Plant Cell Reports
Plant Cell Reports (1982) 1: 108-110
© Springer-Verlag 1982
In vitro P l a n t R e g e n e r a t i o n in L a t e r a l B u d E x p l a n t s o f bromelioides V a r . T r i c o l o r M . B . F o s t e r
Cryptanthus
V. Helena Mathews and P. S. Rao Plant Morphogenesis and Tissue Culture Section, Bio-Organic Division, Bhabha Atomic Research Centre, Bombay 400 085, India Received November 5, 1981 ; January 6, 1982
Abstract
M a t e r i a l s and Methods
L a t e r a l buds of Gryptanthus b r o m e l i o i d e s v a r . t r i c o l o r v a r i e g a t e d ornamental b r o m e l i a d , were c u l t u r e d on Murashige and Skoog's b a s a l medium ( ~ ) supplemented w i t h n a p t h a l e n e a c e t i c a c i d , i n d o l e b u t y r i c a c i d , and k i n e t i n , each at 2 mg/1. The b a s a l p a r t of the c u l t u r e d buds p r o l i f e r a t e d and produced a d v e n t i t i o u s s h o o t s . The s h o o t s developed i n t o p l a n t s of t h r e e d i f f e r e n t phenot y p e s : (a) entirely g r e e n , ( b ) . s t r i a t e d (green
L a t e r a l buds (2 mm l o n g ) were e x c i s e d from mature stems of C r y p t a n t h u s b r o m e l i o i d e s v a t . t r i c o l o r and were s u r f a c e - s t e r i l i z e d i n 0 . 1 p e r e e n t m e r c u r i c c h l o r i d e s o l u t i o n f o r 3 rain. and s u b s e q u e n t l y washed r e p e a t e d l y i n s t e r i l e distilled water. The buds were p l a c e d on MS medium ( ~ u r a s h i g e and Skoog, 1962). The e f f e c t of v a r i o u s auxins (NAA, IAA, IBA, 2 , 4 - D ) and
and yellowish-white) and (c) yellowish-white.
c o m b i n a t i o n s a t v a r i o u s c o n c e n t r a t i o n s , was studied. The pH of the medium was a d j u s t e d t o 5.8 b e f o r e s o l i d i f y i n g w i t h 0.8~ agar ( B a c t e r i o l o g i c a l g r a d e , 0entron Research L a b s . , Bombay). The c u l t u r e s were i n c u b a t e d under c o n t i n u o u s
~ o o t i n g could be induced in (a) and (b) types but s u c c e s s f u l t r a n s p l a n t a t i o n was p o s s i b l e only w i t h g r e e n p l a n t s some of which underwent c o l o r changes a f t e r e s t a b l i s h m e n t in s o i l .
cytokinins (Kn, BA, 2,i-P, Z) alone and in
light (i000 Lux) at 25 + 2°C with a relative A b b r e v i a t i o n s : BA, b e n z y l a d e n i n e ; 2,4-D, 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 ; IAA, i n d o l e a c e t i c a c i d ; IBA, i n d o l e b u t y r i c a c i d ; 2 , i - P , 6 ~ d i m e t h y l a l l y l a m i n o p u r i n e ; Kn, k i n e t i n , NAA, n a p h t h a l e n e a c e t i c a c i d , Z, z e a t i n .
hmnidity of 55-60~* For i n d u c t i o n of r o o t 2stem, t h e r e g e n e r a t e d s h e e t s were c u l t u r e d on s t r e n g t h MS medium w i t h o u t any hormones. Rooted p l a n t s were t r a n s f e r r e d t o s o i l . Besalts
Introduction lateral A r e v i e w of r e c e n t r e s e a r c h u t i l i z i n g the t e c h n i q u e of in v i t r o c u l t u r e of p l a n t organs i n d i c a t e s i t s p o t e n t i a l use in t h e r a p i d c l o n a l m u l t i p l i c a t i o n of d i v e r s e c r o p s , e x p e c i a l l y t h e ornamentals ( H o l d g a t e , 1977). The f a m i l y B r o m e l i a c e a e i s composed of mainly short-stemmed herbaceous e p i p h y t e s , and i n c l u d e s t h e p i n e a p p l e and some c o m m e r c i a l l y i m p o r t a n t o r n a m e n t a l s . The p l a n t s are e i t h e r p r o p a g a t e d by seeds or v e g e t a t i v e l y from ' p u p s ' or d i v i s i o n s , the l a t t e r i s a slow p r o c e s s . The a p p l i c a t i o n of t i s s u e c u l t u r e t e c h n i q u e s to members of t h e Bromeli~ceae may i n c r e a s e s u b s t a n t i a l l y the r a t e s of m u l t i p l i c a t i o n (Murashige, 1974~ Mathews and Banyan, 1979, Mathews e t al., 1974). 0 r y p t a n t h u s b r o m e l i o i d e s v a r . t r i c o l o r i s an ornamental b r o m e l i a d grown f o r i t s v a r i e g a t e d l e a v e s which a r e green i n the m i d d l e , and y e l l o w i s h - w h i t e in the margins. The t e n d e r l e a v e s p o s s e s s a carmine red c o l o u r . The p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n with t h e o b j e c t i v e of e s t a b l i s h i n g a t i s s u e c u l t u r e p r o c e d u r e f o r the m u l t i p l i c a t i o n of t h i s v a r i e g a t e d p l a n t and to i n v e s t i g a t e the p o s s i b i l i t y of o b t a i n i n g new
variants.
0721-7714/82/0001/0108/$ 01.00
(2
buds c u l t u r e d on ~
+ NAA (2 m~/1) ÷ I~k
÷ Kinetin (2 m /1) showed the unfolding
of t h e s c a l e l e a v e s w i t h i n a p e r i o d of 8 weeks. Upon t r a n s f e r of the u n f o l d e d buds t o f r e s h medium of the same c o m p o s i t i o n , t h e b a s a l p a r t of the d e v e l o p i n g shoot underwent p r o l i f e r a t i o n s i m u l t a n e o u s l y producing a l a r g e number of a d v e n t i t i o u s shoots (Fig. 1). I f the a d v e n t i t i o u s s h o o t s were i s o l a t e d and s u b c u l t u r e d on the same medium, numerous new shoots developed again at the base of the e x p l a n t , thus enhancing the r a t e of m u l t i p l i c a t i o n . The a d v e n t i t i o u s s h o o t s were of t h r e e t y p e s : Ca) s t r i a t e d , comparable t o t h e
natural type, (b) entirely green, and (c) yellowish w h i t e ( F i g . 2 ) . In the s t r i a t e d t y p e s , the
degree of g r e e n and y e l l o w i s h - w h i t e s t r i a t i o n s v a r i e d and i n e n t i r e l y g r e e n p l a n t s , the d e g r e e of greenness v a r i e d . The c u l t u r e of d i f f e r e n t phenotypes a g a i n r e s u l t e d in a d v e n t i t i o u s bud p r o d u c t i o n ( F i g . 3). As the number of subc u l t u r e s i n c r e a s e d t h e r e was a tendency f o r green p l a n t s %o dominate. In the i n i t i a l c u l t u r e the bud i n i t i a l s produced at the base of the e x p l a n t were too small to be scored f o r d i f f e r e n t p h e n o t y p e s . Therefore, the p r o l i f e r a t e d mass from each c u l t u r e was i s o l a t e d
109
F i g s . ( 1 - 5 ) 1. A d v e n t i t i o u s bud f o r m a t i o n i n l a t e r a l bud c u l L u r e of C r y p t a n t h u s b r o m e l i o i d e s on ~iS + NAA + IBA + Kn ( e a c h a t 2 m g / 1 ) . 2. E e g e n e r a t e d s h o o t s of d i f f e r e n t p h e n o t y p e s : g r e e n , s t r i a t e d and y e l l o w i s h white respectively. 3. Differentiation of s h o o t buds f r o m t h e b a s e of g r e e n , y e l l o w i s h - w h i t e and s t r i a t e d e x p l a n t s on c u l t u r e . 4. Proliferation and differentiation i n t h e s t e m p o r t i o n of a g r e e n e x p l a n t on c u l t u r e . 5. R o o t i n g i n p l a n t s of d i f f e r e n t p h e n o t y p e s . Green and s t r i a t e d p r o d u c e d good r o o t s y s t e m w h i l e y e l l o w i s h - w h i t e p l a n t was c o m p l e t e l y d e v o i d of r o o t s .
and t h e o r i g i n a l e x p l a n t a s w e l l as p r o l i f e r a t e d t i s s u e w i t h s h o o t buds a t d i f f e r e n t s t a g e s of growth were c u l t u r e d s e p a r a t e l y . I t was f o u n d t h a t by r e p e a t e d s u b c u l t u r e s g r e e n and y e l l o w i s h w h i t e e x p l a n t s could r e p r o d u c e t r u e to t h e i r t y p e w h i l e t h e s t r i p e d e x p l a n t s gave r i s e t o s e g r e g a t e d p r o g e n i e s of v a r y i n g p a t t e r n s ° I n o r d e r t o s t u d y t h e p a t t e r n of r e g e n e r a t i o n in different p h e n o t y p e s , g r e e n , s t r i a t e d and yellowish-white shoot explants were isolated and c u l t u r e d s e p a r a t e l y . I n t h e c a s e of g r e e n , r e g e n e r a t i o n was o b s e r v e d i n a l l t h e e x p l a n t s (100%) w h i l e i n s t r i a t e d i t was 83% and 58% i n yellowish-white explants. Nevertheless, the r a t e of p r o l i f e r a t i o n in different t y p e s was more or l e s s s i m i l a r . A l t h o u g h m o s t of t h e i n v i t r o d e r i v e d p l a n t s d i d n o t p o s s e s s t h e p r o m i n e n t s t e m p o r t i o n of the n a t u r a l p l a n t , o c c a s i o n a l l y g r e e n and
y e l l o w i s h - w h i t e p l a n t s showed t h e r o s e t t e l e a v e s well above the base of the stem. Fig. 4 shows one such plant (green) .in which differentiation of shoot buds was observed throughout the stem portion. dtlen t h e s h o o t s were 3 cm i n s i z e t h e y w e r e transferred individually to half-strength MS medium w i t h o u t h o r m o n e s f o r r o o t i n g . Rooting was i n d u c e d r e a d i l y i n g r e e n and s t r i a t e d p l a n t s ( F i g . 5 ) w h e r e a s i n y e l l o w i s h - w h i t e p l a n t s no r o o t i n g was o b s e r v e d . In s t r i a t e d t y p e s t h e r e was a d e f i n i t e c o r r e l a t i o n b e t w e e n t h e number o f r o o t s and ~he i n t e n s i t y o f g r e e n c o l o u r i n t h e plant. S t r i a t e d t y p e s i n w h i c h g r e e n c o l o u r was pro~ainent p r o d u c e d 1 0 - 1 5 r o o t s s i m i l a r t o t h e totally green types. ?ihereas in those i n Mlich g r e e n n e s s was r e s t r i c t e d to very narrow streaks i n t h e l e a v e s a maximum of 2 - 3 r o o t s w e r e observed. The s ~ r i p e d ones when t r a n s p l a n t e d t o s o i l d i d n e t s u r v i v e f o r more t h a n two w e e k s
110 ~vhereas upto 90~ s u r v i v a l was o b t a i u e d i n c a s e of ~reen ~ - r a n s p l a n t s . Three months a f t e r e s t a b l i s h m e n t i n s o i l , 20~ o~ ~reen t r a n s p l a n t s ~ ~ turned r e d d i s h p u r p l e , .~0~.~ had ~reen and p u r p l e s t r i p e s and 50~ r e t a i n e d t h e green c o l o u r .
system as a result o£ interaction with environment can lead to varied phenotype different from the original plant.
Aclmow~ed~e~ents Discussion The f o r e g o i n g o b s e r v a t i o n s have r e v e a l e d t h a t
the v a r i e g a t e d pheno~ype o~ C r y p t a n t h u s b r o m e l i o i d e s undergoes s e g r e g a t i o n under e x p e rimental conditions. This would p r o b a b l y be due to ~be c h i m e r a l n a t u r e of t h e p l a n t . Similar c h i m e r a l s e g r e g a t i o n has been r e p o r t e d in t i s s u e c u l t u r e s of o t h e r b r o m e l i a d s such a s Aechmea hybrid ( Z i t h e r and P i e p e r , 1976), Aec,h~e~ Baker ' A l b o - m a r g i n a t a ' , Crypt~nthus 'It' nd ~ u r a s h i g e , 1974~, Dracaena deremen~is , 1975), Chrysanthemum m o r i f o , l i u ~ .
~
"Indianapolis" (B'Ush e¢ al.~ 1976) and Pelargo~i ~ spp. ( S k i ~ a ~ J~ick~ 1976). In nature the plants are propagated through stolons ~ d no variation has been observed with the offspring. The segregation pattern observed in in vitro studies s~gest that the plant could be a pe~iclinal chimera. Boxus and D~uar¢ (19S0) have reported that in tissue cultures even meristem explant would dissociate periclinal chimras. In the present study the different phenotypes could have resulted as tollows~ the green plants could have o~iginated from ~he multiplication of the green part of the explan¢; the yellowish-white plant from the meris¢emoids in the non-chlorophyllous cell layers and the striated ones due to the encroachment of d i f f e r ~ cell layers du~ing the adventitious bud ~ormation.
We thank Mr. P. ~lukundan, Madras, for providin~ the plants of Cryvtanthus bromelioides for experimental work.
~eterenees Boxus Ph, D r n a r ~ P h (1980) I n : S a l a F, P a r i s i B, C e l i a R, C e f f e r i 0 ( e d s ) Proc I n t Workshop P1 C e l l C u l t u r e s , pp 265-269 Bush SH, E a r l e ~I~, Lang~ans BW (1976~ 63~ 729-737 ~ a v i d s o n SE, 9annan JA (1977) Herr 8oc 9 0 : 3 0 3 - 3 0 4 Deborah 0 (1975)
Am J Bet
Proc F l a S t a t e
Act. H e r r 5 4 : 8 3 - 9 2
[~oldgate t}P (1977) I n : B e i n e r t J, Bajaj YPS ( e d s ) P l a n t C e l l , T i s s u e and Organ C u l t u r e . S p r i ~ e r - V e r l a g , H e i d e l b e r g , Chap I pp 18-42 Jones JB, ~ u r a s h i g e T (1974) Soc 2 4 : 1 1 7 - 1 2 6
Proc I n t P1 Prep
Mathews Vii, B a ~ a n TS (1979)
Sci Herr 11:319-328
Mathews V~, Itangan TS, Narayanaswamy S (1976) Z Pflanzenphysiol 79:450-454
In Cr~p~anthus ~iv~t.t.atu~., Davidson and Donnan
(1977) r e p o r t e d t h a t a l t h o u g h t i s s u e
culture
derived plants were green to begin ~ i t h , t~ey resumed normal pink brown colour soon after transfer to soil. In the present investigation, a few striated plants obtained in vitro did not survive in soil for more than two weeks while the ~reen plants underwent some colour changes. It is difficult to predict w h e ~ e r these transplants will ultimately develop into different phenotypes. Ho~ver, a segregated new genetic
M u r a s h i g e T (1974) 25:135-166
h~un Bey P l a n t P h y ~ i o l
Murashige T, Skoe~ F (1962) 15:473-497
Physiol Plant
S k i r v i n P~, J a n i c k J (1976) 101:281-290
J A m e r Soc Herr S c i
Zimmer K, P i e p e r W (I9~6)
Acta Herr 6 4 : 2 5 - 2 9
Plant Cell Reports (1982) 1: 108-110
© Springer-Verlag 1982
In vitro P l a n t R e g e n e r a t i o n in L a t e r a l B u d E x p l a n t s o f bromelioides V a r . T r i c o l o r M . B . F o s t e r
Cryptanthus
V. Helena Mathews and P. S. Rao Plant Morphogenesis and Tissue Culture Section, Bio-Organic Division, Bhabha Atomic Research Centre, Bombay 400 085, India Received November 5, 1981 ; January 6, 1982
Abstract
M a t e r i a l s and Methods
L a t e r a l buds of Gryptanthus b r o m e l i o i d e s v a r . t r i c o l o r v a r i e g a t e d ornamental b r o m e l i a d , were c u l t u r e d on Murashige and Skoog's b a s a l medium ( ~ ) supplemented w i t h n a p t h a l e n e a c e t i c a c i d , i n d o l e b u t y r i c a c i d , and k i n e t i n , each at 2 mg/1. The b a s a l p a r t of the c u l t u r e d buds p r o l i f e r a t e d and produced a d v e n t i t i o u s s h o o t s . The s h o o t s developed i n t o p l a n t s of t h r e e d i f f e r e n t phenot y p e s : (a) entirely g r e e n , ( b ) . s t r i a t e d (green
L a t e r a l buds (2 mm l o n g ) were e x c i s e d from mature stems of C r y p t a n t h u s b r o m e l i o i d e s v a t . t r i c o l o r and were s u r f a c e - s t e r i l i z e d i n 0 . 1 p e r e e n t m e r c u r i c c h l o r i d e s o l u t i o n f o r 3 rain. and s u b s e q u e n t l y washed r e p e a t e d l y i n s t e r i l e distilled water. The buds were p l a c e d on MS medium ( ~ u r a s h i g e and Skoog, 1962). The e f f e c t of v a r i o u s auxins (NAA, IAA, IBA, 2 , 4 - D ) and
and yellowish-white) and (c) yellowish-white.
c o m b i n a t i o n s a t v a r i o u s c o n c e n t r a t i o n s , was studied. The pH of the medium was a d j u s t e d t o 5.8 b e f o r e s o l i d i f y i n g w i t h 0.8~ agar ( B a c t e r i o l o g i c a l g r a d e , 0entron Research L a b s . , Bombay). The c u l t u r e s were i n c u b a t e d under c o n t i n u o u s
~ o o t i n g could be induced in (a) and (b) types but s u c c e s s f u l t r a n s p l a n t a t i o n was p o s s i b l e only w i t h g r e e n p l a n t s some of which underwent c o l o r changes a f t e r e s t a b l i s h m e n t in s o i l .
cytokinins (Kn, BA, 2,i-P, Z) alone and in
light (i000 Lux) at 25 + 2°C with a relative A b b r e v i a t i o n s : BA, b e n z y l a d e n i n e ; 2,4-D, 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 ; IAA, i n d o l e a c e t i c a c i d ; IBA, i n d o l e b u t y r i c a c i d ; 2 , i - P , 6 ~ d i m e t h y l a l l y l a m i n o p u r i n e ; Kn, k i n e t i n , NAA, n a p h t h a l e n e a c e t i c a c i d , Z, z e a t i n .
hmnidity of 55-60~* For i n d u c t i o n of r o o t 2stem, t h e r e g e n e r a t e d s h e e t s were c u l t u r e d on s t r e n g t h MS medium w i t h o u t any hormones. Rooted p l a n t s were t r a n s f e r r e d t o s o i l . Besalts
Introduction lateral A r e v i e w of r e c e n t r e s e a r c h u t i l i z i n g the t e c h n i q u e of in v i t r o c u l t u r e of p l a n t organs i n d i c a t e s i t s p o t e n t i a l use in t h e r a p i d c l o n a l m u l t i p l i c a t i o n of d i v e r s e c r o p s , e x p e c i a l l y t h e ornamentals ( H o l d g a t e , 1977). The f a m i l y B r o m e l i a c e a e i s composed of mainly short-stemmed herbaceous e p i p h y t e s , and i n c l u d e s t h e p i n e a p p l e and some c o m m e r c i a l l y i m p o r t a n t o r n a m e n t a l s . The p l a n t s are e i t h e r p r o p a g a t e d by seeds or v e g e t a t i v e l y from ' p u p s ' or d i v i s i o n s , the l a t t e r i s a slow p r o c e s s . The a p p l i c a t i o n of t i s s u e c u l t u r e t e c h n i q u e s to members of t h e Bromeli~ceae may i n c r e a s e s u b s t a n t i a l l y the r a t e s of m u l t i p l i c a t i o n (Murashige, 1974~ Mathews and Banyan, 1979, Mathews e t al., 1974). 0 r y p t a n t h u s b r o m e l i o i d e s v a r . t r i c o l o r i s an ornamental b r o m e l i a d grown f o r i t s v a r i e g a t e d l e a v e s which a r e green i n the m i d d l e , and y e l l o w i s h - w h i t e in the margins. The t e n d e r l e a v e s p o s s e s s a carmine red c o l o u r . The p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n with t h e o b j e c t i v e of e s t a b l i s h i n g a t i s s u e c u l t u r e p r o c e d u r e f o r the m u l t i p l i c a t i o n of t h i s v a r i e g a t e d p l a n t and to i n v e s t i g a t e the p o s s i b i l i t y of o b t a i n i n g new
variants.
0721-7714/82/0001/0108/$ 01.00
(2
buds c u l t u r e d on ~
+ NAA (2 m~/1) ÷ I~k
÷ Kinetin (2 m /1) showed the unfolding
of t h e s c a l e l e a v e s w i t h i n a p e r i o d of 8 weeks. Upon t r a n s f e r of the u n f o l d e d buds t o f r e s h medium of the same c o m p o s i t i o n , t h e b a s a l p a r t of the d e v e l o p i n g shoot underwent p r o l i f e r a t i o n s i m u l t a n e o u s l y producing a l a r g e number of a d v e n t i t i o u s shoots (Fig. 1). I f the a d v e n t i t i o u s s h o o t s were i s o l a t e d and s u b c u l t u r e d on the same medium, numerous new shoots developed again at the base of the e x p l a n t , thus enhancing the r a t e of m u l t i p l i c a t i o n . The a d v e n t i t i o u s s h o o t s were of t h r e e t y p e s : Ca) s t r i a t e d , comparable t o t h e
natural type, (b) entirely green, and (c) yellowish w h i t e ( F i g . 2 ) . In the s t r i a t e d t y p e s , the
degree of g r e e n and y e l l o w i s h - w h i t e s t r i a t i o n s v a r i e d and i n e n t i r e l y g r e e n p l a n t s , the d e g r e e of greenness v a r i e d . The c u l t u r e of d i f f e r e n t phenotypes a g a i n r e s u l t e d in a d v e n t i t i o u s bud p r o d u c t i o n ( F i g . 3). As the number of subc u l t u r e s i n c r e a s e d t h e r e was a tendency f o r green p l a n t s %o dominate. In the i n i t i a l c u l t u r e the bud i n i t i a l s produced at the base of the e x p l a n t were too small to be scored f o r d i f f e r e n t p h e n o t y p e s . Therefore, the p r o l i f e r a t e d mass from each c u l t u r e was i s o l a t e d
109
F i g s . ( 1 - 5 ) 1. A d v e n t i t i o u s bud f o r m a t i o n i n l a t e r a l bud c u l L u r e of C r y p t a n t h u s b r o m e l i o i d e s on ~iS + NAA + IBA + Kn ( e a c h a t 2 m g / 1 ) . 2. E e g e n e r a t e d s h o o t s of d i f f e r e n t p h e n o t y p e s : g r e e n , s t r i a t e d and y e l l o w i s h white respectively. 3. Differentiation of s h o o t buds f r o m t h e b a s e of g r e e n , y e l l o w i s h - w h i t e and s t r i a t e d e x p l a n t s on c u l t u r e . 4. Proliferation and differentiation i n t h e s t e m p o r t i o n of a g r e e n e x p l a n t on c u l t u r e . 5. R o o t i n g i n p l a n t s of d i f f e r e n t p h e n o t y p e s . Green and s t r i a t e d p r o d u c e d good r o o t s y s t e m w h i l e y e l l o w i s h - w h i t e p l a n t was c o m p l e t e l y d e v o i d of r o o t s .
and t h e o r i g i n a l e x p l a n t a s w e l l as p r o l i f e r a t e d t i s s u e w i t h s h o o t buds a t d i f f e r e n t s t a g e s of growth were c u l t u r e d s e p a r a t e l y . I t was f o u n d t h a t by r e p e a t e d s u b c u l t u r e s g r e e n and y e l l o w i s h w h i t e e x p l a n t s could r e p r o d u c e t r u e to t h e i r t y p e w h i l e t h e s t r i p e d e x p l a n t s gave r i s e t o s e g r e g a t e d p r o g e n i e s of v a r y i n g p a t t e r n s ° I n o r d e r t o s t u d y t h e p a t t e r n of r e g e n e r a t i o n in different p h e n o t y p e s , g r e e n , s t r i a t e d and yellowish-white shoot explants were isolated and c u l t u r e d s e p a r a t e l y . I n t h e c a s e of g r e e n , r e g e n e r a t i o n was o b s e r v e d i n a l l t h e e x p l a n t s (100%) w h i l e i n s t r i a t e d i t was 83% and 58% i n yellowish-white explants. Nevertheless, the r a t e of p r o l i f e r a t i o n in different t y p e s was more or l e s s s i m i l a r . A l t h o u g h m o s t of t h e i n v i t r o d e r i v e d p l a n t s d i d n o t p o s s e s s t h e p r o m i n e n t s t e m p o r t i o n of the n a t u r a l p l a n t , o c c a s i o n a l l y g r e e n and
y e l l o w i s h - w h i t e p l a n t s showed t h e r o s e t t e l e a v e s well above the base of the stem. Fig. 4 shows one such plant (green) .in which differentiation of shoot buds was observed throughout the stem portion. dtlen t h e s h o o t s were 3 cm i n s i z e t h e y w e r e transferred individually to half-strength MS medium w i t h o u t h o r m o n e s f o r r o o t i n g . Rooting was i n d u c e d r e a d i l y i n g r e e n and s t r i a t e d p l a n t s ( F i g . 5 ) w h e r e a s i n y e l l o w i s h - w h i t e p l a n t s no r o o t i n g was o b s e r v e d . In s t r i a t e d t y p e s t h e r e was a d e f i n i t e c o r r e l a t i o n b e t w e e n t h e number o f r o o t s and ~he i n t e n s i t y o f g r e e n c o l o u r i n t h e plant. S t r i a t e d t y p e s i n w h i c h g r e e n c o l o u r was pro~ainent p r o d u c e d 1 0 - 1 5 r o o t s s i m i l a r t o t h e totally green types. ?ihereas in those i n Mlich g r e e n n e s s was r e s t r i c t e d to very narrow streaks i n t h e l e a v e s a maximum of 2 - 3 r o o t s w e r e observed. The s ~ r i p e d ones when t r a n s p l a n t e d t o s o i l d i d n e t s u r v i v e f o r more t h a n two w e e k s
110 ~vhereas upto 90~ s u r v i v a l was o b t a i u e d i n c a s e of ~reen ~ - r a n s p l a n t s . Three months a f t e r e s t a b l i s h m e n t i n s o i l , 20~ o~ ~reen t r a n s p l a n t s ~ ~ turned r e d d i s h p u r p l e , .~0~.~ had ~reen and p u r p l e s t r i p e s and 50~ r e t a i n e d t h e green c o l o u r .
system as a result o£ interaction with environment can lead to varied phenotype different from the original plant.
Aclmow~ed~e~ents Discussion The f o r e g o i n g o b s e r v a t i o n s have r e v e a l e d t h a t
the v a r i e g a t e d pheno~ype o~ C r y p t a n t h u s b r o m e l i o i d e s undergoes s e g r e g a t i o n under e x p e rimental conditions. This would p r o b a b l y be due to ~be c h i m e r a l n a t u r e of t h e p l a n t . Similar c h i m e r a l s e g r e g a t i o n has been r e p o r t e d in t i s s u e c u l t u r e s of o t h e r b r o m e l i a d s such a s Aechmea hybrid ( Z i t h e r and P i e p e r , 1976), Aec,h~e~ Baker ' A l b o - m a r g i n a t a ' , Crypt~nthus 'It' nd ~ u r a s h i g e , 1974~, Dracaena deremen~is , 1975), Chrysanthemum m o r i f o , l i u ~ .
~
"Indianapolis" (B'Ush e¢ al.~ 1976) and Pelargo~i ~ spp. ( S k i ~ a ~ J~ick~ 1976). In nature the plants are propagated through stolons ~ d no variation has been observed with the offspring. The segregation pattern observed in in vitro studies s~gest that the plant could be a pe~iclinal chimera. Boxus and D~uar¢ (19S0) have reported that in tissue cultures even meristem explant would dissociate periclinal chimras. In the present study the different phenotypes could have resulted as tollows~ the green plants could have o~iginated from ~he multiplication of the green part of the explan¢; the yellowish-white plant from the meris¢emoids in the non-chlorophyllous cell layers and the striated ones due to the encroachment of d i f f e r ~ cell layers du~ing the adventitious bud ~ormation.
We thank Mr. P. ~lukundan, Madras, for providin~ the plants of Cryvtanthus bromelioides for experimental work.
~eterenees Boxus Ph, D r n a r ~ P h (1980) I n : S a l a F, P a r i s i B, C e l i a R, C e f f e r i 0 ( e d s ) Proc I n t Workshop P1 C e l l C u l t u r e s , pp 265-269 Bush SH, E a r l e ~I~, Lang~ans BW (1976~ 63~ 729-737 ~ a v i d s o n SE, 9annan JA (1977) Herr 8oc 9 0 : 3 0 3 - 3 0 4 Deborah 0 (1975)
Am J Bet
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[~oldgate t}P (1977) I n : B e i n e r t J, Bajaj YPS ( e d s ) P l a n t C e l l , T i s s u e and Organ C u l t u r e . S p r i ~ e r - V e r l a g , H e i d e l b e r g , Chap I pp 18-42 Jones JB, ~ u r a s h i g e T (1974) Soc 2 4 : 1 1 7 - 1 2 6
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Mathews Vii, B a ~ a n TS (1979)
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culture
derived plants were green to begin ~ i t h , t~ey resumed normal pink brown colour soon after transfer to soil. In the present investigation, a few striated plants obtained in vitro did not survive in soil for more than two weeks while the ~reen plants underwent some colour changes. It is difficult to predict w h e ~ e r these transplants will ultimately develop into different phenotypes. Ho~ver, a segregated new genetic
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h~un Bey P l a n t P h y ~ i o l
Murashige T, Skoe~ F (1962) 15:473-497
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