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Energy and osmotic requirement for high frequency regeneration of rice plants from long-term cultures
Plant Science, 48 (1987) 189--194 Elsevier Scientific Publishers Ireland Ltd.
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ENERGY AND O S M O T I C R E Q U I R E M E N T
F O R HIGH FREQUENCY REGENERATION OF RICE PLANTS FROM LONG-TERM CULTURES
P.B. KAVI KISHOR Department of Botany, Kakatiya University, Warangal-506 009 (India) (Received July 28th, 1986) (Revision received October 14th, 1986) (Accepted October 18th, 1986) Rice callus showed optimum growth at a medium osmolarity of 300, but maximum frequency (92--96%) of plantlet production was noticed at 200 mOsmol. Minimum requirement of sucrose for root differentiation was found to be 0.5%, while it was 1% for shoot. Tissues grown on a low sucrose medium produced high percent frequency of shoots only if the medium was supplemented with osmotic agents like sorbitol or mannitol to give the molar sucrose equivalent to 2 or 3%. Callus growing on sucrose as sole carbon source lost its shoot forming ability by 100 days in culture, whereas tissues proliferating on sorbitol or mannitol (besides sucrose) differentiated shoots over a period of 1500 days. Partial replacement of sucrose requirement with sorbitol or mannitol for organogenesis suggested that tissue carbohydrate has an osmoregulatory function. Key words: Oryza sativa L. ; osmotic potentials; long-term cultures; sugar alcohols
Introduction Rice callus is a slow g r o w i n g tissue a n d t h e r e are h a r d l y a n y r e p o r t s o n the increase o f t h e g r o w t h o f t h e callus. O n e o f the m a i n c o n s t r a i n t s in utilizing cell a n d tissue c u l t u r e in cereal b r e e d i n g p r o g r a m m e is the low rate o f r e g e n e r a t i o n o f p l a n t s f r o m c u l t u r e d cells. S h o o t f o r m i n g ability declines w i t h t i m e a n d e x a m p l e s of such species are Brassica oleracea [1] M e d i c a g o sativa [2] rice [3] etc. E m b r y o genic callus o f rice p r o v i d e d l o n g - t e r m ( 3 5 0 d a y s o n l y ) t o t i p o t e n t cultures [4] b u t t h e r e is n o visual d i f f e r e n c e b e t w e e n e m b r y o g e n i c a n d n o n - e m b r y o g e n i c callus in rice. F u r t h e r , s o m e varieties o f rice do n o t p r o d u c e e m b r y o genic callus ( p e r s o n a l o b s e r v a t i o n ) . Sheridan [5] h a d r e c o m m e n d e d t h a t ' E f f o r t s h o u l d f o c u s on the d e v e l o p m e n t o f c u l t u r e m e d i a w h i c h allow the initiation and m a i n t e n a n c e
Abbreviations: IAA, indole-3-acetic acid ; KN, kinetin; LS, Linsmaier and Skoog.
o f p l a n t r e g e n e r a t i o n in l o n g - t e r m cultures'. T o r r e y [6] suggested t h a t the loss in o r g a n f o r m i n g c a p a c i t y in l o n g - t e r m c u l t u r e s of P i s u m s a t i v u m is c o r r e l a t e d w i t h the increase in a b n o r m a l i t y o f c h r o m o s o m a l c o n s t i t u t i o n and n u m b e r . H o w e v e r , we h a v e n o t o b s e r v e d a n y increase in c h r o m o s o m e n u m b e r in l o n g - t e r m c u l t u r e s ( 1 5 0 0 days} o f rice. Besides acting as a s o u r c e o f energy, m e d i u m supplied c a r b o h y d r a t e m a y also f u n c t i o n as an o s m o t i c a g e n t [ 7 , 8 ] . T h e r e are r e p o r t s in t h e r e f e r e n c e s w h e r e mild o s m o t i c stress a f f e c t e d t o b a c c o ]9] a n d s o y b e a n tissue c u l t u r e s [10] b y increa-~ing callus g r o w t h a n d m o d i f y i n g the cellular m o r p h o l o g y . Liu a n d Lai [11] r e p o r t e d high f r e q u e n c y p l a n t r e g e n e r a t i o n in w a t e r stressed c u l t u r e s of rice. T h e i m p o r t a n c e of o s m o t i c c o n d i t i o n s in t h e c u l t u r e of e m b r y o s of H o r d e u m [ 1 2 ] , in l o n g - t e r m r e g e n e r a t i o n o f alfalfa [13] and during r o o t and s h o o t d i f f e r e n t i a t i o n in t o b a c c o [ ! 4 ] have b e e n o u t l i n e d earlier. T h e a i m o f this p a p e r is to increase callus g r o w t h , t o p r o d u c e rice p l a n t s
0618-9452/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
190 in l o n g - t e r m c u l t u r e s and t o e n h a n c e the p e r c e n t f r e q u e n c y o f p l a n t l e t f o r m a t i o n in rice. T h e s e p r o b l e m s are solved w i t h t h e incorporation of appropriate concentrations o f sugar a n d sugar alcohols in t h e n u t r i e n t medium. Materials a n d m e t h o d s R i c e ( O r y z a sativa L. cv. Bala) callus w a s initiated f r o m m a t u r e e m b r y o s o n L i n s m a i e r a n d S k o o g ' s (LS) agar m e d i u m [15] supplem e n t e d w i t h 2 mg/1 2 , 4 - d i c h l o r o p h e n o x y acetic acid (2,4-D) a n d 2% sucrose a n d m a i n t a i n e d on the same. A f t e r o n e or t w o s u b c u l t u r e s , t h e callus was t r a n s f e r r e d o n t o LS basal m e d i u m c o n t a i n i n g 2 mg/1 2,4-D, 2% sucrose a n d 3% s o r b i t o l or 3% m a n n i t o l . F o r r e g e n e r a t i o n o f plants, LS salts supplem e n t e d w i t h 1 mg/1 indole-3-acetic acid ( I A A ) , 4 mg/1 k i n e t i n ( K N ) a n d 2% sucrose was used. All s u p p l e m e n t s w e r e a d d e d t o the media before autoclaving on a weight per v o l u m e basis. C u l t u r e s w e r e i n c u b a t e d at p h o t o p e r i o d s of 24 h in d i f f u s e light ( 4 0 0 lux) f o r callus initiation a n d in b r i g h t light ( 2 0 0 0 lux) f o r p l a n t r e g e n e r a t i o n at a t e m p e r a t u r e o f 25 + 2°C. Cool, w h i t e , f l u o r e s c e n t light was p r o v i d e d f o r all t h e cultures. In each
Table I.
Results and discussion D i f f e r e n t c o n c e n t r a t i o n s o f sucrose ( 0 . 2 5 - 15%) was a d d e d to the LS basal m e d i u m c o n t a i n i n g 2 mg/1 2,4-D a n d its e f f e c t o n g r o w t h as m e a s u r e d b y fresh a n d d r y weights a n d also t o t a l o s m o t i c s t r e n g t h of the m e d i a are r e p r e s e n t e d in T a b l e I. Increasing c o n c e n t r a t i o n s o f sucrose in the m e d i u m increased t h e g r o w t h o f callus till 6% level, b u t g r o w t h declined at higher levels (10 a n d 15%) o f sucrose. V a r i o u s c o n c e n t r a t i o n s of s o r b i t o l and m a n n i t o l w e r e a d d e d to LS basal m e d i u m c o n t a i n i n g 2 mg/1 2,4-D and 2% sucrose a n d their e f f e c t on g r o w t h is p r e s e n t e d in T a b l e II. Callus s h o w e d increasing g r o w t h o n s o r b i t o l or m a n n i t o l u p to an o p t i m u m o f 3% b u t
Effect of sucrose on the growth of embryo callus of rice
Conc. of sucrose (%)
Osmolarity of the medium mOsmol
LS basal (no sucrose)
0.25 0.5 1.0 2.0 3.0 4.0 6.0 i0.0 15.0
c u l t u r e t u b e 15 ml o f the m e d i u m was t a k e n and 150 + 14.5 mg o f fresh callus (22.0 -+ 4.3 m g of d r y wt.) was inoculated. A t o t a l of six replicates f o r g r o w t h m e a s u r e m e n t s a n d 4 5 - - 7 5 replicates f o r r e g e n e r a t i o n of p l a n t s w e r e h a r v e s t e d a f t e r 30 d a y s in culture. E x p e r i m e n t s w e r e r e p e a t e d at least t w o t i m e s w i t h c o n s i s t e n t results. T o t a l o s m o l a r i t y o f t h e m e d i u m was m e a s u r e d b e f o r e a u t o c l a v i n g using a w e s c o r 5 1 0 0 C V a p o r Pressure O s m o m e t e r .
98 106 112 123 149 176 200 249 350 476
Mean fresh wt. mg/culture
Mean dry wt. mg/culture
Bars -2.4 --2.6 --2.7 --3.0 --3.7 --4.3 --4.9 -6.1 -8.6 -11,7
aFigures in parenthesis represent S.E.
Tissues did not survive
210.7 (±14.8) a 270.9 (±16.4) 378.0(±22.0) 441.0(220.5) 599.6(-+26.1) 601.5(±32.6) 782.0 (± 35.9) 452.2(228.8) 401.0(±20.0)
27.3 (_+ 4.6) a 32.1(+ 5.0) 40.8(± 5.2) 55.0(-+ 6.3) 75.9(_+ 7.5) 78.4(± 8.4) 101.2 (± 10.1) 91.4(-+ 7.7) 86.6(± 7.5)
191 higher concentrations (6 and 9%) suppressed the growth on b o t h a fresh and a dr y weight basis. Thus sorbitol and mannitol r e p r o d u c e d the sucrose inhibition of growth (Table II) at higher levels. However, r e pl a c e m ent of 3% sorbitol or 3% mannitol by 8% sucrose (osmolarity of this m e d i u m is equivalent to that of 2% sucrose plus 3% sorbitol or mannitol) failed to evoke an identical response. P r o m o t i o n of callus growth by sugar alcohols has been d e m o n s t r a t e d by Nesius et al. [16] in Pauls Scarlet Rose and by Staudt [17] in Vitis. Studies with Prunus persica (see Ref. 18) suggested, t hat its growth on sorbitol may be adaptive, since the initial growth on the sugar alcohol was much lower than on sucrose and only with time did it come to equal t hat of the disaccharide. E m b r y o callus o f rice (75 days old) proliferating on 2% sucrose containing medium, regenerated plantlets with 15% f r e q u e n c y b u t lost its ability to organise shoots by day 100 in culture. On the ot her hand, callus tissues proliferating on sucrose plus sorbitol or sucrose plus mannitol differentiated whole plants in the same regenerating medium over Table II. Effect of sorbitol and mannitol on the growth of rice callus Conc. of sugar alcohol (%)
Mean fresh wt.
mg/culture
Mean dry wt. rag/culture
2% s u c r o s e alone
(control) Sorbitol 1.5 3.0 6.0 9.0
785.7 (-+30.1) 1214.0 (± 42.6) 500.6(±21.8) 359.0(±17.2)
Mannitol 1.5 3.0 6.0 9.0
615.5 (_'227.4) 926.0(238.3) 515.4(±19.7) 380.0(±18.5)
441.0 (± 20.5) a
aFigures in parenthesis represent S.E.
55.0 (+ 6.3) a 98.9(2 7.6) 155.0 (2 11.6) 78.7 (± 8.2) 67.2(-+ 6.8) 82.9 (± 125.8(2 76.0(2 63.1(±
7.1) 8.9) 7.0) 6.5)
a period of 1500 days with 50--60% freq u e n c y [ 1 9 ] . I n c o r p o r a t i o n of sorbitol or mannitol into the callus proliferating medium (in addition to sucrose) thus helped in retaining the shoot forming ability of tissues in long-term cultures and also in enhancing the p e r c e n t f r e q u e n c y of plantlet formation. The correlations between the percent f r e q u e n c y of r o o t and shoot f o r m a t i o n and the concentration of sucrose and osmotic agents in the medium are shown in Tables III and IV. Minimum requi rem ent of sucrose for r o o t differentiation was f o u n d to be 0.5% and for that of shoot it was 1%. This result could also be reproduced when 0.25% and 0.5% sucrose containing media were supplem e n t e d with sorbitol or mannitol to give the molar sucrose equivalent to that of 0.5 and 1% for r o o t and shoot form at i on respectively (Table III). Maximum r o o t and shoot differentiation was noticed when 2 or 3% sucrose (osmolarity of these media were 142 and 176 mOsmol) was supplied to the medium (Table IV). Tissues grown on a medium with sucrose levels lower or higher than 2% showed a reduced capacity to form roots and shoots. Total suppression of r o o t and shoot formation occurred when no sucrose was supplied or m edi um sucrose concentrations reached a b o u t 15% (Tables III and IV). When .~orbitol or mannitol were added to 0.5% sucrose containing media to give the molar sucrose equivalents to 1,2 and 3%, there was a decline in the r o o t and shoot forming capacities of the tissues. A similar trend was observed with f u r t h e r increase of sucrose c o n t e n t to 1% and sorbitol or mannitol to give the molar sucrose equivalents to 2,3 and 6% (Table III). A c o n c u r r e n t high f r e q u e n c y of r o o t and shoot form at i on (92--96%) was noticed at a medium osmolarity of 200 mOsmol when the medium was supplemented with 2% sucrose and sorbitol or mannitol to give the molar sucrose equivalent to 2.5% (Table IV). It appears from Tables III and IV that sorbitol or mannitol had a promotive effect on shoot f o r m a t i o n at the 2% and 3% sucrose equivalent levels. However, replacement of sorbitol
192 T a b l e III.
E n e r g y a n d o s m o t i c r e q u i r e m e n t s for r o o t a n d s h o o t f o r m a t i o n in rice callus
Conc. of sucrose (%)
M a n n i t o l or s o r b i t o l to give m o l a r sucrose equivalent t o (%)
O s m o l a r i t y of t h e m e d i u m
% f r e q u e n c y of d i f f e r e n t i a t i o n a
mOsmol
Bars
Shoot
Root
0.25 0.25 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0
Nil 0.5 Nil 1.0 2.0 3.0 Nil 2.0 3.0 6.0
106 116 113 159 255 349 123 228 304 592
--2.6 --2.8 --2.8 -3.9 --6.2 -8.6 --3.0 -5.6 --7.5 --14.5
Nil Nil Nil 50--55 (Tiny) 40--42 (Tiny) 20--24 (Tiny) 50--53 76--80 80--85 Nil
Nil 60--65 65--70 75--77 44--46 25--30 80--85 85--90 60--65 30--35
a% f r e q u e n c y of d i f f e r e n t i a t i o n -- [(No. of calli w i t h r o o t s or s h o o t s ) / ( N o , of calli i n o c u l a t e d ) ] x 100.
or mannitol with equimolar concentrations of glycerol, erythritol, myo-inositol, sucrose and sodium chloride failed to evoke an identical response under identical conditions. Increase of sorbitol or mannitol content in 1% and 2% sucrose containing media to give the molar sucrose equivalent to 6% suppressed the production of shoots completely. No difference in percent frequency of shoot or r o o t formation was observed whether mannitol was added or sorbitol, along with sucrose. Table IV.
Non-penetrating osmotic agents like agaragar, polyethyleneglycol (6000 mol. wt.) and dextran (10 000 mol. wt.) were also used in the present study. Polyethyleneglycol (2.5, 5.0 and 7.5% levels) when used as an osmotic agent in the regenerating medium along with 2% sucrose greatly inhibited shoot formation. Similarly, high levels of agar-agar and dextran (2% and 3%) suppressed the shoot forming ability of rice callus tissues significantly.
E n e r g y a n d o s m o t i c r e q u i r e m e n t s for r o o t a n d s h o o t f o r m a t i o n in rice callus
Conc. o f sucrose (%)
M a n n i t o l or sorbitol to give m o l a r sucrose equiv a l e n t t o (%)
O s m o l a r i t y of t h e m e d i u m
% f r e q u e n c y of d i f f e r e n t i a t i o n
mOsmol
Bars
Shoot
Root
2.0 2.0 2.0 2.0 2.0 2.5 3.0 4.0 6.0 10.0 15.0
Nil 2.25 2.5 3.0 6.0 Nil Nil Nil Nil Nil Nil
142 170 200 253 562 161 176 198 255 353 480
--3.5 --4.2 --4.9 --6.2 --13.8 -3.9 -4.3 -4.9 --6.2 -8.6 --11.8
50---60 72--76 92--96 86---90 Nil 52--58 50--58 30--35 6--10 4--5 Nil
85--90 85--90 94--98 90--95 70--72 85--90 85--90 80--82 60--65 55--60 4--5
193 In L i n s m a i e r a n d S k o o g ' s m e d i u m , sucrose a c c o u n t s f o r 57% o f t h e o s m o t i c p o t e n t i a l , w h e r e a s m a j o r inorganic ions add 38% a n d trace e l e m e n t s and organic s u p p l e m e n t s c o n t r i b u t e 5%. Barg and U m i e l [7] i n d i c a t e d t h a t o s m o l a r i t y o f the m e d i u m a f f e c t e d b o t h greening and s h o o t f o r m a t i o n in some t o b a c c o callus cultures. B r o w n et al. [ 2 0 ] s h o w e d t h a t the m e d i u m supplied sucrose is being used in the ratios 1 : 3 to 2 : 3 f o r o s m o r e g u lation a n d as c a r b o n - e n e r g y source respectively. The success in partially replacing the sucrose r e q u i r e m e n t f o r r o o t and s h o o t f o r m a t i o n with sorbitol or m a n n i t o l in o s m o t i c a l l y equivalent levels s u p p o r t e d their view t h a t p a r t o f the sucrose is acting as an o s m o t i c u m . T h u s m e d i u m supplied c a r b o h y d r a t e has a dual role o f acting as an e n e r g y and o s m o t i c source. Cleland [ 2 1 , 2 2 ] and Z i m m e r m a n n [ 2 3 ] are o f the view t h a t high levels o f sucrose and m a n n i t o l in the m e d i u m w o u l d act against the c r e a t i o n o f a critical t u r g o r pressure w h i c h m u s t be established b e f o r e cell e x p a n s i o n can occur. This stress c o n d i t i o n w o u l d inhibit b o t h g r o w t h o f callus tissues and organogenesis. P r o m o t i o n o f s h o o t and r o o t f o r m a t i o n b y sorbitol or mannitol at l o w e r concentrations, may reflect the g e n e r a t i o n o f a t u r g o r w h i c h results in o p t i m u m o r g a n o g e n e t i c response. B u t such a p r o m o t i o n o f s h o o t and r o o t d i f f e r e n t i a t i o n in rice c o u l d n o t be reprod u c e d w i t h o t h e r o s m o t i c agents like m y o inositol or e r y t h r i t o l or glycerol o r s o d i u m chloride. Such a p r o m o t i o n o f s h o o t and r o o t d i f f e r e n t i a t i o n to occur, the o s m o t i c agent responsible for the increase in t u r g o r m u s t be able to p e n e t r a t e the callus tissues and cross the cell m e m b r a n e . T h a t was the reason w h y n o n - p e n e t r a t i n g o s m o t i c agents like agar-agar, p o l y e t h y l e n e g l y c o l and d e x t r a n are ineffective in bringing a b o u t a similar response. However, the existence o f an as y e t u n k n o w n m e c h a n i s m c a n n o t be ruled o u t to explain the p r o m o t i o n o f s h o o t and r o o t differentiat i o n in rice with the a b o v e o s m o t i c agents. Sucrose at 2% level p r o d u c e d 5 0 - - 6 0 % s h o o t d i f f e r e n t i a t i o n b u t a d d i t i o n o f sorbitol or
m a n n i t o l to give m o l a r sucrose equivalent to 2.5% resulted in m a x i m u m p r o d u c t i o n o f shoots. This implies t h a t 2% sucrose m a y be the o p t i m a l level for d i f f e r e n t i a t i o n t o o c c u r b u t an a d d i t i o n a l s u p p l y of sorbitol or mannitol is necessary to p r o d u c e the m a x i m u m t u r g o r and s u b s e q u e n t s h o o t regeneration.
References 1 J. Lustinec and J. Horok, Experientia, 26 (1970) 919. 2 J.W. Saunders and E.T. Bingham, Am. J. Bot., 62 (1975) 850. 3 P.B. Kavi Kishor and G.M. Reddy, SABRAO J., 17 (1985) 181. 4 J.W. Heyser, T.A. Dykes, K.J. De Mott and M.W. Nabors, Plant Sci. Lett., 29 {1983) 175. 5 W.F. Sheridan, Plant regeneration and chromosome stability in tissue cultures, in: L. Ledoux (Ed.), Genetic Manipulations with Plant Material, Plenum Press, New York and London, 1975, p. 263. 6 J.G. Torrey, Physiol. Plant., 20 (1967) 265. 7 R. Barg and N. Umiel, Z. Pflanzenphysiol., 81 (1977) 161. 8 T.A. Thorpe, Meded. Fac. Landbouwwet. Rijks Univ. Gent., 42 (1977) 1681. 9 S. Klenovska, Acta Fac. Rerum Nat. Univ. Comeonianae Physiol. Plant., 11 (1976) 41. 10 S.L. Kimball, W.D. Beversdorf and E.T. Bingham, Crop Sci., 15 (1975) 750. 11 L.F. Liu and K.L. Lai, High frequency plant regeneration from water-stressed rice cultures, in: 1st International Congress of Plant Molecular Biology, Savannah, Georgia, U.S.A., Oct. 27-Nov. 2, 1985, Abstr. p. 11. 12 C.H. Granatek and A.W. Cockerline, In vitro, 14 (1978) 212. 13 S.J. Stavarek, T.P. Croughan and D.W. Rains, Plant Sci. Lett., 19 (1980) 253. 14 P.B. Kavi Kishor and A.R. Mehta, Proc. Ind. Nat. Sci. Acad., B49 (1983) 729. 15 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 16 K.K. Nesius, L.E. Uchytil and J.S. Fletcher, Planta, 106 (1972) 173. 17 G. Staudt, J. Plant Physiol., 116 (1984) 161. 18 A. Maretzki, M. Thorn and L.G. Nickell, Utilization and metabolism of carbohydrates in cell ~nd callus cultures, in: H.E. Street (Ed.), Tissue Culture and Plant Science, Academic Press, New York and London, 1974, p. 329. 19 P.B. Kavi Kishor and G.M. Reddy, 2. Plant Physiol., 126 r1986) 49.
19t
20 D.C.W. Brown, D.W.M. Leung and T.A. Thorpe, Physiol. Plant., 46 (1979) 36. 21 R. Cleland, Annu. Rev. Plant Physiol., 22 (1971) 197. 22 R. Cleland, The control of cell enlargement, in:
D.H. Jennings (Ed.), Integration of Activity in the Higher Plant, S.E.B. Syrup. XXXI University Press, Cambridge, U.K., 1977, p. 101. 23 U. Zimmermann, Annu. Rev. Plant Physiol., 29 (1978) 121.
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ENERGY AND O S M O T I C R E Q U I R E M E N T
F O R HIGH FREQUENCY REGENERATION OF RICE PLANTS FROM LONG-TERM CULTURES
P.B. KAVI KISHOR Department of Botany, Kakatiya University, Warangal-506 009 (India) (Received July 28th, 1986) (Revision received October 14th, 1986) (Accepted October 18th, 1986) Rice callus showed optimum growth at a medium osmolarity of 300, but maximum frequency (92--96%) of plantlet production was noticed at 200 mOsmol. Minimum requirement of sucrose for root differentiation was found to be 0.5%, while it was 1% for shoot. Tissues grown on a low sucrose medium produced high percent frequency of shoots only if the medium was supplemented with osmotic agents like sorbitol or mannitol to give the molar sucrose equivalent to 2 or 3%. Callus growing on sucrose as sole carbon source lost its shoot forming ability by 100 days in culture, whereas tissues proliferating on sorbitol or mannitol (besides sucrose) differentiated shoots over a period of 1500 days. Partial replacement of sucrose requirement with sorbitol or mannitol for organogenesis suggested that tissue carbohydrate has an osmoregulatory function. Key words: Oryza sativa L. ; osmotic potentials; long-term cultures; sugar alcohols
Introduction Rice callus is a slow g r o w i n g tissue a n d t h e r e are h a r d l y a n y r e p o r t s o n the increase o f t h e g r o w t h o f t h e callus. O n e o f the m a i n c o n s t r a i n t s in utilizing cell a n d tissue c u l t u r e in cereal b r e e d i n g p r o g r a m m e is the low rate o f r e g e n e r a t i o n o f p l a n t s f r o m c u l t u r e d cells. S h o o t f o r m i n g ability declines w i t h t i m e a n d e x a m p l e s of such species are Brassica oleracea [1] M e d i c a g o sativa [2] rice [3] etc. E m b r y o genic callus o f rice p r o v i d e d l o n g - t e r m ( 3 5 0 d a y s o n l y ) t o t i p o t e n t cultures [4] b u t t h e r e is n o visual d i f f e r e n c e b e t w e e n e m b r y o g e n i c a n d n o n - e m b r y o g e n i c callus in rice. F u r t h e r , s o m e varieties o f rice do n o t p r o d u c e e m b r y o genic callus ( p e r s o n a l o b s e r v a t i o n ) . Sheridan [5] h a d r e c o m m e n d e d t h a t ' E f f o r t s h o u l d f o c u s on the d e v e l o p m e n t o f c u l t u r e m e d i a w h i c h allow the initiation and m a i n t e n a n c e
Abbreviations: IAA, indole-3-acetic acid ; KN, kinetin; LS, Linsmaier and Skoog.
o f p l a n t r e g e n e r a t i o n in l o n g - t e r m cultures'. T o r r e y [6] suggested t h a t the loss in o r g a n f o r m i n g c a p a c i t y in l o n g - t e r m c u l t u r e s of P i s u m s a t i v u m is c o r r e l a t e d w i t h the increase in a b n o r m a l i t y o f c h r o m o s o m a l c o n s t i t u t i o n and n u m b e r . H o w e v e r , we h a v e n o t o b s e r v e d a n y increase in c h r o m o s o m e n u m b e r in l o n g - t e r m c u l t u r e s ( 1 5 0 0 days} o f rice. Besides acting as a s o u r c e o f energy, m e d i u m supplied c a r b o h y d r a t e m a y also f u n c t i o n as an o s m o t i c a g e n t [ 7 , 8 ] . T h e r e are r e p o r t s in t h e r e f e r e n c e s w h e r e mild o s m o t i c stress a f f e c t e d t o b a c c o ]9] a n d s o y b e a n tissue c u l t u r e s [10] b y increa-~ing callus g r o w t h a n d m o d i f y i n g the cellular m o r p h o l o g y . Liu a n d Lai [11] r e p o r t e d high f r e q u e n c y p l a n t r e g e n e r a t i o n in w a t e r stressed c u l t u r e s of rice. T h e i m p o r t a n c e of o s m o t i c c o n d i t i o n s in t h e c u l t u r e of e m b r y o s of H o r d e u m [ 1 2 ] , in l o n g - t e r m r e g e n e r a t i o n o f alfalfa [13] and during r o o t and s h o o t d i f f e r e n t i a t i o n in t o b a c c o [ ! 4 ] have b e e n o u t l i n e d earlier. T h e a i m o f this p a p e r is to increase callus g r o w t h , t o p r o d u c e rice p l a n t s
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190 in l o n g - t e r m c u l t u r e s and t o e n h a n c e the p e r c e n t f r e q u e n c y o f p l a n t l e t f o r m a t i o n in rice. T h e s e p r o b l e m s are solved w i t h t h e incorporation of appropriate concentrations o f sugar a n d sugar alcohols in t h e n u t r i e n t medium. Materials a n d m e t h o d s R i c e ( O r y z a sativa L. cv. Bala) callus w a s initiated f r o m m a t u r e e m b r y o s o n L i n s m a i e r a n d S k o o g ' s (LS) agar m e d i u m [15] supplem e n t e d w i t h 2 mg/1 2 , 4 - d i c h l o r o p h e n o x y acetic acid (2,4-D) a n d 2% sucrose a n d m a i n t a i n e d on the same. A f t e r o n e or t w o s u b c u l t u r e s , t h e callus was t r a n s f e r r e d o n t o LS basal m e d i u m c o n t a i n i n g 2 mg/1 2,4-D, 2% sucrose a n d 3% s o r b i t o l or 3% m a n n i t o l . F o r r e g e n e r a t i o n o f plants, LS salts supplem e n t e d w i t h 1 mg/1 indole-3-acetic acid ( I A A ) , 4 mg/1 k i n e t i n ( K N ) a n d 2% sucrose was used. All s u p p l e m e n t s w e r e a d d e d t o the media before autoclaving on a weight per v o l u m e basis. C u l t u r e s w e r e i n c u b a t e d at p h o t o p e r i o d s of 24 h in d i f f u s e light ( 4 0 0 lux) f o r callus initiation a n d in b r i g h t light ( 2 0 0 0 lux) f o r p l a n t r e g e n e r a t i o n at a t e m p e r a t u r e o f 25 + 2°C. Cool, w h i t e , f l u o r e s c e n t light was p r o v i d e d f o r all t h e cultures. In each
Table I.
Results and discussion D i f f e r e n t c o n c e n t r a t i o n s o f sucrose ( 0 . 2 5 - 15%) was a d d e d to the LS basal m e d i u m c o n t a i n i n g 2 mg/1 2,4-D a n d its e f f e c t o n g r o w t h as m e a s u r e d b y fresh a n d d r y weights a n d also t o t a l o s m o t i c s t r e n g t h of the m e d i a are r e p r e s e n t e d in T a b l e I. Increasing c o n c e n t r a t i o n s o f sucrose in the m e d i u m increased t h e g r o w t h o f callus till 6% level, b u t g r o w t h declined at higher levels (10 a n d 15%) o f sucrose. V a r i o u s c o n c e n t r a t i o n s of s o r b i t o l and m a n n i t o l w e r e a d d e d to LS basal m e d i u m c o n t a i n i n g 2 mg/1 2,4-D and 2% sucrose a n d their e f f e c t on g r o w t h is p r e s e n t e d in T a b l e II. Callus s h o w e d increasing g r o w t h o n s o r b i t o l or m a n n i t o l u p to an o p t i m u m o f 3% b u t
Effect of sucrose on the growth of embryo callus of rice
Conc. of sucrose (%)
Osmolarity of the medium mOsmol
LS basal (no sucrose)
0.25 0.5 1.0 2.0 3.0 4.0 6.0 i0.0 15.0
c u l t u r e t u b e 15 ml o f the m e d i u m was t a k e n and 150 + 14.5 mg o f fresh callus (22.0 -+ 4.3 m g of d r y wt.) was inoculated. A t o t a l of six replicates f o r g r o w t h m e a s u r e m e n t s a n d 4 5 - - 7 5 replicates f o r r e g e n e r a t i o n of p l a n t s w e r e h a r v e s t e d a f t e r 30 d a y s in culture. E x p e r i m e n t s w e r e r e p e a t e d at least t w o t i m e s w i t h c o n s i s t e n t results. T o t a l o s m o l a r i t y o f t h e m e d i u m was m e a s u r e d b e f o r e a u t o c l a v i n g using a w e s c o r 5 1 0 0 C V a p o r Pressure O s m o m e t e r .
98 106 112 123 149 176 200 249 350 476
Mean fresh wt. mg/culture
Mean dry wt. mg/culture
Bars -2.4 --2.6 --2.7 --3.0 --3.7 --4.3 --4.9 -6.1 -8.6 -11,7
aFigures in parenthesis represent S.E.
Tissues did not survive
210.7 (±14.8) a 270.9 (±16.4) 378.0(±22.0) 441.0(220.5) 599.6(-+26.1) 601.5(±32.6) 782.0 (± 35.9) 452.2(228.8) 401.0(±20.0)
27.3 (_+ 4.6) a 32.1(+ 5.0) 40.8(± 5.2) 55.0(-+ 6.3) 75.9(_+ 7.5) 78.4(± 8.4) 101.2 (± 10.1) 91.4(-+ 7.7) 86.6(± 7.5)
191 higher concentrations (6 and 9%) suppressed the growth on b o t h a fresh and a dr y weight basis. Thus sorbitol and mannitol r e p r o d u c e d the sucrose inhibition of growth (Table II) at higher levels. However, r e pl a c e m ent of 3% sorbitol or 3% mannitol by 8% sucrose (osmolarity of this m e d i u m is equivalent to that of 2% sucrose plus 3% sorbitol or mannitol) failed to evoke an identical response. P r o m o t i o n of callus growth by sugar alcohols has been d e m o n s t r a t e d by Nesius et al. [16] in Pauls Scarlet Rose and by Staudt [17] in Vitis. Studies with Prunus persica (see Ref. 18) suggested, t hat its growth on sorbitol may be adaptive, since the initial growth on the sugar alcohol was much lower than on sucrose and only with time did it come to equal t hat of the disaccharide. E m b r y o callus o f rice (75 days old) proliferating on 2% sucrose containing medium, regenerated plantlets with 15% f r e q u e n c y b u t lost its ability to organise shoots by day 100 in culture. On the ot her hand, callus tissues proliferating on sucrose plus sorbitol or sucrose plus mannitol differentiated whole plants in the same regenerating medium over Table II. Effect of sorbitol and mannitol on the growth of rice callus Conc. of sugar alcohol (%)
Mean fresh wt.
mg/culture
Mean dry wt. rag/culture
2% s u c r o s e alone
(control) Sorbitol 1.5 3.0 6.0 9.0
785.7 (-+30.1) 1214.0 (± 42.6) 500.6(±21.8) 359.0(±17.2)
Mannitol 1.5 3.0 6.0 9.0
615.5 (_'227.4) 926.0(238.3) 515.4(±19.7) 380.0(±18.5)
441.0 (± 20.5) a
aFigures in parenthesis represent S.E.
55.0 (+ 6.3) a 98.9(2 7.6) 155.0 (2 11.6) 78.7 (± 8.2) 67.2(-+ 6.8) 82.9 (± 125.8(2 76.0(2 63.1(±
7.1) 8.9) 7.0) 6.5)
a period of 1500 days with 50--60% freq u e n c y [ 1 9 ] . I n c o r p o r a t i o n of sorbitol or mannitol into the callus proliferating medium (in addition to sucrose) thus helped in retaining the shoot forming ability of tissues in long-term cultures and also in enhancing the p e r c e n t f r e q u e n c y of plantlet formation. The correlations between the percent f r e q u e n c y of r o o t and shoot f o r m a t i o n and the concentration of sucrose and osmotic agents in the medium are shown in Tables III and IV. Minimum requi rem ent of sucrose for r o o t differentiation was f o u n d to be 0.5% and for that of shoot it was 1%. This result could also be reproduced when 0.25% and 0.5% sucrose containing media were supplem e n t e d with sorbitol or mannitol to give the molar sucrose equivalent to that of 0.5 and 1% for r o o t and shoot form at i on respectively (Table III). Maximum r o o t and shoot differentiation was noticed when 2 or 3% sucrose (osmolarity of these media were 142 and 176 mOsmol) was supplied to the medium (Table IV). Tissues grown on a medium with sucrose levels lower or higher than 2% showed a reduced capacity to form roots and shoots. Total suppression of r o o t and shoot formation occurred when no sucrose was supplied or m edi um sucrose concentrations reached a b o u t 15% (Tables III and IV). When .~orbitol or mannitol were added to 0.5% sucrose containing media to give the molar sucrose equivalents to 1,2 and 3%, there was a decline in the r o o t and shoot forming capacities of the tissues. A similar trend was observed with f u r t h e r increase of sucrose c o n t e n t to 1% and sorbitol or mannitol to give the molar sucrose equivalents to 2,3 and 6% (Table III). A c o n c u r r e n t high f r e q u e n c y of r o o t and shoot form at i on (92--96%) was noticed at a medium osmolarity of 200 mOsmol when the medium was supplemented with 2% sucrose and sorbitol or mannitol to give the molar sucrose equivalent to 2.5% (Table IV). It appears from Tables III and IV that sorbitol or mannitol had a promotive effect on shoot f o r m a t i o n at the 2% and 3% sucrose equivalent levels. However, replacement of sorbitol
192 T a b l e III.
E n e r g y a n d o s m o t i c r e q u i r e m e n t s for r o o t a n d s h o o t f o r m a t i o n in rice callus
Conc. of sucrose (%)
M a n n i t o l or s o r b i t o l to give m o l a r sucrose equivalent t o (%)
O s m o l a r i t y of t h e m e d i u m
% f r e q u e n c y of d i f f e r e n t i a t i o n a
mOsmol
Bars
Shoot
Root
0.25 0.25 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0
Nil 0.5 Nil 1.0 2.0 3.0 Nil 2.0 3.0 6.0
106 116 113 159 255 349 123 228 304 592
--2.6 --2.8 --2.8 -3.9 --6.2 -8.6 --3.0 -5.6 --7.5 --14.5
Nil Nil Nil 50--55 (Tiny) 40--42 (Tiny) 20--24 (Tiny) 50--53 76--80 80--85 Nil
Nil 60--65 65--70 75--77 44--46 25--30 80--85 85--90 60--65 30--35
a% f r e q u e n c y of d i f f e r e n t i a t i o n -- [(No. of calli w i t h r o o t s or s h o o t s ) / ( N o , of calli i n o c u l a t e d ) ] x 100.
or mannitol with equimolar concentrations of glycerol, erythritol, myo-inositol, sucrose and sodium chloride failed to evoke an identical response under identical conditions. Increase of sorbitol or mannitol content in 1% and 2% sucrose containing media to give the molar sucrose equivalent to 6% suppressed the production of shoots completely. No difference in percent frequency of shoot or r o o t formation was observed whether mannitol was added or sorbitol, along with sucrose. Table IV.
Non-penetrating osmotic agents like agaragar, polyethyleneglycol (6000 mol. wt.) and dextran (10 000 mol. wt.) were also used in the present study. Polyethyleneglycol (2.5, 5.0 and 7.5% levels) when used as an osmotic agent in the regenerating medium along with 2% sucrose greatly inhibited shoot formation. Similarly, high levels of agar-agar and dextran (2% and 3%) suppressed the shoot forming ability of rice callus tissues significantly.
E n e r g y a n d o s m o t i c r e q u i r e m e n t s for r o o t a n d s h o o t f o r m a t i o n in rice callus
Conc. o f sucrose (%)
M a n n i t o l or sorbitol to give m o l a r sucrose equiv a l e n t t o (%)
O s m o l a r i t y of t h e m e d i u m
% f r e q u e n c y of d i f f e r e n t i a t i o n
mOsmol
Bars
Shoot
Root
2.0 2.0 2.0 2.0 2.0 2.5 3.0 4.0 6.0 10.0 15.0
Nil 2.25 2.5 3.0 6.0 Nil Nil Nil Nil Nil Nil
142 170 200 253 562 161 176 198 255 353 480
--3.5 --4.2 --4.9 --6.2 --13.8 -3.9 -4.3 -4.9 --6.2 -8.6 --11.8
50---60 72--76 92--96 86---90 Nil 52--58 50--58 30--35 6--10 4--5 Nil
85--90 85--90 94--98 90--95 70--72 85--90 85--90 80--82 60--65 55--60 4--5
193 In L i n s m a i e r a n d S k o o g ' s m e d i u m , sucrose a c c o u n t s f o r 57% o f t h e o s m o t i c p o t e n t i a l , w h e r e a s m a j o r inorganic ions add 38% a n d trace e l e m e n t s and organic s u p p l e m e n t s c o n t r i b u t e 5%. Barg and U m i e l [7] i n d i c a t e d t h a t o s m o l a r i t y o f the m e d i u m a f f e c t e d b o t h greening and s h o o t f o r m a t i o n in some t o b a c c o callus cultures. B r o w n et al. [ 2 0 ] s h o w e d t h a t the m e d i u m supplied sucrose is being used in the ratios 1 : 3 to 2 : 3 f o r o s m o r e g u lation a n d as c a r b o n - e n e r g y source respectively. The success in partially replacing the sucrose r e q u i r e m e n t f o r r o o t and s h o o t f o r m a t i o n with sorbitol or m a n n i t o l in o s m o t i c a l l y equivalent levels s u p p o r t e d their view t h a t p a r t o f the sucrose is acting as an o s m o t i c u m . T h u s m e d i u m supplied c a r b o h y d r a t e has a dual role o f acting as an e n e r g y and o s m o t i c source. Cleland [ 2 1 , 2 2 ] and Z i m m e r m a n n [ 2 3 ] are o f the view t h a t high levels o f sucrose and m a n n i t o l in the m e d i u m w o u l d act against the c r e a t i o n o f a critical t u r g o r pressure w h i c h m u s t be established b e f o r e cell e x p a n s i o n can occur. This stress c o n d i t i o n w o u l d inhibit b o t h g r o w t h o f callus tissues and organogenesis. P r o m o t i o n o f s h o o t and r o o t f o r m a t i o n b y sorbitol or mannitol at l o w e r concentrations, may reflect the g e n e r a t i o n o f a t u r g o r w h i c h results in o p t i m u m o r g a n o g e n e t i c response. B u t such a p r o m o t i o n o f s h o o t and r o o t d i f f e r e n t i a t i o n in rice c o u l d n o t be reprod u c e d w i t h o t h e r o s m o t i c agents like m y o inositol or e r y t h r i t o l or glycerol o r s o d i u m chloride. Such a p r o m o t i o n o f s h o o t and r o o t d i f f e r e n t i a t i o n to occur, the o s m o t i c agent responsible for the increase in t u r g o r m u s t be able to p e n e t r a t e the callus tissues and cross the cell m e m b r a n e . T h a t was the reason w h y n o n - p e n e t r a t i n g o s m o t i c agents like agar-agar, p o l y e t h y l e n e g l y c o l and d e x t r a n are ineffective in bringing a b o u t a similar response. However, the existence o f an as y e t u n k n o w n m e c h a n i s m c a n n o t be ruled o u t to explain the p r o m o t i o n o f s h o o t and r o o t differentiat i o n in rice with the a b o v e o s m o t i c agents. Sucrose at 2% level p r o d u c e d 5 0 - - 6 0 % s h o o t d i f f e r e n t i a t i o n b u t a d d i t i o n o f sorbitol or
m a n n i t o l to give m o l a r sucrose equivalent to 2.5% resulted in m a x i m u m p r o d u c t i o n o f shoots. This implies t h a t 2% sucrose m a y be the o p t i m a l level for d i f f e r e n t i a t i o n t o o c c u r b u t an a d d i t i o n a l s u p p l y of sorbitol or mannitol is necessary to p r o d u c e the m a x i m u m t u r g o r and s u b s e q u e n t s h o o t regeneration.
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19t
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