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Growth and sugar uptake of excised root and callus of tomato
Plant Science Letters, 21 (1981) 229-234 © Elsevier/North-Holland Scientific Publishers Ltd.
229
GROWTH A N D S U G A R UPTAKE O F EXCISED ROOT AND CALLUS
O F TOMATO
CHEE-KOK
CHIN, J O A O C. H A A S a and C E C I L C. STILL a
Department of Horticulture and Forestry and aDepartment of Microbiology and Biochemistry, Rutgers -- The State University, New Brunswick, NJ 08903 (U.S.A.)
(Received August 13th, 1980) (Revision received August 12th, 1980) (Accepted August 12th, 1980 )
SUMMARY
Excised r o o t and callus o f t o m a t o ( L y c o p e r s i c o n e s c u l e n t u m cv. Rutgers) differed in their requirements for carbon. While roots strongly preferred sucrose over glucose and fructose, callus grew well in sucrose and glucose and only slightly less well in fructose. R o o t s cultured in sucrose were found to have higher levels o f sucrose and reducing sugars than those cultured in glucose and fructose. In contrast, the levels o f sucrose and reducing sugars o f callus cultures maintained i n sucrose, glucose and fructose did not differ as much. Glucose and fructose were taken up at lower rates than sucrose b y callus and at much lower rates b y excised t o m a t o roots. It appears that inability o f excised t o m a t o roots to take up glucose and fructose efficiently was t h e cause of the failure o f these t w o sugars to support growth o f excised t o m a t o roots.
INTRODUC~ON It has been k n o w n that excised t o m a t o roots have a unique carbon source requirement; t h e y grow well on media with sucrose as a carbon source, b u t n o t with any o f a wide range o f other sugars including glucose and fructose [1,2]. While the inability o f some sugars to support growth of excised t o m a t o roots can be traced t o their failure to be metabolized, the ineffectiveness o f glucose and fructose is n o t due t o their inability to serve as substrates for respiration and other metabolic activities [3]. Recently we observed that in contrast to excised roots, callus cultures o f t o m a t o did well o n b o t h sucrose and glucose and only slightly less well Abbreviations: BA, benzyladenine; NAA, a-naphthalene acetic acid.
280
on fructose. This paper describes an attempt to examine the reasons for the different responses o f excised root and callus of tomato to carbon sources by comparing: (a) the ability o f the cultures to convert glucose and fructose to sucrose and (b) the efficiency of root and callus in taking up sucrose, glucose and fructose. MATERIALS AND METHODS
Root culture. A clone of excised tomato roots was established from a single seed of Lycopersicon esculentum cv. Rutgem, using the techniques of Street and Lowe [4] and was maintained in modified White's medium [ 5 ] in darkness. Callus culture. Stem tissue from a greenhouse-grown tomato plant was sterilized with 0.5% sodium hypochlorite for 15 min and rinsed 5 times in sterile water. Slices of 1 mm thickness were then cut and cultured in modified White's medium [5] containing 1 ~M benzyladenine (BA) and 5 ~M a-naphthalene acetic acid (NAA) solidified with 0.75% agar. The culture was kept at 25°C in darkness. Sugar extraction and determination. Sugars were extracted as reported elsewhere [6]. Reducing sugars were determined by the method of Somogyi [7]. Sucrose was determined by hydrolyzing with yeast invertase (Sigma) followed by measurement of the increase in reducing sugars [6]. Uptake of sugars. One-cm-long tips from 7
RESULTS Growth of root and calluson different sugars. Table I shows that roots of tomato cultivar Rutgem behaved like root of other cuitivars [1,3]; they grow 5--10 times better in sucrose than in glucose and fructose. In contrast,the calluscultures did not exhibit such drastic differences in growth in sucrose, glucose and fructose (Table II). Levels of sugars in root and callus. Irrespective of whether the roots were cultured in sucrose, glucose or fructose, they all contained sucrose
231 TABLE I GROWTH AND S U G A R LEVELS OF TOMATO ROOTS C U L T U R E D IN SUCROSE, GLUCOSE AND F R U C T O S E M E D I A s Sugars
Final fresh wt. mg/20 roots
Sucrose rng/g fresh wt.
Reducing sugars mg/g fresh wt.
1% Sucrose 2% Sucrose 3% Sucrose
320 b b 645 a 612 a
8.22 b 18.24 a 19.12 a
10.48 c 17.22 b 21.12 a
1% Glucose 2% Glucose 3% Glucose
62 c 67 c 68 c
2.11 c 3.74 c 4.22 c
3.60 d 6.42 d 6.71 d
1% Fructose 2% Fructose 3% Fructose
52 d 50 d 42 d
1.96 c 3.78 c 3.12 c
2.91 e 5.12 d 8.11 cd
aseven -day-old roots were used for fresh wt. and sugar determinations. bMean (4 replications) separation within columns by Duncan's multiple range test, 5% level. Values having different letters are significantly different from each other.
(Table I). Roots grown in sucrose, however, possessed m u c h more sucrose. In addition, sucrose-grown roots had more reducing sugars t h a n glucoseor fructose-grown roots. Sucrose was also found in callus cultured in different sugars (Table II). TABLE II GROWTH AND S U G A R LEVELS OF TOMATO CALLI C U L T U R E D IN SUCROSE, GLUCOSE AND F R U C T O S E ~ I E D I A a Sugars
Increase in fresh wt. mg/callus
Sucrose mg/g fresh wt.
Reducing sugars mg/g fresh wt.
1% Sucrose 2% Sucrose 3% Sucrose
318 d b 387 b 402 a
18.35 c 19.68 c 36.98 a
23.25 bc 27.76 b 36.53 a
1% Glucose 2% Glucose 3% Glucose
277 e 412 a 388 b
17.58 e 27.41 b 30.17 b
18.78 cd 27.22 b 34.91 a
1% Fructose 2% Fructose 3% Fructose
271 e 352 c 310 d
16.95 c 21.14 c 27.12 b
16.70 d 23.08 bc 28.37 b
astern segments were weighed and cultured for 28 days. Increase in fresh wt. was obtained by subtracting t h e initial wt. from the final wt. Means were obtained from 4 bexperiments each with ~tO cultures. Mean (4 replications) separation within columns by Duncan's multiple range test, 5% level. Values having different letters are significantly different from each other.
232 ,?
b--25
~t~ lC :3
o
root
S 1
2
3
callus
1
Concentrations
2
3
%
Fig. 1. Uptake of sucrose (o), glucose (.) and fructose (~) by root and callus of tomato. Data points represent mean values of 4 experiments.
Compared with roots, callus possessed m u c h higher levelsof both sucrose and reducing sugars. The levels of sucrose and reducing sugars in calli cultured in different sugars did not differ as greatly as those in roots (Tables I and II).
Uptake of sugars. Rates of uptake of sugars by excised tomato roots varied; sucrose was taken up quickly and glucose very slowly (Fig. la). Segments of callus also took up sucrose preferentially. However, the differences in rate of uptake of sucrose, glucose and fructose were not as great (Fig. lb). The presence of B A and N A A was found to have no appreciable I
I
|
I
!
~A
I£
.5
I
~
F
G
NAA ÷ IA
~]j0
TIME, rain.
Fig. 2. Effect of NAA (5 ~M) and BA (1 pM) on the uptake of giueome (c) and fructose (o) by exci~d tomato rook. Deta points reprewnt mean valuta of 2 expm4ments.
233 effect on the uptake of glucose and fructose by excised tomato roots (Fig. 2).
DISCUSSION The results of this study show that excised root and callus culture o f tomato differed in their requirement for sucrose as a carbon source. While roots grew approx. 10 times better in sucrose than in glucose and fructose, such differences were n o t observed with callus cultures. Sucrose has been found in several instances to have an effect on morphogenesis and metabolism [8,9]; Street [10] expressed the view that tomato roots require a critical level of sucrose in their meristems and that this level is established and maintained only b y the provision of exogenous sucrose. This presupposes that the roots are unable to synthesize sucrose and also that sucrose is absorbed without degradation. This appears to be supported b y the observation o f Chin and Weston [11] that assymetrically labeled sucrose entered excised tomato roots without alteration o f its labeling pattern. The inability of roots cultured in different sugars to synthesize sucrose was, however, not supported b y the observation that sucrose was present in roots cultured in glucose and fructose. Nevertheless, it was found that roots cultured in sucrose had much higher concentrations of sucrose thanroots cultured in glucose or fructose. In fact, the tissues that grew well, i.e., the roots cultured in sucrose and callus cultured on different sugars, not only had higher levels of endogenous sucrose but also of reducing sugars compared with roots cultured in glucose or fructose. This raises the possibility that the low growth rate of roots cultured in glucose and fructose was the result o f carbohydrate deficiency. Street and McGregor [12] found that roots cultured in glucose and fructose resembled those grown on limiting levels of sucrose (0.5% or lower) in their morphology, low growth rate, simple anatomy and low percentage of dry matter. Thus, the data presented here and those o f Street and McGregor both indicate that roots cultured in fructose and glucose are carbohydrate deficient. In contrast to roots, calli cultured in glucose and fructose did not possess greatly lowered levels of sucrose and reducing sugars compared with those cultured in sucrose. In fact, the sugar levels of calli cultured in glucose and fructose were much higher than those of roots cultured in the same sugars. Thus, it appears that calli cultured in glucose and fructose did not suffer a deficiency o f sugars as roots did and consequently they did not display reduced growth rates. The sugar uptake experiments show that sucrose was taken up by callus at a lower rate than it permeated excised tomato roots. Although callus t o o k up glucose and fructose at lower rates than sucrose the differences are smaller than in roots. The media for roots and callus cultures were identical except that the latter also contained BA, NAA and agar. The possibility that the presence o f growth regulators might facilitate the uptake
234 o f glucose arid fructose was examined and f o u n d n o t to be supported b y experimental evidence. Thus, t h e difference in ability t o take up glucose and fructose by r o o t and callus was probably n o t caused by the presence of BA and NAA. It is k n o w n t h a t t h e physiology o f cells o f different differentiation states may differ. Therefore, perhaps it is n o t surprising to find t h a t the callus and excised root of t o m a t o differed in their ability to absorb different sugars. It is speculated t h a t t h e very low efficiency o f excised roots to absorb glucose and fructose led to t h e carbohydrate deficiency and lack o f growth o f roots in these sugars. On the o t h e r hand because callus cultures could absorb glucose and fructose relatively efficiently t h e y grew well on these two sugars. ACKNOWLEDGEMENTS This work was performed as a part o f NJAES project No. 12403. Supported b y the New Jersey Agricultural Experiment Station. REFERENCES 1 2 3 4 5 6 7 8 9 10
P.R. White, Plant Physiol., 15 (1940) 355. J.D. Ferguson, H.E. Street and S.B. David, Ann. Bot., 22 (1958) 513. D.R. Morgan and H.E. Street, Ann. Bot., 23 (1959) 89. H.E. Street and J.S. Lowe, Ann. Bot., 14 (1950) 307. D.E.G. Sheat, B.H. Fletcher and H.E. Street,New Phytol., 58 (1959) 128. C. Chin and J.N. Sacalis, J. Am. Soc. Hort. Sci., 102 (1977) 541. M. Somogyi, J. Biol. Chem., 195 (1952) 19. R.H, Wetmore and J.P. Rier, Am. J. Bot., 50 (1963) 418. J. Edelman and A.D~ Hanson, J. Exp. Bot., 23 (1972) 469. H.E. Street, Growth in organized and unorganized systems, in: F.C. Steward (Ed.), Plant Physiology, Vol. 5B, Academic Press, New York, 1969, P. 11 C. Chin and G.D. Weston, Phytochemistry, 14 (1975) 69. 12 H.E. Street and S.M. McGregor, Ann. Bot., 16 (1952) 185.
229
GROWTH A N D S U G A R UPTAKE O F EXCISED ROOT AND CALLUS
O F TOMATO
CHEE-KOK
CHIN, J O A O C. H A A S a and C E C I L C. STILL a
Department of Horticulture and Forestry and aDepartment of Microbiology and Biochemistry, Rutgers -- The State University, New Brunswick, NJ 08903 (U.S.A.)
(Received August 13th, 1980) (Revision received August 12th, 1980) (Accepted August 12th, 1980 )
SUMMARY
Excised r o o t and callus o f t o m a t o ( L y c o p e r s i c o n e s c u l e n t u m cv. Rutgers) differed in their requirements for carbon. While roots strongly preferred sucrose over glucose and fructose, callus grew well in sucrose and glucose and only slightly less well in fructose. R o o t s cultured in sucrose were found to have higher levels o f sucrose and reducing sugars than those cultured in glucose and fructose. In contrast, the levels o f sucrose and reducing sugars o f callus cultures maintained i n sucrose, glucose and fructose did not differ as much. Glucose and fructose were taken up at lower rates than sucrose b y callus and at much lower rates b y excised t o m a t o roots. It appears that inability o f excised t o m a t o roots to take up glucose and fructose efficiently was t h e cause of the failure o f these t w o sugars to support growth o f excised t o m a t o roots.
INTRODUC~ON It has been k n o w n that excised t o m a t o roots have a unique carbon source requirement; t h e y grow well on media with sucrose as a carbon source, b u t n o t with any o f a wide range o f other sugars including glucose and fructose [1,2]. While the inability o f some sugars to support growth of excised t o m a t o roots can be traced t o their failure to be metabolized, the ineffectiveness o f glucose and fructose is n o t due t o their inability to serve as substrates for respiration and other metabolic activities [3]. Recently we observed that in contrast to excised roots, callus cultures o f t o m a t o did well o n b o t h sucrose and glucose and only slightly less well Abbreviations: BA, benzyladenine; NAA, a-naphthalene acetic acid.
280
on fructose. This paper describes an attempt to examine the reasons for the different responses o f excised root and callus of tomato to carbon sources by comparing: (a) the ability o f the cultures to convert glucose and fructose to sucrose and (b) the efficiency of root and callus in taking up sucrose, glucose and fructose. MATERIALS AND METHODS
Root culture. A clone of excised tomato roots was established from a single seed of Lycopersicon esculentum cv. Rutgem, using the techniques of Street and Lowe [4] and was maintained in modified White's medium [ 5 ] in darkness. Callus culture. Stem tissue from a greenhouse-grown tomato plant was sterilized with 0.5% sodium hypochlorite for 15 min and rinsed 5 times in sterile water. Slices of 1 mm thickness were then cut and cultured in modified White's medium [5] containing 1 ~M benzyladenine (BA) and 5 ~M a-naphthalene acetic acid (NAA) solidified with 0.75% agar. The culture was kept at 25°C in darkness. Sugar extraction and determination. Sugars were extracted as reported elsewhere [6]. Reducing sugars were determined by the method of Somogyi [7]. Sucrose was determined by hydrolyzing with yeast invertase (Sigma) followed by measurement of the increase in reducing sugars [6]. Uptake of sugars. One-cm-long tips from 7
RESULTS Growth of root and calluson different sugars. Table I shows that roots of tomato cultivar Rutgem behaved like root of other cuitivars [1,3]; they grow 5--10 times better in sucrose than in glucose and fructose. In contrast,the calluscultures did not exhibit such drastic differences in growth in sucrose, glucose and fructose (Table II). Levels of sugars in root and callus. Irrespective of whether the roots were cultured in sucrose, glucose or fructose, they all contained sucrose
231 TABLE I GROWTH AND S U G A R LEVELS OF TOMATO ROOTS C U L T U R E D IN SUCROSE, GLUCOSE AND F R U C T O S E M E D I A s Sugars
Final fresh wt. mg/20 roots
Sucrose rng/g fresh wt.
Reducing sugars mg/g fresh wt.
1% Sucrose 2% Sucrose 3% Sucrose
320 b b 645 a 612 a
8.22 b 18.24 a 19.12 a
10.48 c 17.22 b 21.12 a
1% Glucose 2% Glucose 3% Glucose
62 c 67 c 68 c
2.11 c 3.74 c 4.22 c
3.60 d 6.42 d 6.71 d
1% Fructose 2% Fructose 3% Fructose
52 d 50 d 42 d
1.96 c 3.78 c 3.12 c
2.91 e 5.12 d 8.11 cd
aseven -day-old roots were used for fresh wt. and sugar determinations. bMean (4 replications) separation within columns by Duncan's multiple range test, 5% level. Values having different letters are significantly different from each other.
(Table I). Roots grown in sucrose, however, possessed m u c h more sucrose. In addition, sucrose-grown roots had more reducing sugars t h a n glucoseor fructose-grown roots. Sucrose was also found in callus cultured in different sugars (Table II). TABLE II GROWTH AND S U G A R LEVELS OF TOMATO CALLI C U L T U R E D IN SUCROSE, GLUCOSE AND F R U C T O S E ~ I E D I A a Sugars
Increase in fresh wt. mg/callus
Sucrose mg/g fresh wt.
Reducing sugars mg/g fresh wt.
1% Sucrose 2% Sucrose 3% Sucrose
318 d b 387 b 402 a
18.35 c 19.68 c 36.98 a
23.25 bc 27.76 b 36.53 a
1% Glucose 2% Glucose 3% Glucose
277 e 412 a 388 b
17.58 e 27.41 b 30.17 b
18.78 cd 27.22 b 34.91 a
1% Fructose 2% Fructose 3% Fructose
271 e 352 c 310 d
16.95 c 21.14 c 27.12 b
16.70 d 23.08 bc 28.37 b
astern segments were weighed and cultured for 28 days. Increase in fresh wt. was obtained by subtracting t h e initial wt. from the final wt. Means were obtained from 4 bexperiments each with ~tO cultures. Mean (4 replications) separation within columns by Duncan's multiple range test, 5% level. Values having different letters are significantly different from each other.
232 ,?
b--25
~t~ lC :3
o
root
S 1
2
3
callus
1
Concentrations
2
3
%
Fig. 1. Uptake of sucrose (o), glucose (.) and fructose (~) by root and callus of tomato. Data points represent mean values of 4 experiments.
Compared with roots, callus possessed m u c h higher levelsof both sucrose and reducing sugars. The levels of sucrose and reducing sugars in calli cultured in different sugars did not differ as greatly as those in roots (Tables I and II).
Uptake of sugars. Rates of uptake of sugars by excised tomato roots varied; sucrose was taken up quickly and glucose very slowly (Fig. la). Segments of callus also took up sucrose preferentially. However, the differences in rate of uptake of sucrose, glucose and fructose were not as great (Fig. lb). The presence of B A and N A A was found to have no appreciable I
I
|
I
!
~A
I£
.5
I
~
F
G
NAA ÷ IA
~]j0
TIME, rain.
Fig. 2. Effect of NAA (5 ~M) and BA (1 pM) on the uptake of giueome (c) and fructose (o) by exci~d tomato rook. Deta points reprewnt mean valuta of 2 expm4ments.
233 effect on the uptake of glucose and fructose by excised tomato roots (Fig. 2).
DISCUSSION The results of this study show that excised root and callus culture o f tomato differed in their requirement for sucrose as a carbon source. While roots grew approx. 10 times better in sucrose than in glucose and fructose, such differences were n o t observed with callus cultures. Sucrose has been found in several instances to have an effect on morphogenesis and metabolism [8,9]; Street [10] expressed the view that tomato roots require a critical level of sucrose in their meristems and that this level is established and maintained only b y the provision of exogenous sucrose. This presupposes that the roots are unable to synthesize sucrose and also that sucrose is absorbed without degradation. This appears to be supported b y the observation o f Chin and Weston [11] that assymetrically labeled sucrose entered excised tomato roots without alteration o f its labeling pattern. The inability of roots cultured in different sugars to synthesize sucrose was, however, not supported b y the observation that sucrose was present in roots cultured in glucose and fructose. Nevertheless, it was found that roots cultured in sucrose had much higher concentrations of sucrose thanroots cultured in glucose or fructose. In fact, the tissues that grew well, i.e., the roots cultured in sucrose and callus cultured on different sugars, not only had higher levels of endogenous sucrose but also of reducing sugars compared with roots cultured in glucose or fructose. This raises the possibility that the low growth rate of roots cultured in glucose and fructose was the result o f carbohydrate deficiency. Street and McGregor [12] found that roots cultured in glucose and fructose resembled those grown on limiting levels of sucrose (0.5% or lower) in their morphology, low growth rate, simple anatomy and low percentage of dry matter. Thus, the data presented here and those o f Street and McGregor both indicate that roots cultured in fructose and glucose are carbohydrate deficient. In contrast to roots, calli cultured in glucose and fructose did not possess greatly lowered levels of sucrose and reducing sugars compared with those cultured in sucrose. In fact, the sugar levels of calli cultured in glucose and fructose were much higher than those of roots cultured in the same sugars. Thus, it appears that calli cultured in glucose and fructose did not suffer a deficiency o f sugars as roots did and consequently they did not display reduced growth rates. The sugar uptake experiments show that sucrose was taken up by callus at a lower rate than it permeated excised tomato roots. Although callus t o o k up glucose and fructose at lower rates than sucrose the differences are smaller than in roots. The media for roots and callus cultures were identical except that the latter also contained BA, NAA and agar. The possibility that the presence o f growth regulators might facilitate the uptake
234 o f glucose arid fructose was examined and f o u n d n o t to be supported b y experimental evidence. Thus, t h e difference in ability t o take up glucose and fructose by r o o t and callus was probably n o t caused by the presence of BA and NAA. It is k n o w n t h a t t h e physiology o f cells o f different differentiation states may differ. Therefore, perhaps it is n o t surprising to find t h a t the callus and excised root of t o m a t o differed in their ability to absorb different sugars. It is speculated t h a t t h e very low efficiency o f excised roots to absorb glucose and fructose led to t h e carbohydrate deficiency and lack o f growth o f roots in these sugars. On the o t h e r hand because callus cultures could absorb glucose and fructose relatively efficiently t h e y grew well on these two sugars. ACKNOWLEDGEMENTS This work was performed as a part o f NJAES project No. 12403. Supported b y the New Jersey Agricultural Experiment Station. REFERENCES 1 2 3 4 5 6 7 8 9 10
P.R. White, Plant Physiol., 15 (1940) 355. J.D. Ferguson, H.E. Street and S.B. David, Ann. Bot., 22 (1958) 513. D.R. Morgan and H.E. Street, Ann. Bot., 23 (1959) 89. H.E. Street and J.S. Lowe, Ann. Bot., 14 (1950) 307. D.E.G. Sheat, B.H. Fletcher and H.E. Street,New Phytol., 58 (1959) 128. C. Chin and J.N. Sacalis, J. Am. Soc. Hort. Sci., 102 (1977) 541. M. Somogyi, J. Biol. Chem., 195 (1952) 19. R.H, Wetmore and J.P. Rier, Am. J. Bot., 50 (1963) 418. J. Edelman and A.D~ Hanson, J. Exp. Bot., 23 (1972) 469. H.E. Street, Growth in organized and unorganized systems, in: F.C. Steward (Ed.), Plant Physiology, Vol. 5B, Academic Press, New York, 1969, P. 11 C. Chin and G.D. Weston, Phytochemistry, 14 (1975) 69. 12 H.E. Street and S.M. McGregor, Ann. Bot., 16 (1952) 185.