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Nicotine accumulation in callus and small plants of tobacco (Nicotiana tabacum L.) grown in media supplemented with nicotine
Plant Science Letters, 23 (1981) 315--319 Elsevier/North-Holland Scientific Publishers Ltd.
315
NICOTINE ACCUMULATION IN CALLUS AND SMALL PLANTS OF TOBACCO ( N I C O T I A N A T A B A C U M L.) GROWN IN MEDIA SUPPLEMENTED WITH NICOTINE*
J.W. SAUNDERS**, H.J. PUDLINER and L.P. BUSH***
Department of Agronomy, University of Kentucky, Lexington, KY 40546 (U.S.A.)
(Received March 30th, 1981) (Revision received June 30th, 1981) (Accepted July 6th, 1981)
SUMMARY Callus of Nicotiana tabacum L. cv. Burley 21 was grown on MurashigeSkoog medium with 0.03 rag/1 kinetin and 2.0 rag/1 ~-indoleacetic acid and nicotine levels of 0--32 raM. With increased nicotine concentrations in the medium, callus nicotine accumulation increased from nearly zero as fresh weight decreased. Nicotine accumulation was as great as 6.2% with 60% of the dry weight accumulation compared with the control to which no nicotine was added. Burley 21 callus accumulated only nornicotine if nornicotine was the sole supplemental alkaloid in the medium. Callus of Robinson Medium Broadleaf accumulated only nicotine when grown on a medium supplemented only with nicotine, although both green and cured leaves of this cultivar demethylate nicotine to nornicotine. Burley 21 plants at the 6--8-leaf stage grown hydroponically with 8 mM nicotine accumulated 2.4% nicotine in 10 days compared with 0.3% in the controls.
INTRODUCTION Nicotine accumulation in callus or suspension cultures of Nicotiana species seems to be variable and influenced by genetic predisposition, nature and rate of growth, lighting, growth regulator combinations and concentrations and the presence of protein synthesis inhibitors [1,2]. It is possible to *The investigation reported in this paper (No. 81-34S) is in connection with a project of the Kentue]o, Tobacco Research Board and the Kentucky Agricultural Expm~,ment Station and k published with approval of the Director. e°USDA Sutarbeet Invmti~tions, P.O. Box 1688, Fast Lansing, M148828. eee'ro whom any con'espondenee should be addressed.
316 obtain cultures with either high or negligible nicotine concentrations by the proper selection of cell strain and growth conditions. The absence of nicotine in at least one culture has been associated with the absence of activity of several nicotine biosynthetic enzymes [3]. This paper reports the potential to alter tissue levels of nicotine in callus and small plants by growth in the presence of relatively high levels of nicotine. METHODS AND MATERIALS Callus was initiated from 1 c m long sterile hypocotyl segments from seedlings of cultivars 'Burley 21', 'Low Alkaloid Burley 21', and 'Robinson M e d i u m Broadleaf'. Nicotine is the principal alkaloid in Burley 21 and L o w Alkaloid Burley 21 plants and nornicotine is the principal alkaloid in Robinson M e d i u m Broadleaf plants. Seeds were surface sterilized by washing 3 min in 9 5 % ethanol, 5 min in 0.8% sodium hypochlorite, followed by 6 rinses in steriledistilledwater. Callus used as inoculum for experiments was taken through three 28
317 1.6' 41
~
•
•
•
•
=.2
•
It
6 ~ Z
m [.I- t/I
• O
'<
•
u
0 (~
4
~
1'2
16
2~,
32
0o
MMOLAR NICOTINE IN MEDIUM
Fig. 1. Effect of exogenous nicotine in the nutrient medium on 28-day callus yield and tissue nicotine content of 'Burley 21' callus.
nicotine, fresh weight of the tissue was 6 0 % of the control (no nicotine added to the medium). Nicotine concentration in the callus increased with increased nicotine level in the medium, up to 6.2% o f dry weight at 32 mM nicotine in the medium. Nicotine was the only alkaloid detected in the Burley 21 callus. To determine if accumulation o f nicotine by tissue grown in the presence of nicotine was a more general phenomenon in tobacco~ plants of Burley 21 at the 6--8-leaf stage were grown in 9.5% nutrient solution with 0, 2 or 8 mM nicotine. Nutrient solutions were replaced dally. Five plants from the 8 mM nicotine treatment were harvested at 1--2-day intervals for leaf nicotine determination. Nicotine levels in the leaves of plants grown in 8 mM nicotine increased steadily over a 10
2.4 Ld Z (~2.0 U 7 1.6,
~
8 MMOLAR NICOT INE
1.2.
~O.B N
2 MMOLAR NICOTINE
0.4
~) NO ADDED NICOTINE: 60
HOURS
120
180
240
IN SOLUTION
Fig. 2. Temporal pattern o f nicotine accumulation of sn~ll plants grown in 8 mM nicotine in 25% Hoa~land solution.
318
Burley 21 callus accumulated nornicotine when 20 mM nornicotine was in the medium as the only alkaloid (data n o t shown). Other alkaloids were n o t detected in the callus. Callus of Robinson Medium Broadleaf accumulated nicotine as the sole alkaloid when grown in the presence of 20 mM nicotine. Robinson Medium Broadleaf is a cultivar classified as a converter, with relatively high nornicotine levels in green and cured leaf. Conversion involves a demethylation of nicotine in green and cured leaf, a process which accounts for most of the biosynthesis of nornicotine [6]. As callus of Robinson Medium Broadleaf accumulated nicotine b u t n o t nornicotine when grown in the presence of high levels of nicotine, the most likely explanation is that the demethylation process is n o t operative in callus tissue as grown in this experiment. Nicotine accumulation was negligible in this callus grown in the absence of exogenous nicotine. However, our data do n o t eliminate the possibility of equal rates of endogenous nicotine biosynthesis and degradation. Nicotine as an additive to t o b a c c o callus culture medium in low concentrations has been reported to stimulate growth in the absence of cytokinin [7]. The accumulation and minimal toxicity of nicotine in tobacco callus grown in the presence of nicotine has been n o t e d previously for nicotine concentrations of up to 5 mM [ 8 ] , b u t our results indicate that at nicotine concentrations in the medium up to 32 mM callus continued to grow. NeUes and Muller [9] reported that the nicotine content of t o b a c c o leaf discs floated on a nicotine-containing solution reached equilibrium with the solution in 12 h. Most of the nicotine in t o b a c c o leaves is in the vacuole [ 10] and consequently the accumulation of alkaloids in callus tissue is probably in the vacuole and is at equilibrium with the alkaloid level in the medium at medium levels below a lethal dose. Accumulation in the vacuole would account for the minimal toxicity observed from the high levels of nicotine in callus. Callus derived from L o w Alkaloid Burley 21 (which was TABLE I GROWTH AND NICOTINE CONTENT IN BURLEY 21 AND LOW ALKALOID BURLEY 21 CALLUS GROWTH ON MURASHIGE-SKOOG MEDIUM WITH 1.0 mg/l a-NAPHTHALENEACETIC ACID AND 0.2 mgfl KINETIN N i c o t i n e levels (Media) O,m)
0 5 10 30 100 500
Alkaloid c o n t e n t
Callus dry wt. Burley 21 (rag)
L A Burley 21 (mg)
Burley 21 (%)
L A Burley 21 (%)
166 231 239 160 9-09 185
243 230 214 168 284 195
0.004 0.019 0.018 0.008 0.084 0.215
0.008 0.008 0.004 0.011 0.0"/0 0.150
± ± ± ± ± ±
48 52 49 86 45 37
± ± ± ± ± ±
36 38 54 94 85 49.
± ± ± ± ± ±
0.003 0.002 0.005 0.008 0.022 0.040
+ ± ± ± ± *
0.001 0.001 0.008 0.009 0.020 0.028
319
developed for minimal endogenous nicotine production and accumulation) grows as well and accumulates as much nicotine as the near isogenic Burley 21 callus when both are grown on a m e d i u m containing nicotine (Table I). However, Burley 21 and L o w Alkaloid Burley 21 callus cultured on a m e d i u m designed for o p t i m u m endogenous nicotine production showed t h a t the levels of nicotine in the callus reflected the nicotine levels f o u n d in the leaves of the respective lines. The observations of 'loading' callus tissue with alkaloids suggests t h a t callus would be suitable for metabolic studies of the alkaloids. However, because most of the alkaloids are apparently in the vacuoles and since Robinson Medium Broadleaf callus did n o t demethylate nicotine to nornicotine, it is important to k n o w the pool sizes and equilibria o f alkaloids in a tissue in order to interpret data obtained from metabolic studies appropriately. REFERENCES
1 S. Ohta, O. Matsui and M. Yatazawa, Agric. Biol. Chem., 42 (1978) 1245. 2 D. Neumann and E. Muller, Biochem. Physiol. Pflanzen., 162 (1971) 503. 3 S. Mizusaki, Y. Tanabe, M. Noguchi and E. Tamaki, Plant Cell Physiol., 14 (1973) 103.
4 5 6 7
Technicon Industrial Systems, Ind. Method No. 117--71A (1972). L.P. Bush, J. Chromatogr., 73 (1972) 243. W. Stepka and L.J. Dewey, Plant Physiol., 36 (1961) 592. J.E. Peters, P.H.L. Wu, W.R. Sharp and E.F. Paddock, Physiol. Plant., 31 (1974) 97. 8 S. Ohta, Y. Kojirna and M. Yatazawa, Agric. Biol. Chem., 42 (1978) 1733. 9 A. Nelles and E. Muller, Biochem. Physiol. Pflanzen., 162 (1971) 495. 10 J.A. Saunders, Plant Physiol., 64 (1979) 74.
315
NICOTINE ACCUMULATION IN CALLUS AND SMALL PLANTS OF TOBACCO ( N I C O T I A N A T A B A C U M L.) GROWN IN MEDIA SUPPLEMENTED WITH NICOTINE*
J.W. SAUNDERS**, H.J. PUDLINER and L.P. BUSH***
Department of Agronomy, University of Kentucky, Lexington, KY 40546 (U.S.A.)
(Received March 30th, 1981) (Revision received June 30th, 1981) (Accepted July 6th, 1981)
SUMMARY Callus of Nicotiana tabacum L. cv. Burley 21 was grown on MurashigeSkoog medium with 0.03 rag/1 kinetin and 2.0 rag/1 ~-indoleacetic acid and nicotine levels of 0--32 raM. With increased nicotine concentrations in the medium, callus nicotine accumulation increased from nearly zero as fresh weight decreased. Nicotine accumulation was as great as 6.2% with 60% of the dry weight accumulation compared with the control to which no nicotine was added. Burley 21 callus accumulated only nornicotine if nornicotine was the sole supplemental alkaloid in the medium. Callus of Robinson Medium Broadleaf accumulated only nicotine when grown on a medium supplemented only with nicotine, although both green and cured leaves of this cultivar demethylate nicotine to nornicotine. Burley 21 plants at the 6--8-leaf stage grown hydroponically with 8 mM nicotine accumulated 2.4% nicotine in 10 days compared with 0.3% in the controls.
INTRODUCTION Nicotine accumulation in callus or suspension cultures of Nicotiana species seems to be variable and influenced by genetic predisposition, nature and rate of growth, lighting, growth regulator combinations and concentrations and the presence of protein synthesis inhibitors [1,2]. It is possible to *The investigation reported in this paper (No. 81-34S) is in connection with a project of the Kentue]o, Tobacco Research Board and the Kentucky Agricultural Expm~,ment Station and k published with approval of the Director. e°USDA Sutarbeet Invmti~tions, P.O. Box 1688, Fast Lansing, M148828. eee'ro whom any con'espondenee should be addressed.
316 obtain cultures with either high or negligible nicotine concentrations by the proper selection of cell strain and growth conditions. The absence of nicotine in at least one culture has been associated with the absence of activity of several nicotine biosynthetic enzymes [3]. This paper reports the potential to alter tissue levels of nicotine in callus and small plants by growth in the presence of relatively high levels of nicotine. METHODS AND MATERIALS Callus was initiated from 1 c m long sterile hypocotyl segments from seedlings of cultivars 'Burley 21', 'Low Alkaloid Burley 21', and 'Robinson M e d i u m Broadleaf'. Nicotine is the principal alkaloid in Burley 21 and L o w Alkaloid Burley 21 plants and nornicotine is the principal alkaloid in Robinson M e d i u m Broadleaf plants. Seeds were surface sterilized by washing 3 min in 9 5 % ethanol, 5 min in 0.8% sodium hypochlorite, followed by 6 rinses in steriledistilledwater. Callus used as inoculum for experiments was taken through three 28
317 1.6' 41
~
•
•
•
•
=.2
•
It
6 ~ Z
m [.I- t/I
• O
'<
•
u
0 (~
4
~
1'2
16
2~,
32
0o
MMOLAR NICOTINE IN MEDIUM
Fig. 1. Effect of exogenous nicotine in the nutrient medium on 28-day callus yield and tissue nicotine content of 'Burley 21' callus.
nicotine, fresh weight of the tissue was 6 0 % of the control (no nicotine added to the medium). Nicotine concentration in the callus increased with increased nicotine level in the medium, up to 6.2% o f dry weight at 32 mM nicotine in the medium. Nicotine was the only alkaloid detected in the Burley 21 callus. To determine if accumulation o f nicotine by tissue grown in the presence of nicotine was a more general phenomenon in tobacco~ plants of Burley 21 at the 6--8-leaf stage were grown in 9.5% nutrient solution with 0, 2 or 8 mM nicotine. Nutrient solutions were replaced dally. Five plants from the 8 mM nicotine treatment were harvested at 1--2-day intervals for leaf nicotine determination. Nicotine levels in the leaves of plants grown in 8 mM nicotine increased steadily over a 10
2.4 Ld Z (~2.0 U 7 1.6,
~
8 MMOLAR NICOT INE
1.2.
~O.B N
2 MMOLAR NICOTINE
0.4
~) NO ADDED NICOTINE: 60
HOURS
120
180
240
IN SOLUTION
Fig. 2. Temporal pattern o f nicotine accumulation of sn~ll plants grown in 8 mM nicotine in 25% Hoa~land solution.
318
Burley 21 callus accumulated nornicotine when 20 mM nornicotine was in the medium as the only alkaloid (data n o t shown). Other alkaloids were n o t detected in the callus. Callus of Robinson Medium Broadleaf accumulated nicotine as the sole alkaloid when grown in the presence of 20 mM nicotine. Robinson Medium Broadleaf is a cultivar classified as a converter, with relatively high nornicotine levels in green and cured leaf. Conversion involves a demethylation of nicotine in green and cured leaf, a process which accounts for most of the biosynthesis of nornicotine [6]. As callus of Robinson Medium Broadleaf accumulated nicotine b u t n o t nornicotine when grown in the presence of high levels of nicotine, the most likely explanation is that the demethylation process is n o t operative in callus tissue as grown in this experiment. Nicotine accumulation was negligible in this callus grown in the absence of exogenous nicotine. However, our data do n o t eliminate the possibility of equal rates of endogenous nicotine biosynthesis and degradation. Nicotine as an additive to t o b a c c o callus culture medium in low concentrations has been reported to stimulate growth in the absence of cytokinin [7]. The accumulation and minimal toxicity of nicotine in tobacco callus grown in the presence of nicotine has been n o t e d previously for nicotine concentrations of up to 5 mM [ 8 ] , b u t our results indicate that at nicotine concentrations in the medium up to 32 mM callus continued to grow. NeUes and Muller [9] reported that the nicotine content of t o b a c c o leaf discs floated on a nicotine-containing solution reached equilibrium with the solution in 12 h. Most of the nicotine in t o b a c c o leaves is in the vacuole [ 10] and consequently the accumulation of alkaloids in callus tissue is probably in the vacuole and is at equilibrium with the alkaloid level in the medium at medium levels below a lethal dose. Accumulation in the vacuole would account for the minimal toxicity observed from the high levels of nicotine in callus. Callus derived from L o w Alkaloid Burley 21 (which was TABLE I GROWTH AND NICOTINE CONTENT IN BURLEY 21 AND LOW ALKALOID BURLEY 21 CALLUS GROWTH ON MURASHIGE-SKOOG MEDIUM WITH 1.0 mg/l a-NAPHTHALENEACETIC ACID AND 0.2 mgfl KINETIN N i c o t i n e levels (Media) O,m)
0 5 10 30 100 500
Alkaloid c o n t e n t
Callus dry wt. Burley 21 (rag)
L A Burley 21 (mg)
Burley 21 (%)
L A Burley 21 (%)
166 231 239 160 9-09 185
243 230 214 168 284 195
0.004 0.019 0.018 0.008 0.084 0.215
0.008 0.008 0.004 0.011 0.0"/0 0.150
± ± ± ± ± ±
48 52 49 86 45 37
± ± ± ± ± ±
36 38 54 94 85 49.
± ± ± ± ± ±
0.003 0.002 0.005 0.008 0.022 0.040
+ ± ± ± ± *
0.001 0.001 0.008 0.009 0.020 0.028
319
developed for minimal endogenous nicotine production and accumulation) grows as well and accumulates as much nicotine as the near isogenic Burley 21 callus when both are grown on a m e d i u m containing nicotine (Table I). However, Burley 21 and L o w Alkaloid Burley 21 callus cultured on a m e d i u m designed for o p t i m u m endogenous nicotine production showed t h a t the levels of nicotine in the callus reflected the nicotine levels f o u n d in the leaves of the respective lines. The observations of 'loading' callus tissue with alkaloids suggests t h a t callus would be suitable for metabolic studies of the alkaloids. However, because most of the alkaloids are apparently in the vacuoles and since Robinson Medium Broadleaf callus did n o t demethylate nicotine to nornicotine, it is important to k n o w the pool sizes and equilibria o f alkaloids in a tissue in order to interpret data obtained from metabolic studies appropriately. REFERENCES
1 S. Ohta, O. Matsui and M. Yatazawa, Agric. Biol. Chem., 42 (1978) 1245. 2 D. Neumann and E. Muller, Biochem. Physiol. Pflanzen., 162 (1971) 503. 3 S. Mizusaki, Y. Tanabe, M. Noguchi and E. Tamaki, Plant Cell Physiol., 14 (1973) 103.
4 5 6 7
Technicon Industrial Systems, Ind. Method No. 117--71A (1972). L.P. Bush, J. Chromatogr., 73 (1972) 243. W. Stepka and L.J. Dewey, Plant Physiol., 36 (1961) 592. J.E. Peters, P.H.L. Wu, W.R. Sharp and E.F. Paddock, Physiol. Plant., 31 (1974) 97. 8 S. Ohta, Y. Kojirna and M. Yatazawa, Agric. Biol. Chem., 42 (1978) 1733. 9 A. Nelles and E. Muller, Biochem. Physiol. Pflanzen., 162 (1971) 495. 10 J.A. Saunders, Plant Physiol., 64 (1979) 74.