 - 2

3 . E R N E R Y , 0 R E u v E N I , EE G O L D s c H m D T 1 9 7 5 P a r t i a l p u r i f i c a t i o n o f a g r o w t h f a c t o r f ro r a n g e j u i c e w h i c h a f f e c t s c i t r u s t i s s u e c u l t u r e a n d i t s r e p l a c e m e n t b y c i t r i c a c i d . P lP h y s i o l 5 6 : 2 7 9 - 2 8 2

4 . M U R A S H I G E T 1 9 7 4 P l a n t p r o p a g a t i o n t h r o u g h t i s s u e c u l t u r e s . A n n u R e v P l a n t P h y s i o l1 3 5 - 1 6 6

5 . M U R A S H I G E T , F S K O O G 1 9 6 2 A r e v i s e d m e d i u m f o r r a p i d g r o w t h a n d b i o a s s a y s w i t h t o b a c

t i s s u e c u l t u r e s . P h y s i o l P l a n t 1 5 : 4 7 3 - 4 9 7

6 . M U R A S H I G E T , DPH T u c K E a 1 9 6 9 G r o w t h f a c t o r r e q u i r e m e n t s o f c i t r u s t i s s u e c u l t u r e . P r

F i r s t I n t C i t r u s S y m p 3 : 1 1 5 5 - 1 1 6 1

7 . NAVARRo L , CN R O I S T A C H E R , T M U R A S s c G E 1 9 7 5 I m p r o v e m e n t o f s h o o t - t i p g r a f t i n g i n v i

f o r v i r u s - f r e e c i t r u s . J Am S o c H o r t S c i 1 0 0 : 4 7 1 - 4 7 9

8 . U L R I C H R 1 9 7 0 O r g a n i c a c i d s . I n AC H u l m e , e d , T h e B i o ch e m is t r y o f F r u i t s a n d T h

P r o d u c t s , V o l I . A c a d e m i c P r e s s , L o n d o n , p p . 8 9 - 1 1 8

9 . VMDI A , P S P i E G E L - R o Y , E GALUN 1 9 7 5 C i t r u s c e l l c u l t u r e : i s o l a t i o n o f p r o t o p l a s t s , p l a td e n s i t i e s , e f f e c t o f m u t a g e n s a n d r e g e n e r a t i o n o f e m b r y o s . P l a n t S c i L e t t 4 : 2 3 1 - 2 3 6