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Utilization and accumulation of 14C-sucrose in sour cherry shoots rooted in vitro
Scientia Horticulturae, 44 ( 1990 ) 261-267 Elsevier Science Publishers B.V., A m s t e r d a m
261
Utilization and accumulation of 14C-sucrose in sour cherry shoots rooted in vitro Bozena Borkowska and Marek Kubik Institute of Pomology and Floriculture, 96-100 Skierniewice (Poland) (Accepted for publication 8 February 1990)
ABSTRACT Borkowska, B. and Kubik, M., 1990. Utilization and accumulation of ~4C-sucrose in sour cherry shoots rooted in vitro. Scientia Hortic., 44:261-267. Single shoots of sour cherry (Prunus cerasus L. ) cultivar 'Schattenmorelle' were grown on a rooting medium supplemented with 14C-sucrose. Uptake of radioactive sucrose from the medium and its distribution in the stems, leaves and roots during the course of their development were investigated. Radioactivity of shoots was detected as early as 3 days after excision, and both total and specific radioactivity rose continuously throughout the experimental period. After 5 weeks, the radioactivity of the agar medium had decreased to 77% of the original value, but only 5% of this activity was found in the shoots. At the beginning of shoot development, the radioactivity was distributed equally between the stem and leaves. Later, the radioactivity of the leaves was higher than that of the stem. The higher the leaf position, the higher was its radioactivity. At the time of root formation, radioactivity of leaves and stems decreased simultaneously with the appearance of radioactivity in the roots. Keywords: Prunus cerasus; rooting medium; sour cherry; sucrose absorption; tissue culture; in vitro. Abbreviations: IBA-NH4 = ammonium salt ofindole-3-butyric acid; MS = Murashige and Skoog's medium; NCS=N-chlorosuccinimide; POPOP=l,4-bis[2-(5-phenyl)-oxazolyl]benzol; PPO=2,5diphenyloxazole.
INTRODUCTION
Intact plants meet their energy requirements autotrophicaUy. In cultured plant tissue, the normal functions of chloroplasts are reduced. Donnelly et al. (1984) found that cultured plantlets had CO2 uptake five times lower than control plants. The newest growth of the plantlets removed from test tubes achieved 160-200% CO2 uptake of culture-formed leaves. Thus, it is necessary to substitute for those carbohydrates normally produced photosynthetically by others added to the medium (Donnelly et al., 1984; HisaJima et al., 1985; Longford and Wainwright, 1986 ). Sucrose is the usual carbohydrate present in plant tissue culture media and 0304-4238/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
262
B. BORKOWSKA AND M. KUBIK
it is added at concentrations of 2-3%. Growth of cultured tissue depends on the utilization of the sucrose as a carbon and energy source (Maretzki et al., 1974; Borkowska, 1986; Lapera et al., 1988 ). The purpose of the present study was to determine the requirement for sugar, and to examine the uptake of sucrose from the m e d i u m and its translocation/ accumulation in sour cherry shoots cultured in vitro under standard conditions, depending on growth and morphogenesis. MATERIALS AND METHODS
Single shoots of sour cherry (Prunus cerasus L. ) cultivar "Schattenmorelle', 1.5 cm long, were separated from proliferated cultures (Borkowska, 1990) and placed on a rooting m e d i u m containing half-strength MS major salts (Murashige and Skoog, 1962), full-strength microelements and vitamins with 2 mg 1-1 a m m o n i u m salt of indole-3-butyric acid (IBA-NH4). The m e d i u m was solidified with Bacto Difco agar (7.2 g 1-1 ) and the pH adjusted to 5.2 before autoclaving. U-14C-sucrose with specific activity 12 000 MBq m M - 1 (4 MBq in total) was added to 250 ml of the rooting medium. The final concentration of sucrose was 20 g 1-1. Each test tube contained 5 ml of the agar medium. The shoots were cultured at 24°C under 16 h fluorescent light at 4 W m -2. The shoots were analysed after 3 and 5 days, and later after 1, 2, 3, 4, 5 and 8 weeks of cultivation. At each sampling time, nine individual shoots were analysed. At the 5th week, both unrooted and rooted shoots were analysed and, at the 8th week only, rooted plantlets were sampled. The parts of the shoots grown above the agar surface were divided into stem, apex and individual leaves. The roots cut off the shoots were rinsed several times with warm water until t h e radioactivity of the last portion of water was close to background. Each leaf, stem, apex and samples of m e d i u m was weighed separately. After digestion in NCS (200/tl ) plus water ( 100/tl) at 45 °C for 20 h, 100/zl of acetic acid and 10 ml of scintillation cocktail (7 g 2,5-diphenyloxazole ( P P O ) , 100 mg 1,4-bis [ 2- (5-phenyl)-oxazolyl ]benzol ( P O P O P ) in 1 1 of dioxane) were added. The radioactivity was measured in a Beckman LS- 1701 liquid scintillation counter. The radioactivity of samples was calculated per mg of fresh weight (specific activity) or presented as a total. The experiment was repeated twice. The first series was treated as preliminary and only results from the second one (similar to those obtained earlier) are presented in this paper. The results were subjected to statistical analysis and standard deviations are presented in the figures as vertical bars (half of full value).
14C-SUCROSEIN VITRO ROOTED SOURCHERRY SHOOTS
263
RESULTS
Characterization o f shoot development. - During the first week of culturing, no signs of shoot growth were observed. Starting in the second week, the height of the shoots, and the number and weight of leaves increased continuously until the 5th week. The roots were formed between the 4th and 5th week of cultivation (Table 1 ), and ~ 70% of shoots were rooted. Generally, 8 weeks was too long a time for regular development because of the small volume of the medium. Utilization o f sucrose by shoots. - Radioactivity was detected in shoots as early as 3 days after excision. Total and specific radioactivity of the shoots (from the 5th week including the roots) increased throughout the culturing period (Fig. 1 ). After 5 weeks, the radioactivity of the agar medium had decreased to 77% of the original value, showing that ~ 25% of the sucrose was taken up by the cultivated shoots. However, the activity of the shoots represented only 5% of the original activity of the agar medium, measured as total activity per glass tube (Table 2).
D&tribution of radioactivity in cultured shoots.- Starting with the first sampiing date, radioactivity was detected in the shoots, and during the first week it was equally distributed between stem and leaves. Later on, the radioactivity TABLE1 Characteristics of shoot development Days ( d ) / weeks (w) in culture
Shoots
Roots
Height (mm)
Leaves per shoot
Weight (mg fresh weight)
Unrooted shoots 3d 5d 1w 2w 3w 4w 5w
20.8 21.1 22,4 23.7 24.7 26.3 25.4
4.1 4.3 4.2 5.4 6.1 7.3 7.1
10.1 11.4 10.6 12.9 18.1 22.9 25.8
Rooted plantlets 5w 8w
24.0 27.2
7.0 7.4
21.0 33.6
Length (mm)
Number per shoot
Weight (mg fresh weight)
12.8 32.3
2.9 2.6
26.0 33.0
264
B. BORKOWSKAANDM. KUBIK
6O
~ 5000 I
,~0. t..,
~ 3000 20 .~ 100G
I
2
3
~ 5 6 7 weeks of cultivefion
8
Fig. 1. Increase in total and specific radioactivity of the plantlets during the growth period. Vertical bars show half of standard deviations.
TABLE 2 Changes in the radioactivity of the agar medium and the shoots during the growth period Weeks in culture
Agar medium Bq rag- 1 dry weight
Radioactivity (%)
0
619.8 557.4 476.3
100.0 90.3 77.1
3 5
Shoots Bq mg- 1 dry weight
0
252.8 363.8
Percent of initial activity of medium of one tube 0
2.6 5.1
of the leaves continuously increased and became higher than the radioactivity of the stems (Table 3). After 3 days of culture, the radioactivity of the leaves was low, but upper
14C-SUCROSE IN VITRO ROOTED SOUR CHERRY SHOOTS
265
TABLE 3 Distribution of radioactivity in cultured shoots (% of total ) Days ( d ) / weeks (w) in culture
Leaves
Stem
Unrooted shoots 3d 5d 1w 2w 3w 4w 5w
48.6 54.2 51.8 56.8 65.2 69.9 72.8
51.3 45.7 48.2 43.2 34.8 30.1 27.2
Rooted plantlets 5w 8w
31.2 41.0
14.0 19.7
Roots
54.8 39.3
80
"~ ~
ks 60
.0
~ ~o
I week
20 ~
-?-----T
3 days
J, consecutiveleoves
Fig. 2. Specific radioactivity o f i n d i v i d u a l leaves t a k e n f r o m the base ( 1 ) to the apex ( 6 ) a n d m e a s u r e d after 3 days, 1 or 4 weeks o f culture.
266
B. BORKOWSKA AND M. KUBIK
leaves showed a tendency to have a higher level than the lower ones. Later on, the radioactivity of all leaves increased and there was a distinct gradient in their activity: the higher the leaf position, the higher its specific radioactivity (Fig. 2). All remaining sampling dates showed the same characteristics as those presented in Fig. 2. The radioactivity of the stem was lower than that detected in the apex (Fig. 3). Newly formed roots (in the 5th week of cultivation) showed the largest portion of radioactivity, decreasing that in the "green parts" of the plantlets. Later, the leaves partly regained their radioactivity (see Table 3; Fig. 3 ). DISCUSSION
Leaves formed in vitro, as well as persistent leaves (the leaves explanted together with shoot), differ anatomically and physiologically from leaves "in vivo", and they show a low capacity for autotrophic metabolism (Donnelly and Vidaver, 1984; Donnelly et al., 1984). Thus, the growth of tissues cultured in vitro depends primarily on carbohydrate utilization from the medium. As reported by Maretzki et al. (1974), disappearance of exogenous carbohydrates from the medium is rapid. In their cell cultures of Saccharum sp., 50% of the initial sucrose content (2%) remained in the medium after 12 days, but for Rosa sp., all of the carbohydrates in the medium disappeared by the 8th to 12th day. Accumulation of sucrose by sour cherry shoots, expressed as a level of reducing sugars, has also been documented in earlier experiments (Borkowska, 1986 and unpublished data). In the present experiments, ~ 25% of the initial sucrose content was ab-
_~1oo
(.J
.~ so
20
1
2
3
4
5
6
7
8
weeks of cuifivctfion
Fig. 3. Specific radioactivity of stem (open circles) and apex (black circles) unrooted plants (solid lines) and rooted plants (broken lines).
14C-SUCROSE IN VITRO ROOTED SOUR CHERRY SHOOTS
267
sorbed from the m e d i u m by the shoots by the 5th week, but only 5% of this was accumulated as cell components. This suggests that the majority of 14C-sucrose must have been released as 14CO2. Increasing radioactivity of leaves with their position on the shoot could be related to their degree of maturity. U p p e r (young) leaves are actively growing and carbon is used m u c h more as a cell c o m p o n e n t than as a source of energy. "Mature" lower leaves have completed their growth and in their metabolism respiration would be more dominant. As a result, the radioactivity of older tissue was proportionally low. The uptake of sucrose by sour cherry shoots was rapid very early in the incubation period and its upward translocation was shown by the radioactivity detectable in them. A later decrease in the radioactivity of the "green parts" of plantlets at the time of root formation suggests a transmission of radioactive compounds from every leaf to the roots. Thus, the roots could initially be treated as users of carbon from two sources, the m e d i u m and the green shoots. Later, the leaves have recovered the role of a physiological sink, probably because of their higher growth rate than that of roots. ACKNOWLEDGEMENTS
The authors are grateful to Mr. Janusz Szczerba for his assistance. REFERENCES Borkowska, B., 1986. Development and biochemical characteristics of sour cherry tissue cultures, cv. Schattenmorelle. Gartenbauwissenschaft, 51: 14-17. Borkowska, B., 1990. Rate of proliferation and efficiency of rhizogenesis of sour cherry cultures recultured in vitro for different periods of time. Fruit Sci. Rep., 4, in press. Donnelly, D.J. and Vidaver, W.E., 1984. Leaf anatomy of red raspberry transferred from culture to soil. J. Am. Soc. Hortic. Sci., 109: 172-176. Donnelly, D.J., Vidaver, W.E. and Colbow, K., 1984. Fixation of 14CO2 in tissue cultured red raspberry prior to and after transfer to soil. Plant Cell Tissue Organ Cult., 3:313-317. Hisajima, S., Arai, Y. and Thorpe, A., 1985. Sucrose synthesis in callus cultures. Biol. Plant., 27: 74-77. Lapera, L., Perez-Bermudez, P. and Segura, J., 1988. Influence of carbohydrates on morphogenesis in hypocotyl cultures of Digitalis obscura. 6th Congress Federation of European Societies of Plant Physiology, Split, Yugoslavia, pp. 14-21. Longford, P.J. and Wainwright, H., 1986. Photosynthetic ability of in vitro grown rose shoots in relation to media components. VI Congress of Plant Tissue and Cell Cultures, Minneapolis, Minnesota, p. 433. Maretzki, A., Thorn, M. and Nickell, L.G., 1974. Utilization and metabolism of carbohydrates in cell and callus cultures. In: H.E. Street (Editor), Tissue Culture and Plant Science. Academic Press, New York, pp. 329-361. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.
261
Utilization and accumulation of 14C-sucrose in sour cherry shoots rooted in vitro Bozena Borkowska and Marek Kubik Institute of Pomology and Floriculture, 96-100 Skierniewice (Poland) (Accepted for publication 8 February 1990)
ABSTRACT Borkowska, B. and Kubik, M., 1990. Utilization and accumulation of ~4C-sucrose in sour cherry shoots rooted in vitro. Scientia Hortic., 44:261-267. Single shoots of sour cherry (Prunus cerasus L. ) cultivar 'Schattenmorelle' were grown on a rooting medium supplemented with 14C-sucrose. Uptake of radioactive sucrose from the medium and its distribution in the stems, leaves and roots during the course of their development were investigated. Radioactivity of shoots was detected as early as 3 days after excision, and both total and specific radioactivity rose continuously throughout the experimental period. After 5 weeks, the radioactivity of the agar medium had decreased to 77% of the original value, but only 5% of this activity was found in the shoots. At the beginning of shoot development, the radioactivity was distributed equally between the stem and leaves. Later, the radioactivity of the leaves was higher than that of the stem. The higher the leaf position, the higher was its radioactivity. At the time of root formation, radioactivity of leaves and stems decreased simultaneously with the appearance of radioactivity in the roots. Keywords: Prunus cerasus; rooting medium; sour cherry; sucrose absorption; tissue culture; in vitro. Abbreviations: IBA-NH4 = ammonium salt ofindole-3-butyric acid; MS = Murashige and Skoog's medium; NCS=N-chlorosuccinimide; POPOP=l,4-bis[2-(5-phenyl)-oxazolyl]benzol; PPO=2,5diphenyloxazole.
INTRODUCTION
Intact plants meet their energy requirements autotrophicaUy. In cultured plant tissue, the normal functions of chloroplasts are reduced. Donnelly et al. (1984) found that cultured plantlets had CO2 uptake five times lower than control plants. The newest growth of the plantlets removed from test tubes achieved 160-200% CO2 uptake of culture-formed leaves. Thus, it is necessary to substitute for those carbohydrates normally produced photosynthetically by others added to the medium (Donnelly et al., 1984; HisaJima et al., 1985; Longford and Wainwright, 1986 ). Sucrose is the usual carbohydrate present in plant tissue culture media and 0304-4238/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
262
B. BORKOWSKA AND M. KUBIK
it is added at concentrations of 2-3%. Growth of cultured tissue depends on the utilization of the sucrose as a carbon and energy source (Maretzki et al., 1974; Borkowska, 1986; Lapera et al., 1988 ). The purpose of the present study was to determine the requirement for sugar, and to examine the uptake of sucrose from the m e d i u m and its translocation/ accumulation in sour cherry shoots cultured in vitro under standard conditions, depending on growth and morphogenesis. MATERIALS AND METHODS
Single shoots of sour cherry (Prunus cerasus L. ) cultivar "Schattenmorelle', 1.5 cm long, were separated from proliferated cultures (Borkowska, 1990) and placed on a rooting m e d i u m containing half-strength MS major salts (Murashige and Skoog, 1962), full-strength microelements and vitamins with 2 mg 1-1 a m m o n i u m salt of indole-3-butyric acid (IBA-NH4). The m e d i u m was solidified with Bacto Difco agar (7.2 g 1-1 ) and the pH adjusted to 5.2 before autoclaving. U-14C-sucrose with specific activity 12 000 MBq m M - 1 (4 MBq in total) was added to 250 ml of the rooting medium. The final concentration of sucrose was 20 g 1-1. Each test tube contained 5 ml of the agar medium. The shoots were cultured at 24°C under 16 h fluorescent light at 4 W m -2. The shoots were analysed after 3 and 5 days, and later after 1, 2, 3, 4, 5 and 8 weeks of cultivation. At each sampling time, nine individual shoots were analysed. At the 5th week, both unrooted and rooted shoots were analysed and, at the 8th week only, rooted plantlets were sampled. The parts of the shoots grown above the agar surface were divided into stem, apex and individual leaves. The roots cut off the shoots were rinsed several times with warm water until t h e radioactivity of the last portion of water was close to background. Each leaf, stem, apex and samples of m e d i u m was weighed separately. After digestion in NCS (200/tl ) plus water ( 100/tl) at 45 °C for 20 h, 100/zl of acetic acid and 10 ml of scintillation cocktail (7 g 2,5-diphenyloxazole ( P P O ) , 100 mg 1,4-bis [ 2- (5-phenyl)-oxazolyl ]benzol ( P O P O P ) in 1 1 of dioxane) were added. The radioactivity was measured in a Beckman LS- 1701 liquid scintillation counter. The radioactivity of samples was calculated per mg of fresh weight (specific activity) or presented as a total. The experiment was repeated twice. The first series was treated as preliminary and only results from the second one (similar to those obtained earlier) are presented in this paper. The results were subjected to statistical analysis and standard deviations are presented in the figures as vertical bars (half of full value).
14C-SUCROSEIN VITRO ROOTED SOURCHERRY SHOOTS
263
RESULTS
Characterization o f shoot development. - During the first week of culturing, no signs of shoot growth were observed. Starting in the second week, the height of the shoots, and the number and weight of leaves increased continuously until the 5th week. The roots were formed between the 4th and 5th week of cultivation (Table 1 ), and ~ 70% of shoots were rooted. Generally, 8 weeks was too long a time for regular development because of the small volume of the medium. Utilization o f sucrose by shoots. - Radioactivity was detected in shoots as early as 3 days after excision. Total and specific radioactivity of the shoots (from the 5th week including the roots) increased throughout the culturing period (Fig. 1 ). After 5 weeks, the radioactivity of the agar medium had decreased to 77% of the original value, showing that ~ 25% of the sucrose was taken up by the cultivated shoots. However, the activity of the shoots represented only 5% of the original activity of the agar medium, measured as total activity per glass tube (Table 2).
D&tribution of radioactivity in cultured shoots.- Starting with the first sampiing date, radioactivity was detected in the shoots, and during the first week it was equally distributed between stem and leaves. Later on, the radioactivity TABLE1 Characteristics of shoot development Days ( d ) / weeks (w) in culture
Shoots
Roots
Height (mm)
Leaves per shoot
Weight (mg fresh weight)
Unrooted shoots 3d 5d 1w 2w 3w 4w 5w
20.8 21.1 22,4 23.7 24.7 26.3 25.4
4.1 4.3 4.2 5.4 6.1 7.3 7.1
10.1 11.4 10.6 12.9 18.1 22.9 25.8
Rooted plantlets 5w 8w
24.0 27.2
7.0 7.4
21.0 33.6
Length (mm)
Number per shoot
Weight (mg fresh weight)
12.8 32.3
2.9 2.6
26.0 33.0
264
B. BORKOWSKAANDM. KUBIK
6O
~ 5000 I
,~0. t..,
~ 3000 20 .~ 100G
I
2
3
~ 5 6 7 weeks of cultivefion
8
Fig. 1. Increase in total and specific radioactivity of the plantlets during the growth period. Vertical bars show half of standard deviations.
TABLE 2 Changes in the radioactivity of the agar medium and the shoots during the growth period Weeks in culture
Agar medium Bq rag- 1 dry weight
Radioactivity (%)
0
619.8 557.4 476.3
100.0 90.3 77.1
3 5
Shoots Bq mg- 1 dry weight
0
252.8 363.8
Percent of initial activity of medium of one tube 0
2.6 5.1
of the leaves continuously increased and became higher than the radioactivity of the stems (Table 3). After 3 days of culture, the radioactivity of the leaves was low, but upper
14C-SUCROSE IN VITRO ROOTED SOUR CHERRY SHOOTS
265
TABLE 3 Distribution of radioactivity in cultured shoots (% of total ) Days ( d ) / weeks (w) in culture
Leaves
Stem
Unrooted shoots 3d 5d 1w 2w 3w 4w 5w
48.6 54.2 51.8 56.8 65.2 69.9 72.8
51.3 45.7 48.2 43.2 34.8 30.1 27.2
Rooted plantlets 5w 8w
31.2 41.0
14.0 19.7
Roots
54.8 39.3
80
"~ ~
ks 60
.0
~ ~o
I week
20 ~
-?-----T
3 days
J, consecutiveleoves
Fig. 2. Specific radioactivity o f i n d i v i d u a l leaves t a k e n f r o m the base ( 1 ) to the apex ( 6 ) a n d m e a s u r e d after 3 days, 1 or 4 weeks o f culture.
266
B. BORKOWSKA AND M. KUBIK
leaves showed a tendency to have a higher level than the lower ones. Later on, the radioactivity of all leaves increased and there was a distinct gradient in their activity: the higher the leaf position, the higher its specific radioactivity (Fig. 2). All remaining sampling dates showed the same characteristics as those presented in Fig. 2. The radioactivity of the stem was lower than that detected in the apex (Fig. 3). Newly formed roots (in the 5th week of cultivation) showed the largest portion of radioactivity, decreasing that in the "green parts" of the plantlets. Later, the leaves partly regained their radioactivity (see Table 3; Fig. 3 ). DISCUSSION
Leaves formed in vitro, as well as persistent leaves (the leaves explanted together with shoot), differ anatomically and physiologically from leaves "in vivo", and they show a low capacity for autotrophic metabolism (Donnelly and Vidaver, 1984; Donnelly et al., 1984). Thus, the growth of tissues cultured in vitro depends primarily on carbohydrate utilization from the medium. As reported by Maretzki et al. (1974), disappearance of exogenous carbohydrates from the medium is rapid. In their cell cultures of Saccharum sp., 50% of the initial sucrose content (2%) remained in the medium after 12 days, but for Rosa sp., all of the carbohydrates in the medium disappeared by the 8th to 12th day. Accumulation of sucrose by sour cherry shoots, expressed as a level of reducing sugars, has also been documented in earlier experiments (Borkowska, 1986 and unpublished data). In the present experiments, ~ 25% of the initial sucrose content was ab-
_~1oo
(.J
.~ so
20
1
2
3
4
5
6
7
8
weeks of cuifivctfion
Fig. 3. Specific radioactivity of stem (open circles) and apex (black circles) unrooted plants (solid lines) and rooted plants (broken lines).
14C-SUCROSE IN VITRO ROOTED SOUR CHERRY SHOOTS
267
sorbed from the m e d i u m by the shoots by the 5th week, but only 5% of this was accumulated as cell components. This suggests that the majority of 14C-sucrose must have been released as 14CO2. Increasing radioactivity of leaves with their position on the shoot could be related to their degree of maturity. U p p e r (young) leaves are actively growing and carbon is used m u c h more as a cell c o m p o n e n t than as a source of energy. "Mature" lower leaves have completed their growth and in their metabolism respiration would be more dominant. As a result, the radioactivity of older tissue was proportionally low. The uptake of sucrose by sour cherry shoots was rapid very early in the incubation period and its upward translocation was shown by the radioactivity detectable in them. A later decrease in the radioactivity of the "green parts" of plantlets at the time of root formation suggests a transmission of radioactive compounds from every leaf to the roots. Thus, the roots could initially be treated as users of carbon from two sources, the m e d i u m and the green shoots. Later, the leaves have recovered the role of a physiological sink, probably because of their higher growth rate than that of roots. ACKNOWLEDGEMENTS
The authors are grateful to Mr. Janusz Szczerba for his assistance. REFERENCES Borkowska, B., 1986. Development and biochemical characteristics of sour cherry tissue cultures, cv. Schattenmorelle. Gartenbauwissenschaft, 51: 14-17. Borkowska, B., 1990. Rate of proliferation and efficiency of rhizogenesis of sour cherry cultures recultured in vitro for different periods of time. Fruit Sci. Rep., 4, in press. Donnelly, D.J. and Vidaver, W.E., 1984. Leaf anatomy of red raspberry transferred from culture to soil. J. Am. Soc. Hortic. Sci., 109: 172-176. Donnelly, D.J., Vidaver, W.E. and Colbow, K., 1984. Fixation of 14CO2 in tissue cultured red raspberry prior to and after transfer to soil. Plant Cell Tissue Organ Cult., 3:313-317. Hisajima, S., Arai, Y. and Thorpe, A., 1985. Sucrose synthesis in callus cultures. Biol. Plant., 27: 74-77. Lapera, L., Perez-Bermudez, P. and Segura, J., 1988. Influence of carbohydrates on morphogenesis in hypocotyl cultures of Digitalis obscura. 6th Congress Federation of European Societies of Plant Physiology, Split, Yugoslavia, pp. 14-21. Longford, P.J. and Wainwright, H., 1986. Photosynthetic ability of in vitro grown rose shoots in relation to media components. VI Congress of Plant Tissue and Cell Cultures, Minneapolis, Minnesota, p. 433. Maretzki, A., Thorn, M. and Nickell, L.G., 1974. Utilization and metabolism of carbohydrates in cell and callus cultures. In: H.E. Street (Editor), Tissue Culture and Plant Science. Academic Press, New York, pp. 329-361. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.