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Regeneration of Isolated Protoplasts of Vicia faba L.
Botanisches Institut der Christian-Albrechts-Universität, Kiel
Regeneration of Isolated Protoplasts of Vicia faha L. HORST BINDING
and
REINHARD NEHLS
With 2 Figures Received 30 January 1978 . Accepted 22 February 1978
Summary Protoplasts of Vicia faba L. were isolated from tissue of the shoot apex and from leaves of various ages. They regenerated to calluses in media KM (KAo and MICHAYLUK, 1975) and V-KM, composed of the inorganic salts of V-47 (BINDING, 1974 a) and the organic ingredients of KM. Important factors were low titres of the Vicia protoplasts and temperatures of 22-26 oe. Highest plating efficiencies were achieved if less than 3 . 103 protoplasts per ml of Vicia faba were co-cultured with 0.5-1.0·10' protoplasts of Petunia hybrida. The propagation of regenerated calluses was possible on media MS (MURASHIGE and SKOOG, 1962), NN (NITSCH and NITSCH, 1969) and NT (NAGATA and TAKEBE, 1971) if the concentrations of cytokinins did not exceed 1 ""M. Key words: Protoplasts, co-culture, callus formation.
Introduction The field be an, Vicia faba, is of great interest in general and applied genetics. As a crop it is characterized by symbiosis with nitrogen fixing bacteria and by its seeds being rich of protein of high quality. Large chromosomes of. characteristic morphology have made Vicia faba to a favoured object in cytology and cytogenetics. Protoplast regeneration is becoming increasingly important in genetics and plant breeding programmes. Therefore, it has been intended to obtain regenerants from isolated protoplasts of Vicia faba.
Materials and Methods Plants of Vicia faba L. cv. STAYGREEN were grown under greenhouse conditions. Shoot tips, premeiotic flower buds, and leaves of different ages were taken for protoplast isolation. Axenic shoot cultures yielding good protoplast preparations in species of the Solanaceae (BINDING, 1974 b, 1975; BINDING and NEHLS, 1977; SCHIEDER, 1975, 1977) could not be established in Vicia faba. The plant material was sterilized by incubation in an aqueous solution of 0.1 Ufo HgC12 and 0.1 Ufo sodiumlauryl sulphate for 5 to 8 minutes and were washed with sterile tap water
z.
Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
328
HORST BINDING and REINHARD NEHLS
(BINDING, 1974 a). Leasions easily visible by browning were excised. Leaflets of more than 10 mm in length were made accessible to cell wall lysing enzymes by pealing off the lower epiderm. Younger plant material was dissected into slices of less than 2 mm in square and preincubated in solutions of 0.5 M mannito1l5 mM CaCl2 for 1 hour. The enzyme solution for wall digestion contained 0.3 % Macerozyme, 1.5 % Meicellase, 0.5 M mannitol, and 5 mM CaCI 2 • Leaflets were floated on the surface of the solution directly after pealing and incubated at room temperature and about 1000 lux of incandescent light. Dissected plant material was suspended in the enzyme solution and submitted to vacuum infiltration. The incubation was carried out either at 30°C for 4-5 hours or at about 25°C over-night. The purification of the protoplast suspensions by filtration and sedimentation was performed according to standard procedures (BrNDING, 1974 b, BINDING and NAGY, 1976). The nutrient solutions for protoplast culture were original and modified media KM (KAo and MrCHAYLUK, 1975), DPD/V-47 (DURAND et al., 1973; modification V-47: BINDING, 1974 a), or medium V-KM, a combination of both (BrNDING and NEHLS, 1977). In aseries of experiments, conditioned media were used which were obtained either by preincubating protoplasts of Petunia hybrida in the media or by co-culturing protoplasts of Vicia and Petunia simultaneously. Cell suspensions in semisolid media were prepared by mixing liquid suspensions with equal amounts of 0.25 % agar media at room temperature. The cultures were maintained in growth chambers at various temperatures and irradiated continuously by white fluorescent light of various intensities. The estimation of protoplast titres was based on countings in droplets of 50 .ul. The titres at plating were taken as references for the calculation of plating efficiencies. Regenerated calluses were transfered onto solid agar media (0.8 Ofo agar) MS (MURASHIGE and SKOOG, 1962), NN (NITSCH and NrTscH, 1969) or NT (NAGATA and TAKEBE, 1971) 15-20 days after protoplast isolation. The Petri dishes were placed in a tissue culture room in which the temperature was adjusted to 26 ± 1 oe. The cultures were irradiated by about 2000 lux of cool white fluorescent light (Osram L 22).
Results
The stability of protoplasts isolated from expanded leaves (fig. 1 a) varied considerably in different experiments, whereas reproducibly high yields of stable protoplasts have been obtained from not fully developed plant organs (fig. 1 b). Plant material of not more than 5 mg fresh weight had to' be incubated in one ml of enzyme solution to prevent inactivation of enzymes. Low plating efficiencies not exceeding 10-2 were obtained when the isolated protoplasts were cultured in defined, non-conditioned culture media. Cell divisions were found at plating densities between 1 and 3· 103 protoplasts per ml. The protoplasts extended and budded at higher densities. Useful nutrient solutions rendering sustained cell divisions were media KM and V-KM containing 2.5 f-lM benzyladenin, 5 f-lM a-naphthylacetic acid and 0.5 f-lM 2,4-dichlorophenoxyacetic acid. Favourable temperatures ranged between 22 and 26 oe. No significant differences in response of the protoplasts to light intensities varied between 800 and 2000 lux have been observed. The efficacy of conditioned culture media on Vicia protoplasts was investigated using media V-47 and V-KM conditioned by protoplasts of Petunia hybrida. Z. Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
Regeneration of protoplasts of Vicia
329
1a Fig. 1: Protoplasts of Vicia faba immediate1y after the isolation. a) Protoplast isolated frorn mesophyll of an expanded leaflet. b) Protoplasts of the shoot apex characterized by large nuc1ei, juvenile plastids, and small vacuoles. X 500.
Preliminary experiments had revealed particular qualification of V-KM for the regeneration of Petunia protoplasts. The mitotic activities of Vicia protoplasts were not improved by media in which Petunia protoplasts had been grown for 1-5 days prior to the inoculation of the Vicia protoplasts. On the contrary, the co-culture of both protoplast types in medium V-KM revealed significantly higher plating efficiencies of Vicia reaching values of around 0.2. The hormone content was the same as in non-conditioned media. Best results were obtained if the Petunia protoplasts were plated at densities of 5.10 3 to 104 per ml and the Vicia protoplasts at densities of less than 2· 103 • Higher densities of Vicia affected both Vicia and Petunia cells. The suspensions were diluted every 5 days by adding equal volumes of soft agar media. Appropriate environmental conditions in the growth chamber were the same as those for protoplasts in defined media. Regenerating protoplasts in single-type and co-culture got oval shape after about three days of culture indicating cell wall formation (BINDING, 1966). Lipid droplets became visible after 4-5 days in most of the cells (fig. 2 a). By means of these droplets, it was possible to distinguish cells of Vicia from those of Petunia in co-cultures. First ceIl divisions could be observed in 3-6 days old cultures. Often, a chain of a few ceIls was formed (fig. 2) before globular callus arose from one terminal cell. Callus propagation was unlimited on media MS, NN and NT if they contained not more than 2.5 .uM benzyladenin or kinetin. Cytokinins could be omitted completely whithout aHecting the growth rates significantly. a-naphthylacetic acid favoured the growth in concentrations of 2-15 ,uM. 2,4-Dichlorophenoxyacetic acid could be left out of the media. The calluses had to be held in a well growing state to avoid senescence of parts of the tissue resulting in the death of the whole callus. This has been achieved by transfers after every three weeks. Tissue wh ich had been damaged by partitition to subcultures could be also deleterious. Analysis of mitotic cells Z. Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
330
HORST BINDING
and
REINHARD NEHLS
2a Fig. 2 : Two regenerants of isolated protoplasts of Vicia faba. a) Six days old, containing characteristic lipid droplets. b) Two weeks old, young globular callus being formed by one cell of a bicellular regenerant. X400 (2 a); X 200 (2 b).
revealed diploid, tetraploid and a few octoploid cells. A lot of tracheidal cells were found in the tissue. At the present, oldest regenerants have reached an age of eight months. No organogenetic activity has been observed, until now. Discussion Suspensions of isolated protoplasts of Vicia /aba have been prepared successfully following standard procedures. Restitution of cell walls and mitotic activity .was achieved in culture media KM and V-KM either of which had been proved to be appropriate in a couple of other plant species (Vicia hajastana: KAO and MICHAYLUK, 1975; Solanum tuberosum: MELCHERs, pers. comm., BINDING et al., 1978; Solanum dulcamara: BINDING and NEHLS, 1977; Solanum nigrum: NEHLS, in press; Petunia hybrida: this paper; Petunia hybrida + Vicia /aba: BINDING and NEHLS, in press). However, in Vicia /aba special attention must be payed to deleterious compounds delivered from damaged cells. It has been observed that wall digesoing enzymes were
z. Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
Regeneration of protoplasts of Vicia
331
inactivated by high er amounts of plant material, that protoplasts survived only at low plating densities, and that calluses were affected extremely by damaged parts. The favourable prinoiple provided by he protoplasts of Petunia in co-cultures is not yet known. It appeared to be different from the conditioning effect which had been found to favour growth of Petunia protoplasts at low plating densities (BINDING, 1974 b) in that pre-conditioning of the media was not efficient. The co-culture effect is probably more congruent to the feeding layer system described by RAVEH et a1. (1973) for tobacco protoplasts at low densities. In this manner, it may help to overcome the poor plating efficiencies at the low densities of Vicia protoplasts which are required for their surviva1. The separation of both protoplast types in different layers was not needed because the regenerants were unequivocally distinguished from one another by the formation of lipid droplets at early stages and later by browning of damaged cells of the calluses. Another explanation for the promoting effect of co-culture may be that the Petunia protoplasts eliminated deleterious substances delivered from Vicia cells. However, the plating densities of Vicia protoplasts could not be raised up by co-culture. Furthermore, the action of the Petunia protoplasts could be explained by the absorption of special components of the culture media in a way comparable to the uptake of magnesium by suspension cultures of Ipomoea (VELIKY et a1., 1977). Experiments with Vicia faba aiming at further improvement of the culture conditions of isolated protoplasts, induction of organogenesis, and at parasexual recombination are under investigation. Acknowledgements The authors wish to thank Miss]. EGGERS and Miss G. MORDHORST for teehnieal assistanee and Mrs. R. SCHULZ for the preparation of the photographs. The experiments have been supported by the Deutsche Forsehungsgemeinsehaft.
References BINDING, H.: Z. Pflanzenphysiol. 55,305 (1966). - Plant Sei. Lett. 2,185 (1974 a). - Z. Pflanzenphysiol. 74, 327 (1974 b). - Physiol. Plant. 35, 225 (1975). - In: W. R. SHARP, P. O. LARsEN, E. F. PADDOCK, and V. RAGHAVEN (Eds.), Plant Cell and Tissue Culture: Principles and Applieations, Ohio State Univ. Biosei. Coll. Ser., Vol. IV, in press. BINDING, H. and ]. I. NAGY: In: D. DUDITs, G. L. FARKAS, and P. MALIGA (Eds.), Cell Geneties in Higher Plants, Akademiai Kiad6, Budapest, 1976. - Molee. gen. Genet., in press. BINDING, H. and R. NEHLS: Z. Pflanzenphysiol. 85, 279 (1977). BINDING, H., R. NEHLS, O. SCHIEDER, S. K. SOPORY, and G. WENZEL: Physiol. Plant., 43, 52 (1978). DURAND, ]., I. POTRYKUS et G. DONN: Z. Pflanzenphysiol. 69, 26 (1973). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Exp. Cell Res. 50,151 (1968). KAO, K. N. and M. R. MICHAYLUK: Planta (Berl.) 126,105 (1975).
Z. Pjlanzenphysiol. Bd. 88. S. 327-332. 1978.
332
HORST BINDING and REINHARD NEHLS
MELCHERS, G.: pers. comm. (1976). MURASHIGE, T. and F. SKOOG: Physiol. Plant. 15, 473 (1962). NAGATA, T., and 1. TAKEBE: Planta 99,12 (1971). NEHLS, R.: Plant Sei. Lett., in press. NITSCH, J. P. and C. NITSCH: Science 163, 85 (1969). RAvEH, D., E. HABERMAN, and E. GALUN: In vitra 9, 216 (1973). SCHIEDER, 0.: Z. Pflanzenphysiol. 76,462 (1975). - Z. Pflanzenphysiol. 84, 275 (1977). VELlKY, 1. A., D. RosE, and M. W. ZINK: Can. J. Bot. 55, 1143 (1977).
HORST BINDING, Botanisches Institut der Christian-Albrechts-Universität, Düsternbrooker Weg 17, D-2300 Kielt.
Z. Pjlanzenphysial. Bd. 88. S. 327-332. 1978.
Regeneration of Isolated Protoplasts of Vicia faha L. HORST BINDING
and
REINHARD NEHLS
With 2 Figures Received 30 January 1978 . Accepted 22 February 1978
Summary Protoplasts of Vicia faba L. were isolated from tissue of the shoot apex and from leaves of various ages. They regenerated to calluses in media KM (KAo and MICHAYLUK, 1975) and V-KM, composed of the inorganic salts of V-47 (BINDING, 1974 a) and the organic ingredients of KM. Important factors were low titres of the Vicia protoplasts and temperatures of 22-26 oe. Highest plating efficiencies were achieved if less than 3 . 103 protoplasts per ml of Vicia faba were co-cultured with 0.5-1.0·10' protoplasts of Petunia hybrida. The propagation of regenerated calluses was possible on media MS (MURASHIGE and SKOOG, 1962), NN (NITSCH and NITSCH, 1969) and NT (NAGATA and TAKEBE, 1971) if the concentrations of cytokinins did not exceed 1 ""M. Key words: Protoplasts, co-culture, callus formation.
Introduction The field be an, Vicia faba, is of great interest in general and applied genetics. As a crop it is characterized by symbiosis with nitrogen fixing bacteria and by its seeds being rich of protein of high quality. Large chromosomes of. characteristic morphology have made Vicia faba to a favoured object in cytology and cytogenetics. Protoplast regeneration is becoming increasingly important in genetics and plant breeding programmes. Therefore, it has been intended to obtain regenerants from isolated protoplasts of Vicia faba.
Materials and Methods Plants of Vicia faba L. cv. STAYGREEN were grown under greenhouse conditions. Shoot tips, premeiotic flower buds, and leaves of different ages were taken for protoplast isolation. Axenic shoot cultures yielding good protoplast preparations in species of the Solanaceae (BINDING, 1974 b, 1975; BINDING and NEHLS, 1977; SCHIEDER, 1975, 1977) could not be established in Vicia faba. The plant material was sterilized by incubation in an aqueous solution of 0.1 Ufo HgC12 and 0.1 Ufo sodiumlauryl sulphate for 5 to 8 minutes and were washed with sterile tap water
z.
Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
328
HORST BINDING and REINHARD NEHLS
(BINDING, 1974 a). Leasions easily visible by browning were excised. Leaflets of more than 10 mm in length were made accessible to cell wall lysing enzymes by pealing off the lower epiderm. Younger plant material was dissected into slices of less than 2 mm in square and preincubated in solutions of 0.5 M mannito1l5 mM CaCl2 for 1 hour. The enzyme solution for wall digestion contained 0.3 % Macerozyme, 1.5 % Meicellase, 0.5 M mannitol, and 5 mM CaCI 2 • Leaflets were floated on the surface of the solution directly after pealing and incubated at room temperature and about 1000 lux of incandescent light. Dissected plant material was suspended in the enzyme solution and submitted to vacuum infiltration. The incubation was carried out either at 30°C for 4-5 hours or at about 25°C over-night. The purification of the protoplast suspensions by filtration and sedimentation was performed according to standard procedures (BrNDING, 1974 b, BINDING and NAGY, 1976). The nutrient solutions for protoplast culture were original and modified media KM (KAo and MrCHAYLUK, 1975), DPD/V-47 (DURAND et al., 1973; modification V-47: BINDING, 1974 a), or medium V-KM, a combination of both (BrNDING and NEHLS, 1977). In aseries of experiments, conditioned media were used which were obtained either by preincubating protoplasts of Petunia hybrida in the media or by co-culturing protoplasts of Vicia and Petunia simultaneously. Cell suspensions in semisolid media were prepared by mixing liquid suspensions with equal amounts of 0.25 % agar media at room temperature. The cultures were maintained in growth chambers at various temperatures and irradiated continuously by white fluorescent light of various intensities. The estimation of protoplast titres was based on countings in droplets of 50 .ul. The titres at plating were taken as references for the calculation of plating efficiencies. Regenerated calluses were transfered onto solid agar media (0.8 Ofo agar) MS (MURASHIGE and SKOOG, 1962), NN (NITSCH and NrTscH, 1969) or NT (NAGATA and TAKEBE, 1971) 15-20 days after protoplast isolation. The Petri dishes were placed in a tissue culture room in which the temperature was adjusted to 26 ± 1 oe. The cultures were irradiated by about 2000 lux of cool white fluorescent light (Osram L 22).
Results
The stability of protoplasts isolated from expanded leaves (fig. 1 a) varied considerably in different experiments, whereas reproducibly high yields of stable protoplasts have been obtained from not fully developed plant organs (fig. 1 b). Plant material of not more than 5 mg fresh weight had to' be incubated in one ml of enzyme solution to prevent inactivation of enzymes. Low plating efficiencies not exceeding 10-2 were obtained when the isolated protoplasts were cultured in defined, non-conditioned culture media. Cell divisions were found at plating densities between 1 and 3· 103 protoplasts per ml. The protoplasts extended and budded at higher densities. Useful nutrient solutions rendering sustained cell divisions were media KM and V-KM containing 2.5 f-lM benzyladenin, 5 f-lM a-naphthylacetic acid and 0.5 f-lM 2,4-dichlorophenoxyacetic acid. Favourable temperatures ranged between 22 and 26 oe. No significant differences in response of the protoplasts to light intensities varied between 800 and 2000 lux have been observed. The efficacy of conditioned culture media on Vicia protoplasts was investigated using media V-47 and V-KM conditioned by protoplasts of Petunia hybrida. Z. Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
Regeneration of protoplasts of Vicia
329
1a Fig. 1: Protoplasts of Vicia faba immediate1y after the isolation. a) Protoplast isolated frorn mesophyll of an expanded leaflet. b) Protoplasts of the shoot apex characterized by large nuc1ei, juvenile plastids, and small vacuoles. X 500.
Preliminary experiments had revealed particular qualification of V-KM for the regeneration of Petunia protoplasts. The mitotic activities of Vicia protoplasts were not improved by media in which Petunia protoplasts had been grown for 1-5 days prior to the inoculation of the Vicia protoplasts. On the contrary, the co-culture of both protoplast types in medium V-KM revealed significantly higher plating efficiencies of Vicia reaching values of around 0.2. The hormone content was the same as in non-conditioned media. Best results were obtained if the Petunia protoplasts were plated at densities of 5.10 3 to 104 per ml and the Vicia protoplasts at densities of less than 2· 103 • Higher densities of Vicia affected both Vicia and Petunia cells. The suspensions were diluted every 5 days by adding equal volumes of soft agar media. Appropriate environmental conditions in the growth chamber were the same as those for protoplasts in defined media. Regenerating protoplasts in single-type and co-culture got oval shape after about three days of culture indicating cell wall formation (BINDING, 1966). Lipid droplets became visible after 4-5 days in most of the cells (fig. 2 a). By means of these droplets, it was possible to distinguish cells of Vicia from those of Petunia in co-cultures. First ceIl divisions could be observed in 3-6 days old cultures. Often, a chain of a few ceIls was formed (fig. 2) before globular callus arose from one terminal cell. Callus propagation was unlimited on media MS, NN and NT if they contained not more than 2.5 .uM benzyladenin or kinetin. Cytokinins could be omitted completely whithout aHecting the growth rates significantly. a-naphthylacetic acid favoured the growth in concentrations of 2-15 ,uM. 2,4-Dichlorophenoxyacetic acid could be left out of the media. The calluses had to be held in a well growing state to avoid senescence of parts of the tissue resulting in the death of the whole callus. This has been achieved by transfers after every three weeks. Tissue wh ich had been damaged by partitition to subcultures could be also deleterious. Analysis of mitotic cells Z. Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
330
HORST BINDING
and
REINHARD NEHLS
2a Fig. 2 : Two regenerants of isolated protoplasts of Vicia faba. a) Six days old, containing characteristic lipid droplets. b) Two weeks old, young globular callus being formed by one cell of a bicellular regenerant. X400 (2 a); X 200 (2 b).
revealed diploid, tetraploid and a few octoploid cells. A lot of tracheidal cells were found in the tissue. At the present, oldest regenerants have reached an age of eight months. No organogenetic activity has been observed, until now. Discussion Suspensions of isolated protoplasts of Vicia /aba have been prepared successfully following standard procedures. Restitution of cell walls and mitotic activity .was achieved in culture media KM and V-KM either of which had been proved to be appropriate in a couple of other plant species (Vicia hajastana: KAO and MICHAYLUK, 1975; Solanum tuberosum: MELCHERs, pers. comm., BINDING et al., 1978; Solanum dulcamara: BINDING and NEHLS, 1977; Solanum nigrum: NEHLS, in press; Petunia hybrida: this paper; Petunia hybrida + Vicia /aba: BINDING and NEHLS, in press). However, in Vicia /aba special attention must be payed to deleterious compounds delivered from damaged cells. It has been observed that wall digesoing enzymes were
z. Pflanzenphysiol. Bd. 88. S. 327-332. 1978.
Regeneration of protoplasts of Vicia
331
inactivated by high er amounts of plant material, that protoplasts survived only at low plating densities, and that calluses were affected extremely by damaged parts. The favourable prinoiple provided by he protoplasts of Petunia in co-cultures is not yet known. It appeared to be different from the conditioning effect which had been found to favour growth of Petunia protoplasts at low plating densities (BINDING, 1974 b) in that pre-conditioning of the media was not efficient. The co-culture effect is probably more congruent to the feeding layer system described by RAVEH et a1. (1973) for tobacco protoplasts at low densities. In this manner, it may help to overcome the poor plating efficiencies at the low densities of Vicia protoplasts which are required for their surviva1. The separation of both protoplast types in different layers was not needed because the regenerants were unequivocally distinguished from one another by the formation of lipid droplets at early stages and later by browning of damaged cells of the calluses. Another explanation for the promoting effect of co-culture may be that the Petunia protoplasts eliminated deleterious substances delivered from Vicia cells. However, the plating densities of Vicia protoplasts could not be raised up by co-culture. Furthermore, the action of the Petunia protoplasts could be explained by the absorption of special components of the culture media in a way comparable to the uptake of magnesium by suspension cultures of Ipomoea (VELIKY et a1., 1977). Experiments with Vicia faba aiming at further improvement of the culture conditions of isolated protoplasts, induction of organogenesis, and at parasexual recombination are under investigation. Acknowledgements The authors wish to thank Miss]. EGGERS and Miss G. MORDHORST for teehnieal assistanee and Mrs. R. SCHULZ for the preparation of the photographs. The experiments have been supported by the Deutsche Forsehungsgemeinsehaft.
References BINDING, H.: Z. Pflanzenphysiol. 55,305 (1966). - Plant Sei. Lett. 2,185 (1974 a). - Z. Pflanzenphysiol. 74, 327 (1974 b). - Physiol. Plant. 35, 225 (1975). - In: W. R. SHARP, P. O. LARsEN, E. F. PADDOCK, and V. RAGHAVEN (Eds.), Plant Cell and Tissue Culture: Principles and Applieations, Ohio State Univ. Biosei. Coll. Ser., Vol. IV, in press. BINDING, H. and ]. I. NAGY: In: D. DUDITs, G. L. FARKAS, and P. MALIGA (Eds.), Cell Geneties in Higher Plants, Akademiai Kiad6, Budapest, 1976. - Molee. gen. Genet., in press. BINDING, H. and R. NEHLS: Z. Pflanzenphysiol. 85, 279 (1977). BINDING, H., R. NEHLS, O. SCHIEDER, S. K. SOPORY, and G. WENZEL: Physiol. Plant., 43, 52 (1978). DURAND, ]., I. POTRYKUS et G. DONN: Z. Pflanzenphysiol. 69, 26 (1973). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Exp. Cell Res. 50,151 (1968). KAO, K. N. and M. R. MICHAYLUK: Planta (Berl.) 126,105 (1975).
Z. Pjlanzenphysiol. Bd. 88. S. 327-332. 1978.
332
HORST BINDING and REINHARD NEHLS
MELCHERS, G.: pers. comm. (1976). MURASHIGE, T. and F. SKOOG: Physiol. Plant. 15, 473 (1962). NAGATA, T., and 1. TAKEBE: Planta 99,12 (1971). NEHLS, R.: Plant Sei. Lett., in press. NITSCH, J. P. and C. NITSCH: Science 163, 85 (1969). RAvEH, D., E. HABERMAN, and E. GALUN: In vitra 9, 216 (1973). SCHIEDER, 0.: Z. Pflanzenphysiol. 76,462 (1975). - Z. Pflanzenphysiol. 84, 275 (1977). VELlKY, 1. A., D. RosE, and M. W. ZINK: Can. J. Bot. 55, 1143 (1977).
HORST BINDING, Botanisches Institut der Christian-Albrechts-Universität, Düsternbrooker Weg 17, D-2300 Kielt.
Z. Pjlanzenphysial. Bd. 88. S. 327-332. 1978.