Plant Cell Reports (1985) 4:78-80
Plant Cell Reports © Springer-Vedag 1985
Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.) Minal Mhatre, V. A. Bapat, and P. S. Rao Plant Biotechnology Section, Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India Received December 26, 1984 / Revised version received February 21, 1985 - Communicated by I. K. Vasil
ABSTRACT S t e m segments, axillary buds and leaves excised f r o m established shoot cultures of M o r u s indica w e r e soaked in M S liquid m e d i u m containing benzyladenine (0.5, I, 2 rag/l) and w e r e cultured subsequently on semi solid m e d i u m of the s a m e composition. N u m e r o u s shoot buds differentiated f r o m leaf and axillary buds but stem segments w e r e unresponsive. T h e shoot buds on isolation and culture developed into plantlets. Callus tissues which developed at the base of the leaf explant upon subculture also differentiated n u m e r o u s shoot buds. Abbreviations : BA, benzyl adenine; C M , coconut milk, 2, 4-D, 2, 4 dichlorophenoxy acetic acid, Kn, kinetin, MS, M u r a s h i g e and Skoog, Z, zeatin. INTRODUCTION
T h e m a s s p r o p a g a t i o n o f m o s t f o r e s t and cultivated trees including plantation crops, u s i n g t i s s u e c u l t u r e t e c h n i q u e has b e e n e x t e n s i v e l y a t t e m p t e d in r e c e n t y e a r s ( B o n g a 1977, 1981). I n a n e a r l i e r r e p o r t ( P a t e l et al. 1983) we h a v e d e s c r i b e d the r e g e n e r a t i o n of s i n g l e s h o o t s i n a x i l l a r y bud c u l t u r e s o f M o r u s i n d i c a . This communication reports observations of e x t e n d e d i n v e s t i g a t i o n s on m o r p h o g e n e t i c r e s p o n s e s o f l e a f , a x i l l a r y bud and s t e m segments. MATERIALS
AND
NiETHODS
Aseptically g r o w n shoot cultures of m u l b e r r y w e r e used as experimental source. T h e y w e r e established by culturing resting
Offprint requests to: M. Mhatre
vegetative axillary buds obtained f r o m I0 year old, sexually mature trees of M o r u s indica. T h e axillary buds w e r e surface-sterilized with 0.1% H g C I Z for 8 rains, w a s h e d six times with sterile distilled water and w e r e cultured on h o r m o n e - f r e e Murashige and Skoog's basal m e d i u m (1962) in which they produced extensive shoots and roots. F r o m such established shoot cultures, various explants such as stem segments, leaves and axillary buds w e r e isolated and cultured on the basal m e d i u m consisting of m a c r o - and micro-nutrients of Murashige and Skoog (196Z), vitamin composition as in G a m b o r g ' s B5 m e d i u m ( G a m b o r g et al. 1968) and 3 % sucrose. T h e basal m e d i u m w a s fortified with various growth substances (such as 2. 4-D, BA, Kn, and Z (0.5 to 2 rag/l), and CM). P o w d e r e d activated charcoal (0.1%) w a s also used in s o m e experiments. T h e p H of the m e d i u m w a s adjusted to 5.8 before jellying the m e d i u m with 0.8% agar (Bacteriological grade, S I S C O R e s e a r c h Laboratories, B o m b a y ) . All cultures w e r e maintained in a culture r o o m at Z 5 + Z ° C at a relative humidity of 5 0 - 6 0 % and w e r e exposed to continuous illumination of 950 lux. E a c h treatm e n t consisted of Z4 replicates and each experiment w a s repeated twice. In pretreatment studies nodal and internodal segments, sessile leaves and leaves with petiole w e r e excised f r o m shoot cultures and soaked individually in M S liquid m e d i u m with either B A K n or Z at three different concentrations (0.5, 1 and 2 rag/l), for 48 hours. Following pretreatment the explants w e r e cultured on solid m e d i u m of the s a m e composition. Pretreated, I c m long, healthy green leaves (sessile and with petiole), w e r e cultured in an upright position with only half its lower portion dipped in the m e d i u m . Nodal portions w e r e also cultured in the upright position w h e r e a s internodal segments w e r e placed horizontally on the surface of the m e d i u m .
79 RESULTS Culture
DISCUSSION
of Axillary Buds
About 6 weeks after culture, shoot buds w e r e d i f f e r e n t i a t e d f r o m t h e a x i l l a r y b u d s on c y t o k i n i n - s u p p l e m e n t e d MS m e d i u m . A m o n g t h e t h r e e c y t o k i n i n s t e s t e d , BA p r o v e d to be s u p e r i o r t o Kn a n d Z ( T a b l e 1). On M S + B A (0.5, 1 a n d Z rag/l) m e d i u m , the p r e s o a k e d a x i l l a r y buds p r o d u c e d m u l t i p l e s h o o t s (6 t o 8 p e r b u d ) ( F i g . 3, 4) w h e r e a s t h e n o n - s o a k e d o n e s d i d not. In c o n t r a s t to t h i s , o n M S + K n o r M S + Z (0.5, 1 o r 2 r a g / l ) , o n l y a s i n g l e p l a n t l e t d e v e l o p e d p e r bud, irrespective of the p r e t r e a t m e n t . The multiple s h o o t s p r o d u c e d on BA c o n t a i n i n g m e d i u m , w e r e e x c i s e d , i s o l a t e d a n d c u l t u r e d on h o r m o n e - f r e e MS m e d i u m i n w h i c h t h e y d e v e l o p e d r o o t s a n d g r e w to a h e i g h t o f 6 to 8 i n c h e s w i t h i n 3 to 4 w e e k s . On f u r t h e r t r a n s f e r to p a p e r c u p s , t h e rooted shoots developed rapidly into robust plants ( F i g . 5). T h e m o r t a l i t y r a t e o f t i s s u e c u l t u r e d e r i v e d p l a n t s w a s l o w a n d a b o u t 90% p l a n t s c o u l d s u r v i v e on t r a n s f e r to t h e s o i l . A x i l l a r y buds cultured in liquid medium turned brown.
Clonal selection and propagation of tree s p e c i e s by t i s s u e a n d o r g a n c u l t u r e t e c h n i q u e s h a s c o n s i d e r a b l e p o t e n t i a l in b r e e d i n g a n d improvement. T h e p r e s e n t f i n d i n g s on M o r u s i n d i c a d e m o n s t r a t e the p o s s i b i l i t y for m a s s propagation of mulberry through culture of leaves and axillary buds. This would also be of great advantage for multiplying certain cultivars of M o r u s which are difficult to root. T h e high percentage (90%) of survival of tissue culture derived plants upon transfer to soil points to the feasibility of the technique. The induction of adventitious buds from l e a v e s h a s b e e n r e p o r t e d f r o m a few a n g i o s p e r m i c woody g e n e r a viz, b a l s a m and p o p l a r ( B y c h e n k o v a a n d David, 1978, F a v r e 1977). In t h e p r e s e n t s t u d y on M. i n d i c a m u l t i p l e s h o o t b u d s o r i g i n a t e d d e n o v o f r o m t h e b a s e o f the l e a f p e t i o l e a s w e l l as in sessile leaves. However, the frequency of bud i n d u c t i o n w a s h i g h e r i n l e a v e s w i t h p e t i o l e i n c o n t r a s t to r e s u l t s in M. a l b a (Oka a n d O h y a m a , 1981) w h e r e s e s s i l e l e a v e s y i e l d e d m o r e number of buds.
Culture of L e a v e s T h e upright position of the leaf explant proved favourable for the m a x i m u m induction of shoot buds. After 5 to 7 w e e k s the basal portion of the petiole enlarged and f o r m e d primordia which developed into green healthy shoot buds (Fig. i). Sessile leaves "also showed differentiation of n u m e r o u s shoot buds under similar conditions (Fig. Z). Generally 3 to 4 shoots developed per explant. This response w a s observed only with leaves soaked in B A and not with those soaked either in K n or in Z. N o n soaked leaves did not s h o w multiple bud induction and necrosed after 6-8 w e e k s of culture. Alongwith the differentiation of shoot buds extensive callus development occurred at the base of the leaf (Fig. I). Callus w a s green, nodular and compact. This callus differentiated n u m e r o u s shoot buds which w h e n subcultured on h o r m o n e - f r e e m e d i u m developed into plantlets. O f the three concentrations of B A tested, m a x i m u m response with respect to shoot development occurred on M S + B A (2 rag/l) (Table I). Occasionally a few leaf explants f o r m e d adventitious shoots if liquid m e d i u m w a s used. M o s t of the explants in liquid m e d i u m turned brown. Culture
In the present investigation soaking of explants prior to culture w a s beneficial for shoot bud induction, l~ossibly the pre-treatment confers a physiological status on the explant that is favourable for shoot bud initiation.
Table
I.
R e s p o n s e s of axillary buds and leaves of M o r u s indica to various cytokinins*
A d d i t i o n to MS m e d i u m
No. o f explant s cultured
No. o f e x p l a n t s s h o w i n g m u l t i p l e bud i n d u c t i o n Leaves
BA BA BA Kn Kn Kn Z
(0.5) (1) (Z) (0. 5) (1) (2) (0.5)
(1) Z (Z) Z
Axillar y buds
48 48 48 48 48 48
14 18 20 ---~
48
--
**
48 48
--
** **
--
34 36 40 ** ** **
of S t e r n S e g m e n t s
As compared with axillary buds and leaves, stem segments soaked in cytokinins as well as n o n s o a k e d o n e s p r o v e d r e c a l c i t r a n t to a n y h o r m o n a l t r e a t m e n t f o r i n d u c i n g s h o o t bud differentiation. Callus was formed from excised ends of explants o n M S + C M 1 0 % (v/v) + 2, 4 - D (2 rag/l), and did not show any regeneration.
*
The explants were pretreated with corresp o n d i n g c y t o k i n i n f o r 48 h o u r s . C o n c e n t r a t i o n s : mg/I ; ** On K n a n d Z, o n l y s i n g l e bud regenerated.
80 LEGEND
I n v e s t i g a t i o n s on t i s s u e c u l t u r e s of Morus indica Fig. 1. D i f f e r e n t i a t i o n of shoot buds f r o m the b a s e of p e t i o l e ; a r r o w i n d i c a t e s the o r i g i n a l l e a f explant. Fig. 2.- I n i t i a t i o n o f shoot buds f r o m the b a s e of a s e s s i l e leaf. Fig. 3 . A x i l l a r y bud a f t e r two w e e k s of culture. Fig. 4 . - A x i l l a r y bud c o v e r e d by n u m e r o u s d i f f e r e n t i a t e d p l a n t l e t s a f t e r 8 w e e k s of c u l t u r e . Fig. 5 . - E s t a b l i s h m e n t of plantlet.
REFERENCES Bonga J M (1977) I n : A p p l i e d and F u n d a m e n t a l A s p e c t s of F l a n t Cell, T i s s u e and O r g a n C u l t u r e , 5 R e i n e r t and Y 1r S B a j a j (eds) S p r i n g e r V e r l a g , New York, pp 93-108 Bonga J M (1981) I n : T i s s u e C u l t u r e o f E c o n o m i c a l l y I m p o r t a n t F l a n t s , A N Rao (ed), P r o c . of the COSTED Syrup, Singapore, 1981, pp 191-196 B y c h e n k o v a E A, D a v i d A (1978) Soy. P l a n t P h y s i o l . Z5 : 216-2Z2
F a v r e J M (1977) Ann. A m e l i o r Plant. 169 G a m b o r g O L, M i l l e r R A, C e l l R e s . 50 : 151-158 M u r a s h i g e T, 15 : 473-497 Oka S,
27 : 151-
O j i m a K (1968) Exp.
Skoog F (1962) P h y s i o l . P l a n t
O h y a m a K (1981) Can. J. Bot. 5 9 : 6 8 - 7 4
Patel G K, Bapat V A, R a o P S (1983) Z. Pflanzenphysiol. III : 465-469
Plant Cell Reports © Springer-Vedag 1985
Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.) Minal Mhatre, V. A. Bapat, and P. S. Rao Plant Biotechnology Section, Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India Received December 26, 1984 / Revised version received February 21, 1985 - Communicated by I. K. Vasil
ABSTRACT S t e m segments, axillary buds and leaves excised f r o m established shoot cultures of M o r u s indica w e r e soaked in M S liquid m e d i u m containing benzyladenine (0.5, I, 2 rag/l) and w e r e cultured subsequently on semi solid m e d i u m of the s a m e composition. N u m e r o u s shoot buds differentiated f r o m leaf and axillary buds but stem segments w e r e unresponsive. T h e shoot buds on isolation and culture developed into plantlets. Callus tissues which developed at the base of the leaf explant upon subculture also differentiated n u m e r o u s shoot buds. Abbreviations : BA, benzyl adenine; C M , coconut milk, 2, 4-D, 2, 4 dichlorophenoxy acetic acid, Kn, kinetin, MS, M u r a s h i g e and Skoog, Z, zeatin. INTRODUCTION
T h e m a s s p r o p a g a t i o n o f m o s t f o r e s t and cultivated trees including plantation crops, u s i n g t i s s u e c u l t u r e t e c h n i q u e has b e e n e x t e n s i v e l y a t t e m p t e d in r e c e n t y e a r s ( B o n g a 1977, 1981). I n a n e a r l i e r r e p o r t ( P a t e l et al. 1983) we h a v e d e s c r i b e d the r e g e n e r a t i o n of s i n g l e s h o o t s i n a x i l l a r y bud c u l t u r e s o f M o r u s i n d i c a . This communication reports observations of e x t e n d e d i n v e s t i g a t i o n s on m o r p h o g e n e t i c r e s p o n s e s o f l e a f , a x i l l a r y bud and s t e m segments. MATERIALS
AND
NiETHODS
Aseptically g r o w n shoot cultures of m u l b e r r y w e r e used as experimental source. T h e y w e r e established by culturing resting
Offprint requests to: M. Mhatre
vegetative axillary buds obtained f r o m I0 year old, sexually mature trees of M o r u s indica. T h e axillary buds w e r e surface-sterilized with 0.1% H g C I Z for 8 rains, w a s h e d six times with sterile distilled water and w e r e cultured on h o r m o n e - f r e e Murashige and Skoog's basal m e d i u m (1962) in which they produced extensive shoots and roots. F r o m such established shoot cultures, various explants such as stem segments, leaves and axillary buds w e r e isolated and cultured on the basal m e d i u m consisting of m a c r o - and micro-nutrients of Murashige and Skoog (196Z), vitamin composition as in G a m b o r g ' s B5 m e d i u m ( G a m b o r g et al. 1968) and 3 % sucrose. T h e basal m e d i u m w a s fortified with various growth substances (such as 2. 4-D, BA, Kn, and Z (0.5 to 2 rag/l), and CM). P o w d e r e d activated charcoal (0.1%) w a s also used in s o m e experiments. T h e p H of the m e d i u m w a s adjusted to 5.8 before jellying the m e d i u m with 0.8% agar (Bacteriological grade, S I S C O R e s e a r c h Laboratories, B o m b a y ) . All cultures w e r e maintained in a culture r o o m at Z 5 + Z ° C at a relative humidity of 5 0 - 6 0 % and w e r e exposed to continuous illumination of 950 lux. E a c h treatm e n t consisted of Z4 replicates and each experiment w a s repeated twice. In pretreatment studies nodal and internodal segments, sessile leaves and leaves with petiole w e r e excised f r o m shoot cultures and soaked individually in M S liquid m e d i u m with either B A K n or Z at three different concentrations (0.5, 1 and 2 rag/l), for 48 hours. Following pretreatment the explants w e r e cultured on solid m e d i u m of the s a m e composition. Pretreated, I c m long, healthy green leaves (sessile and with petiole), w e r e cultured in an upright position with only half its lower portion dipped in the m e d i u m . Nodal portions w e r e also cultured in the upright position w h e r e a s internodal segments w e r e placed horizontally on the surface of the m e d i u m .
79 RESULTS Culture
DISCUSSION
of Axillary Buds
About 6 weeks after culture, shoot buds w e r e d i f f e r e n t i a t e d f r o m t h e a x i l l a r y b u d s on c y t o k i n i n - s u p p l e m e n t e d MS m e d i u m . A m o n g t h e t h r e e c y t o k i n i n s t e s t e d , BA p r o v e d to be s u p e r i o r t o Kn a n d Z ( T a b l e 1). On M S + B A (0.5, 1 a n d Z rag/l) m e d i u m , the p r e s o a k e d a x i l l a r y buds p r o d u c e d m u l t i p l e s h o o t s (6 t o 8 p e r b u d ) ( F i g . 3, 4) w h e r e a s t h e n o n - s o a k e d o n e s d i d not. In c o n t r a s t to t h i s , o n M S + K n o r M S + Z (0.5, 1 o r 2 r a g / l ) , o n l y a s i n g l e p l a n t l e t d e v e l o p e d p e r bud, irrespective of the p r e t r e a t m e n t . The multiple s h o o t s p r o d u c e d on BA c o n t a i n i n g m e d i u m , w e r e e x c i s e d , i s o l a t e d a n d c u l t u r e d on h o r m o n e - f r e e MS m e d i u m i n w h i c h t h e y d e v e l o p e d r o o t s a n d g r e w to a h e i g h t o f 6 to 8 i n c h e s w i t h i n 3 to 4 w e e k s . On f u r t h e r t r a n s f e r to p a p e r c u p s , t h e rooted shoots developed rapidly into robust plants ( F i g . 5). T h e m o r t a l i t y r a t e o f t i s s u e c u l t u r e d e r i v e d p l a n t s w a s l o w a n d a b o u t 90% p l a n t s c o u l d s u r v i v e on t r a n s f e r to t h e s o i l . A x i l l a r y buds cultured in liquid medium turned brown.
Clonal selection and propagation of tree s p e c i e s by t i s s u e a n d o r g a n c u l t u r e t e c h n i q u e s h a s c o n s i d e r a b l e p o t e n t i a l in b r e e d i n g a n d improvement. T h e p r e s e n t f i n d i n g s on M o r u s i n d i c a d e m o n s t r a t e the p o s s i b i l i t y for m a s s propagation of mulberry through culture of leaves and axillary buds. This would also be of great advantage for multiplying certain cultivars of M o r u s which are difficult to root. T h e high percentage (90%) of survival of tissue culture derived plants upon transfer to soil points to the feasibility of the technique. The induction of adventitious buds from l e a v e s h a s b e e n r e p o r t e d f r o m a few a n g i o s p e r m i c woody g e n e r a viz, b a l s a m and p o p l a r ( B y c h e n k o v a a n d David, 1978, F a v r e 1977). In t h e p r e s e n t s t u d y on M. i n d i c a m u l t i p l e s h o o t b u d s o r i g i n a t e d d e n o v o f r o m t h e b a s e o f the l e a f p e t i o l e a s w e l l as in sessile leaves. However, the frequency of bud i n d u c t i o n w a s h i g h e r i n l e a v e s w i t h p e t i o l e i n c o n t r a s t to r e s u l t s in M. a l b a (Oka a n d O h y a m a , 1981) w h e r e s e s s i l e l e a v e s y i e l d e d m o r e number of buds.
Culture of L e a v e s T h e upright position of the leaf explant proved favourable for the m a x i m u m induction of shoot buds. After 5 to 7 w e e k s the basal portion of the petiole enlarged and f o r m e d primordia which developed into green healthy shoot buds (Fig. i). Sessile leaves "also showed differentiation of n u m e r o u s shoot buds under similar conditions (Fig. Z). Generally 3 to 4 shoots developed per explant. This response w a s observed only with leaves soaked in B A and not with those soaked either in K n or in Z. N o n soaked leaves did not s h o w multiple bud induction and necrosed after 6-8 w e e k s of culture. Alongwith the differentiation of shoot buds extensive callus development occurred at the base of the leaf (Fig. I). Callus w a s green, nodular and compact. This callus differentiated n u m e r o u s shoot buds which w h e n subcultured on h o r m o n e - f r e e m e d i u m developed into plantlets. O f the three concentrations of B A tested, m a x i m u m response with respect to shoot development occurred on M S + B A (2 rag/l) (Table I). Occasionally a few leaf explants f o r m e d adventitious shoots if liquid m e d i u m w a s used. M o s t of the explants in liquid m e d i u m turned brown. Culture
In the present investigation soaking of explants prior to culture w a s beneficial for shoot bud induction, l~ossibly the pre-treatment confers a physiological status on the explant that is favourable for shoot bud initiation.
Table
I.
R e s p o n s e s of axillary buds and leaves of M o r u s indica to various cytokinins*
A d d i t i o n to MS m e d i u m
No. o f explant s cultured
No. o f e x p l a n t s s h o w i n g m u l t i p l e bud i n d u c t i o n Leaves
BA BA BA Kn Kn Kn Z
(0.5) (1) (Z) (0. 5) (1) (2) (0.5)
(1) Z (Z) Z
Axillar y buds
48 48 48 48 48 48
14 18 20 ---~
48
--
**
48 48
--
** **
--
34 36 40 ** ** **
of S t e r n S e g m e n t s
As compared with axillary buds and leaves, stem segments soaked in cytokinins as well as n o n s o a k e d o n e s p r o v e d r e c a l c i t r a n t to a n y h o r m o n a l t r e a t m e n t f o r i n d u c i n g s h o o t bud differentiation. Callus was formed from excised ends of explants o n M S + C M 1 0 % (v/v) + 2, 4 - D (2 rag/l), and did not show any regeneration.
*
The explants were pretreated with corresp o n d i n g c y t o k i n i n f o r 48 h o u r s . C o n c e n t r a t i o n s : mg/I ; ** On K n a n d Z, o n l y s i n g l e bud regenerated.
80 LEGEND
I n v e s t i g a t i o n s on t i s s u e c u l t u r e s of Morus indica Fig. 1. D i f f e r e n t i a t i o n of shoot buds f r o m the b a s e of p e t i o l e ; a r r o w i n d i c a t e s the o r i g i n a l l e a f explant. Fig. 2.- I n i t i a t i o n o f shoot buds f r o m the b a s e of a s e s s i l e leaf. Fig. 3 . A x i l l a r y bud a f t e r two w e e k s of culture. Fig. 4 . - A x i l l a r y bud c o v e r e d by n u m e r o u s d i f f e r e n t i a t e d p l a n t l e t s a f t e r 8 w e e k s of c u l t u r e . Fig. 5 . - E s t a b l i s h m e n t of plantlet.
REFERENCES Bonga J M (1977) I n : A p p l i e d and F u n d a m e n t a l A s p e c t s of F l a n t Cell, T i s s u e and O r g a n C u l t u r e , 5 R e i n e r t and Y 1r S B a j a j (eds) S p r i n g e r V e r l a g , New York, pp 93-108 Bonga J M (1981) I n : T i s s u e C u l t u r e o f E c o n o m i c a l l y I m p o r t a n t F l a n t s , A N Rao (ed), P r o c . of the COSTED Syrup, Singapore, 1981, pp 191-196 B y c h e n k o v a E A, D a v i d A (1978) Soy. P l a n t P h y s i o l . Z5 : 216-2Z2
F a v r e J M (1977) Ann. A m e l i o r Plant. 169 G a m b o r g O L, M i l l e r R A, C e l l R e s . 50 : 151-158 M u r a s h i g e T, 15 : 473-497 Oka S,
27 : 151-
O j i m a K (1968) Exp.
Skoog F (1962) P h y s i o l . P l a n t
O h y a m a K (1981) Can. J. Bot. 5 9 : 6 8 - 7 4
Patel G K, Bapat V A, R a o P S (1983) Z. Pflanzenphysiol. III : 465-469
