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Continuous propagation of tomato plants by means of callus cultures
Scientia Horticulturae, 4 (1976) 221--227
221
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CONTINUOUS PROPAGATION OF TOMATO PLANTS BY MEANS OF CALLUS CULTURES
E. DE LANGHE and E. DE BRUIJNE
Laboratory of Tropical Crop Husbandry and Physiology, Faculty of Agronomy, State University of Ghent, Coupure, 533, 9000 Ghent (Belgium) (First received 6 December 1974; in final form 12 May 1975)
ABSTRACT De Langhe, E. and de Bruijne, E., 1976. Continuous propagation of tomato plants by means of callus cultures. Scientia Hort., 4: 221--227. Callus obtained in vitro from stem internode tissue was used to investigate the possibilities for accelerated vegetative propagation of Lycopersicum esculentum Mill. and Lycopersicum peruvianum (L). The results of different tests with phytohormones pointed to a high endogenous auxin level in the original stem explants of L. peruvianum. In L. esculentum shoots were obtained when high amounts of zeatin or coconut-milk were applied. Explants of CCC-pretreated (2 chloroethyl trimethyl-ammonium chloride} tomato plantlets showed a significant increase in shoot formation as compared to the untreated ones.
Callus tissue that was more than 2 years old and had been used in 30 transfers still had the capacity to produce normal shoots and roots. More than 200 resulting plants were observed in glasshouse conditions for possible genetic variations. No striking deviations from the original phenotype occurred. Seeds harvested from the fruits of self-fertilized flowers on these plants were sown under normal growth conditions. Some plants of one particular cultivar showed signs of accentuated vegetative development.
INTRODUCTION
Rapid vegetative multiplication is a boon to the plant breeder. It appears, however, that instability and (particularly) the variation in ploidy which was observed in a number of tissues (Reinert, 1973), are serious liabilities in the use of this technique. Fukami and Mackinney (1967), as well as Warren and Rontley (1970), worked with tomato callus to investigate specific processes. They pointed to the importance of trials to obtain whole plants; their metabolism being different in some respects from that of the callus. The present study aimed firstly at accomplishing the rapid vegetative multiplication of tomato plants by means of in vitro culture and, secondly, at determining any deviations from the original morphological pattern.
222
MATERIALS AND METHODS
Seedlings of Lycopersicum esculentum Mill. of the Mediterranean "oblong" cultivars 'Canatella', 'Roma' and 'Ventura', of 'King Plus', a round deep red tomato of moderate climates, and of Lycopersicum peruvianum (L) were grown in the following greenhouse conditions: a temperature of 23°C by day and 20°C by night; a 14-hour photoperiod with additional light from Philips HPLR 700-watt lamps; a relative humidity of about 60--70 %. Defoliated stem pieces were put in denaturated alcohol for one minute, immersed in sodium hypochlorite (8 %) for 25 min, and rinsed in sterile water; finally the internode was cut into 4-mm pieces. These pieces were inoculated upside down into the medium of Linsmaier and Skoog (1965) (called L.S. in this text) with or without the addition of coconut-milk (CCM) at 20 %, and (or) supplemented with indoleacetic acid (IAA), indolebutyric acid (IBA), 6-furfurylamino purine (KIN) and 6(7,7-dimethylallylamino) purine (DMMA). The carbon source was sucrose at 2 %. The pH of the media was adjusted to 5.6 and the media were gelled with 0.6 % agar. Each treatment covered at least one basket of 24 pieces. The cultures were maintained at a temperature of 25°C, 70 % relative humidity and constant light of about 2000 lux (Philips TL 40 W/32). The resulting plantlets were transferred into jiffy pots filled with peat, and observed in the greenhouse. Seeds from self-fertilized flowers on these plants were sown in normal growth conditions in Tunisia (Station Exp~rimentale de Manouba) and in Belgium (B.V.O.-station, Sint Kathelijne-Waver).
Fig.1. Cross-section of shoot formation originating in the subepidermal layers of the explant
223 RESULTS T h r o u g h o u t the various treatments, callus and shoot f o r m a t i o n occurred in almost the same way, callus originating f r om sub-epidermal tissue of the explant (Fig.l). The new meristem s t a r t e d f r om the peripheral layers o f this callus. Influence o f hormones in the culture medium. -- Explants o f L. peruvianum clearly r es p o n d ed with organogenesis in the presence o f auxins and cytokinins (Table I). The ratio o f t he concentrations o f auxins and cytokinins determined the differentiation as already described in t he literature (Gautheret, 1966): increase o f KIN and DMAA in regard t o the same level of auxins stimulated the initiation of s hoot f o r m a t i o n ; higher auxin c o n t e n t enhanced r o o t formation. In t r e a t m e n t No. 2 the early r o o t f o r m a t i o n was competitive with callus growth. T r e a t m e n t No. 4 is considered t o have p r o d u c e d the best response since enough callus was p r o d u c e d for subculturing as well as for y o u n g shoots. TABLE I Influence of auxins and cytokinins on root and shoot formation in explants of L. peruvianum, expressed as the number of tubes containing tissue with neo-formation in proportion to the total number of tubes per treatment. Treatment
Medium
Root formation
Shoot formation
1 2 3
L.S. L.S. + IBA 10-6M, IAA 10-~M L.S. + IBA 10-~M, IAA 10-6M KIN 10-6M L.S. + IBA 10-6M, IAA 10-6M KIN 10-6M, DMAA 10-~M L.S. + IBA 10-6M, IAA 10-6M KIN 10-SM, DMAA 10-SM
0.00 0.60 0.25
0.10 0.40 0.50
0.00
0.75
0.00
0.60
4 5
R o o t f o r m a t i o n was obtained by transferring t he developed shoots into the L.S. m e d i u m r ed u ced t o half its original concent rat i on, w i t h o u t h o r m o n e s and with a pH o f 5.2 (Fig.2). Well-shaped plantlets arose which could easily be transplanted in normal greenhouse conditions. Trials with an undiluted m e d i u m and addition o f auxins, such as IBA 10-SM or 10-6M, resulted in r o o t f o r m a t i o n b u t the plantlets failed t o grow f u r t h e r in the greenhouse. The results obtained with L. peruvianum could n o t be r e p r o d u c e d with any o f the L. esculentum cultivars. Addition in an L.S. m e d i u m of CCM at a conc e n t r atio n o f 20 % and (or) 6-(4-hydroxy-3-methyl-2-butenylamino) purine (zeatin) 10-SM was tried on L. esculentum 'Canatella' and 'King Plus' with the following results: (a) n o r o o t f o r m a t i o n occurred; (b) s h o o t f o r m a t i o n appeared in less t han 40 % of t he 'Canatella' pieces even with zeatin alone and s h o o t - f r e q u e n c y in 'King Plus' was a b o u t 20 %;
224
Fig.2. Young plantlets growing on a reduced basic medium without hormones and with pH 5.2 to induce root formation. (c) several shoots were observed on the same explant o f 'Canatella'; (d) a good p r o d u c t i o n of shoot-forming callus was seen in all the treatments; (e) the presence o f sucrose in the media with 20 % CCM inhibited s h o o t formation. It was co n clu d e d t h a t the multiplication rate for the different cultivars of L. esculentum was 2--3 times lower than for L. peruvianum. Addition o f gibberellic acid (GA3) 10-~M or 10-SM t o t he L.S. m edi um caused a high p r o d u c t i o n of callus. The effect of abscisic acid (ABA) was confusing: in some tr eat m e nt s a real stimulation of shoot f o r m a t i o n could be observed, while in others ABA lowered the shoot-forming capacity of explant and callus.
Stimulation o f shoot formation in the callus by pretreatment o f the tomato plants with chlormequat (CCC). -- The above results suggested that, while
225 t o m a t o stem tissues of L. peruvianum resemble tobacco pith tissues in their effect upon the auxin/cytokinin balance (Skoog and Miller, 1957), the system in L. esculentum might be masked by the presence of too much endogenous GA. Incorporation of CCC (1000 p.p.m.) in the L.S. medium had no effect on the growth of the explants, but pretreatment with CCC on the plants in the greenhouse stimulated the shoot-forming ability of their explants (Table II). The plants were pretreated as follows: an aqueous solution of CCC (2000 p.p.m.) was sprayed daffy upon y o u n g t o m a t o plants 'Canatella' and 'King Plus'. The new leaves were smaller and dark green, while the length of petioles and new internodes was reduced. These morphological s y m p t o m s are similar to those described by Klapwijk (1966), Will (1966) and Abdalla and Verkerk (1968). The callus was kept alive, w i t h o u t notable shoot formation, by transfer every 3 weeks into an L.S. medium with Kin 10-6M. Shoot formation could be stimulated at any time by transplanting the callus into a medium with Z 10-6M or CCM at 20 % (Fig.3). So far, the callus has maintained an undiminished shoot-forming capacity for as m a n y as 30 transplantations. Induction of root formation t o o k place in the same way as previously described for L. peruvianum. TABLE II Influence of pretreatment of tomato plants with chlormequat on the shoot-forming ability of the explant-callus, expressed as total number of shoots/total number of explants. Experiment number 1 2 3
Daysbetween pre-treatment and inoculation 2 5 8
'Canatella'
'King Plus'
Control
CCC
Control
CCC
0.43 0.10 0.54
1.65 0.43 1.14
-0.21 0.15
-0.29 0.64
It may be calculated t h a t one plant can produce more than 5 million plantlets after one year of subculturing, as follows: one branch of a t o m a t o plant easily gives 8 explants; out of each explant 6 plantlets will arise; one explant yields enough callus for 2 new tubes; this callus can be transferred every 3 weeks and will in turn produce 6 plantlets and 2 new tubes with callus. Genetic stability. -- Two hundred and thirty-four plants from in vitro material were observed in the greenhouse. They belonged to the cultivars 'King Plus', 'Canatella', 'Ventura', ' R o m a ' , and L. peruvianum, and numbered 25, 165, 29, 12 and 3 plants respectively. No phenotypical variation could be detected. Norton and Boll (1954) could n o t induce fruit-setting on L. peruvianum plants obtained in vitro. In our experiments the 3 L. peruvianum plants had their origin in tissue of different seedlings, and normal fruits were produced.
226
Fig.3. Shoot-forming callus after 20 transplantations on an L.S. medium with CCM 20 %.
About 30 L. esculentum plants were self-fertilized. In normal growth conditions the seeds developed into identical plants, except for 'King Plus' where 3 plants showed a common kind of deviation with leaf-curling, thick, dark green leaves, delayed fruiting and partial vegetative outgrowth of the inflorescences. These symptoms are probably linked to the same phenomenon: a strong vegetative growth. The possibility of polyploidisation is now being studied. CONCLUSION
The conditions governing reproducible in vitro tissue multiplication and organogenesis in various tomato cultivars have been established. L. peruvianum reacts in a well known way to the auxin/cytokinin balance of organogenesis. Results obtained with CCC-pretreatment point to the possibility that (at least in tissue of L. esculentum) this balance may be obscured
227
by a variable presence of sometimes high amounts of GA in the original tissue. Visual observation of the numerous t o m a t o plants obtained has led to the conclusion that the genetic stability of the tissue was very high. Yet the occurrence of activated vegetative growth in some segregated seedlings of the original clones, calls for further study. ACKN OWLEDGEMENTS
This study was made possible b y a grant of the A.G.C.D. (Administration Gdndrale de la Coopdration au Ddveloppement) in the framework of a BelgoTunisian project for agricultural research. Thanks are due to the research teams of the experimental stations of Manouba and of Sint Kathelijne-Waver for their careful culture and observations. We are much indebted to Mrs. Thea Versluys and Mr. Johan Geirnaert for their valuable help in the culturing of numerous tissues.
REFERENCES Abdalla, A. and Verkerk, K., 1968. Growth, flowering and fruitset of the t o m a t o at high temperature. Neth. J. Agr. Sci., 16: 71--76. Fukami, T. and Mackinney, G., 1967. Culture of t o m a t o callus tissue. Nature (London), 273: 944--945. Gautheret, R.J., 1966. Factors affecting differentiation of plant tissues grown in vitro. In: W. Beermann, C.W. Wardlaw, R.J. Gautheret, V.B. Wigglesworth, P.D. Nieuwkoop, E. Wolff and J.A.D. Zeevaart, Cell differentiation and morphogenesis. North-Holland, Amsterdam, pp. 55--95. Klapwijk, D., 1966. Het effect van CCC op de groei van jonge tomatenplanten. Meded. Dir. Tuinbouw (Neth.), 29: 272--279. Linsmaier, E. and Skoog, F., 1965. Organic growth factor requirements of tobacco tissue culture. Physiol. Plant., 18: 100--127. Norton, J. and Boll, W., 1954. Callus and shoot formation from t o m a t o roots in vitro. Science, 119: 220--221. Reinert, J., 1973. Aspects of organization, organogenesis and embryogenesis. In: H.E. Street, Plant Tissue and Cell Culture. Blackwell, Oxford, pp. 338--355. Skoog, F. and Miller, C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol., 11: 118--130. Warren, R.S. and Rontley, D.G., 1970. The use of tissue culture in the study of single gene resistance of t o m a t o to Phytophthora infestans. J. Amer. Soc. Hort. Sci., 95 : 266--269. Will, H., 1966. Erste versuchsergebnisse mit cycocel zu tomaten. Gartenbauwissenschaft, 31 : 115--123.
221
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CONTINUOUS PROPAGATION OF TOMATO PLANTS BY MEANS OF CALLUS CULTURES
E. DE LANGHE and E. DE BRUIJNE
Laboratory of Tropical Crop Husbandry and Physiology, Faculty of Agronomy, State University of Ghent, Coupure, 533, 9000 Ghent (Belgium) (First received 6 December 1974; in final form 12 May 1975)
ABSTRACT De Langhe, E. and de Bruijne, E., 1976. Continuous propagation of tomato plants by means of callus cultures. Scientia Hort., 4: 221--227. Callus obtained in vitro from stem internode tissue was used to investigate the possibilities for accelerated vegetative propagation of Lycopersicum esculentum Mill. and Lycopersicum peruvianum (L). The results of different tests with phytohormones pointed to a high endogenous auxin level in the original stem explants of L. peruvianum. In L. esculentum shoots were obtained when high amounts of zeatin or coconut-milk were applied. Explants of CCC-pretreated (2 chloroethyl trimethyl-ammonium chloride} tomato plantlets showed a significant increase in shoot formation as compared to the untreated ones.
Callus tissue that was more than 2 years old and had been used in 30 transfers still had the capacity to produce normal shoots and roots. More than 200 resulting plants were observed in glasshouse conditions for possible genetic variations. No striking deviations from the original phenotype occurred. Seeds harvested from the fruits of self-fertilized flowers on these plants were sown under normal growth conditions. Some plants of one particular cultivar showed signs of accentuated vegetative development.
INTRODUCTION
Rapid vegetative multiplication is a boon to the plant breeder. It appears, however, that instability and (particularly) the variation in ploidy which was observed in a number of tissues (Reinert, 1973), are serious liabilities in the use of this technique. Fukami and Mackinney (1967), as well as Warren and Rontley (1970), worked with tomato callus to investigate specific processes. They pointed to the importance of trials to obtain whole plants; their metabolism being different in some respects from that of the callus. The present study aimed firstly at accomplishing the rapid vegetative multiplication of tomato plants by means of in vitro culture and, secondly, at determining any deviations from the original morphological pattern.
222
MATERIALS AND METHODS
Seedlings of Lycopersicum esculentum Mill. of the Mediterranean "oblong" cultivars 'Canatella', 'Roma' and 'Ventura', of 'King Plus', a round deep red tomato of moderate climates, and of Lycopersicum peruvianum (L) were grown in the following greenhouse conditions: a temperature of 23°C by day and 20°C by night; a 14-hour photoperiod with additional light from Philips HPLR 700-watt lamps; a relative humidity of about 60--70 %. Defoliated stem pieces were put in denaturated alcohol for one minute, immersed in sodium hypochlorite (8 %) for 25 min, and rinsed in sterile water; finally the internode was cut into 4-mm pieces. These pieces were inoculated upside down into the medium of Linsmaier and Skoog (1965) (called L.S. in this text) with or without the addition of coconut-milk (CCM) at 20 %, and (or) supplemented with indoleacetic acid (IAA), indolebutyric acid (IBA), 6-furfurylamino purine (KIN) and 6(7,7-dimethylallylamino) purine (DMMA). The carbon source was sucrose at 2 %. The pH of the media was adjusted to 5.6 and the media were gelled with 0.6 % agar. Each treatment covered at least one basket of 24 pieces. The cultures were maintained at a temperature of 25°C, 70 % relative humidity and constant light of about 2000 lux (Philips TL 40 W/32). The resulting plantlets were transferred into jiffy pots filled with peat, and observed in the greenhouse. Seeds from self-fertilized flowers on these plants were sown in normal growth conditions in Tunisia (Station Exp~rimentale de Manouba) and in Belgium (B.V.O.-station, Sint Kathelijne-Waver).
Fig.1. Cross-section of shoot formation originating in the subepidermal layers of the explant
223 RESULTS T h r o u g h o u t the various treatments, callus and shoot f o r m a t i o n occurred in almost the same way, callus originating f r om sub-epidermal tissue of the explant (Fig.l). The new meristem s t a r t e d f r om the peripheral layers o f this callus. Influence o f hormones in the culture medium. -- Explants o f L. peruvianum clearly r es p o n d ed with organogenesis in the presence o f auxins and cytokinins (Table I). The ratio o f t he concentrations o f auxins and cytokinins determined the differentiation as already described in t he literature (Gautheret, 1966): increase o f KIN and DMAA in regard t o the same level of auxins stimulated the initiation of s hoot f o r m a t i o n ; higher auxin c o n t e n t enhanced r o o t formation. In t r e a t m e n t No. 2 the early r o o t f o r m a t i o n was competitive with callus growth. T r e a t m e n t No. 4 is considered t o have p r o d u c e d the best response since enough callus was p r o d u c e d for subculturing as well as for y o u n g shoots. TABLE I Influence of auxins and cytokinins on root and shoot formation in explants of L. peruvianum, expressed as the number of tubes containing tissue with neo-formation in proportion to the total number of tubes per treatment. Treatment
Medium
Root formation
Shoot formation
1 2 3
L.S. L.S. + IBA 10-6M, IAA 10-~M L.S. + IBA 10-~M, IAA 10-6M KIN 10-6M L.S. + IBA 10-6M, IAA 10-6M KIN 10-6M, DMAA 10-~M L.S. + IBA 10-6M, IAA 10-6M KIN 10-SM, DMAA 10-SM
0.00 0.60 0.25
0.10 0.40 0.50
0.00
0.75
0.00
0.60
4 5
R o o t f o r m a t i o n was obtained by transferring t he developed shoots into the L.S. m e d i u m r ed u ced t o half its original concent rat i on, w i t h o u t h o r m o n e s and with a pH o f 5.2 (Fig.2). Well-shaped plantlets arose which could easily be transplanted in normal greenhouse conditions. Trials with an undiluted m e d i u m and addition o f auxins, such as IBA 10-SM or 10-6M, resulted in r o o t f o r m a t i o n b u t the plantlets failed t o grow f u r t h e r in the greenhouse. The results obtained with L. peruvianum could n o t be r e p r o d u c e d with any o f the L. esculentum cultivars. Addition in an L.S. m e d i u m of CCM at a conc e n t r atio n o f 20 % and (or) 6-(4-hydroxy-3-methyl-2-butenylamino) purine (zeatin) 10-SM was tried on L. esculentum 'Canatella' and 'King Plus' with the following results: (a) n o r o o t f o r m a t i o n occurred; (b) s h o o t f o r m a t i o n appeared in less t han 40 % of t he 'Canatella' pieces even with zeatin alone and s h o o t - f r e q u e n c y in 'King Plus' was a b o u t 20 %;
224
Fig.2. Young plantlets growing on a reduced basic medium without hormones and with pH 5.2 to induce root formation. (c) several shoots were observed on the same explant o f 'Canatella'; (d) a good p r o d u c t i o n of shoot-forming callus was seen in all the treatments; (e) the presence o f sucrose in the media with 20 % CCM inhibited s h o o t formation. It was co n clu d e d t h a t the multiplication rate for the different cultivars of L. esculentum was 2--3 times lower than for L. peruvianum. Addition o f gibberellic acid (GA3) 10-~M or 10-SM t o t he L.S. m edi um caused a high p r o d u c t i o n of callus. The effect of abscisic acid (ABA) was confusing: in some tr eat m e nt s a real stimulation of shoot f o r m a t i o n could be observed, while in others ABA lowered the shoot-forming capacity of explant and callus.
Stimulation o f shoot formation in the callus by pretreatment o f the tomato plants with chlormequat (CCC). -- The above results suggested that, while
225 t o m a t o stem tissues of L. peruvianum resemble tobacco pith tissues in their effect upon the auxin/cytokinin balance (Skoog and Miller, 1957), the system in L. esculentum might be masked by the presence of too much endogenous GA. Incorporation of CCC (1000 p.p.m.) in the L.S. medium had no effect on the growth of the explants, but pretreatment with CCC on the plants in the greenhouse stimulated the shoot-forming ability of their explants (Table II). The plants were pretreated as follows: an aqueous solution of CCC (2000 p.p.m.) was sprayed daffy upon y o u n g t o m a t o plants 'Canatella' and 'King Plus'. The new leaves were smaller and dark green, while the length of petioles and new internodes was reduced. These morphological s y m p t o m s are similar to those described by Klapwijk (1966), Will (1966) and Abdalla and Verkerk (1968). The callus was kept alive, w i t h o u t notable shoot formation, by transfer every 3 weeks into an L.S. medium with Kin 10-6M. Shoot formation could be stimulated at any time by transplanting the callus into a medium with Z 10-6M or CCM at 20 % (Fig.3). So far, the callus has maintained an undiminished shoot-forming capacity for as m a n y as 30 transplantations. Induction of root formation t o o k place in the same way as previously described for L. peruvianum. TABLE II Influence of pretreatment of tomato plants with chlormequat on the shoot-forming ability of the explant-callus, expressed as total number of shoots/total number of explants. Experiment number 1 2 3
Daysbetween pre-treatment and inoculation 2 5 8
'Canatella'
'King Plus'
Control
CCC
Control
CCC
0.43 0.10 0.54
1.65 0.43 1.14
-0.21 0.15
-0.29 0.64
It may be calculated t h a t one plant can produce more than 5 million plantlets after one year of subculturing, as follows: one branch of a t o m a t o plant easily gives 8 explants; out of each explant 6 plantlets will arise; one explant yields enough callus for 2 new tubes; this callus can be transferred every 3 weeks and will in turn produce 6 plantlets and 2 new tubes with callus. Genetic stability. -- Two hundred and thirty-four plants from in vitro material were observed in the greenhouse. They belonged to the cultivars 'King Plus', 'Canatella', 'Ventura', ' R o m a ' , and L. peruvianum, and numbered 25, 165, 29, 12 and 3 plants respectively. No phenotypical variation could be detected. Norton and Boll (1954) could n o t induce fruit-setting on L. peruvianum plants obtained in vitro. In our experiments the 3 L. peruvianum plants had their origin in tissue of different seedlings, and normal fruits were produced.
226
Fig.3. Shoot-forming callus after 20 transplantations on an L.S. medium with CCM 20 %.
About 30 L. esculentum plants were self-fertilized. In normal growth conditions the seeds developed into identical plants, except for 'King Plus' where 3 plants showed a common kind of deviation with leaf-curling, thick, dark green leaves, delayed fruiting and partial vegetative outgrowth of the inflorescences. These symptoms are probably linked to the same phenomenon: a strong vegetative growth. The possibility of polyploidisation is now being studied. CONCLUSION
The conditions governing reproducible in vitro tissue multiplication and organogenesis in various tomato cultivars have been established. L. peruvianum reacts in a well known way to the auxin/cytokinin balance of organogenesis. Results obtained with CCC-pretreatment point to the possibility that (at least in tissue of L. esculentum) this balance may be obscured
227
by a variable presence of sometimes high amounts of GA in the original tissue. Visual observation of the numerous t o m a t o plants obtained has led to the conclusion that the genetic stability of the tissue was very high. Yet the occurrence of activated vegetative growth in some segregated seedlings of the original clones, calls for further study. ACKN OWLEDGEMENTS
This study was made possible b y a grant of the A.G.C.D. (Administration Gdndrale de la Coopdration au Ddveloppement) in the framework of a BelgoTunisian project for agricultural research. Thanks are due to the research teams of the experimental stations of Manouba and of Sint Kathelijne-Waver for their careful culture and observations. We are much indebted to Mrs. Thea Versluys and Mr. Johan Geirnaert for their valuable help in the culturing of numerous tissues.
REFERENCES Abdalla, A. and Verkerk, K., 1968. Growth, flowering and fruitset of the t o m a t o at high temperature. Neth. J. Agr. Sci., 16: 71--76. Fukami, T. and Mackinney, G., 1967. Culture of t o m a t o callus tissue. Nature (London), 273: 944--945. Gautheret, R.J., 1966. Factors affecting differentiation of plant tissues grown in vitro. In: W. Beermann, C.W. Wardlaw, R.J. Gautheret, V.B. Wigglesworth, P.D. Nieuwkoop, E. Wolff and J.A.D. Zeevaart, Cell differentiation and morphogenesis. North-Holland, Amsterdam, pp. 55--95. Klapwijk, D., 1966. Het effect van CCC op de groei van jonge tomatenplanten. Meded. Dir. Tuinbouw (Neth.), 29: 272--279. Linsmaier, E. and Skoog, F., 1965. Organic growth factor requirements of tobacco tissue culture. Physiol. Plant., 18: 100--127. Norton, J. and Boll, W., 1954. Callus and shoot formation from t o m a t o roots in vitro. Science, 119: 220--221. Reinert, J., 1973. Aspects of organization, organogenesis and embryogenesis. In: H.E. Street, Plant Tissue and Cell Culture. Blackwell, Oxford, pp. 338--355. Skoog, F. and Miller, C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol., 11: 118--130. Warren, R.S. and Rontley, D.G., 1970. The use of tissue culture in the study of single gene resistance of t o m a t o to Phytophthora infestans. J. Amer. Soc. Hort. Sci., 95 : 266--269. Will, H., 1966. Erste versuchsergebnisse mit cycocel zu tomaten. Gartenbauwissenschaft, 31 : 115--123.