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Regeneration of Plants from Cotyledon Fragments of Buckwheat (Fagopyrum esculentum Moench)
Regeneration of Plants from Cotyledon Fragments of Buckwheat (Fagopyrum esculentum Moench.) VEROSLAVA SREJOVIC and MIRJANA NESKOVIC Institute of Biology, Faculty of Science, Kragujevac Institute of Botany, Faculty of Science, Beograd, Yugoslavia Received January 5, 1981 . Accepted May 15, 1981
Summary Fragments excised from cotyledons of imbibed seeds were initially cultivated on media with high auxin (2,4-D) and low cytokinin (kinetin) content. Cell division in spongy parenchyma cells was induced within 5 days and the fragments were then transferred to different media. Employing various types and concentrations of auxins and cytokinins, it was possible to direct further development and obtain undifferentiated calluses, or calluses bearing either roots or shoots. Whole plantlets were obtained when the shoots were stimulated to produce adventitious roots. Flowering was observed in all rooted plants. When transferred from sterile culture conditions to peat and soil, most plantlets were able to survive. It has been concluded that the regeneration of whole buckwheat plants is feasible from differentiated cotyledon cells, provided three different media were subsequently used, each one suitable for a specific phase of development.
Key words: Fagopyrum esculentum, cotyledon explants, organogenesis, plant regeneration in
vitro.
Introduction Vegetative propagation of many economic plants has so far been achieved through tissue culture methods (Murashige, 1978). Buckwheat is a plant of economic importance, as a food plant, due to its starch reserves in the seeds. Yamane (1974) showed that isolated hypocotyls and cotyledons of buckwheat could be induced to develop calluses, with the capacity for organogenesis and restoration of plantlets. With the exception of Yamane's paper, it seems that neither buckwhest, nor any other species of Polygonaceae has much been used for tissue culture up to now (Murashige, 1978; Narayanaswamy, 1977). While the general conditions for organ formation seem to be well established (Skoog and Miller, 1957; Murashige, 1977), there are differences between species in regard to the optimal hormone concentrations or balance. Many plant tissues require a proper sequence of hormones, in order to regenerate whole plants, either via embryogenesis (Steward et aI., 1967) or organogenesis (Chen and Galston, 1967). The purpose of the present investigation was to study the capacity of buckwheat cells to undergo the change in their differentiation program and to develop into Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
38
VEROSLAVA SREJOVIC and MIRJANA NESKOVIC
whole plantlets. It seems that buckwheat tissue provides a suitable system for studying the sequential role of hormones in organ redifferentiation.
Material and Methods Fruits of Fagopyrum esculentum Moench. cv. Pennquad, of a tetraploid line, were surface sterilized for 2 min with ethanol, the pericarp was removed and the peeled seeds further sterilized for 15 min in 5% calcium hypochlorite solution. After washing, they were left in sterile water for 3 h to imbibe. Then the endosperm was removed under sterile conditions, the embryo axis cut off and the folded cotyledons cut transversely into apical and basal parts, which were put into culture. Three segments were cultivated on 40 ml of agar media, in 100 ml Erlenmayer flasks. Each experiment was started with 12 flasks and was repeated at least three times. Basal medium (BM) for the cultivation of tissue contained the mineral solution Bs {Gam borg et aI., 1968),3% sucrose, 1% agar and (in mg I-I): thiamine 2, pyridoxine 1, nicotinic acid 1, minositol 100 and casein hydrolysate 2000. Auxins 2,4-dichlorophenoxyacetic acid (2,4-D), unaphtaleneacetic acid (NAA), indolyl-3-acetic acid (IAA) and indolyl-3-butyric acid (IBA), as well as cytokinins kinetin (KIN) and benzylaminopurine (BAP) were added in different concentrations, as specified in the text. All cultures were maintained in diffuse light of 4500 K «Tesla» fluorescent tubes, at light intensity of 1500 Ix. The day length was 16 h, and the temperature was regulated at 25 ± 1 0c. Callus pieces in the course of organogenesis were fixed in Carnoy fixative, embedded in paraffin and stained with gallocyanin, hematoxylin, safranin or light green.
Results Induction of cell division in cotyledon explants In mature seeds cotyledons have a large, thin, tightly folded blade, with the anatomy of a typical leaf. At the time of excision, vascular bundles were not fully differentiated and 2 - 3 layers of cambial cells were visible between xylem and phloem. Cotyledon explants were cultivated for the first 5 days on a medium suitable to induce cell division, and containing BM supplemented with 2,4-D 5 mg 1-1 and KIN 0.1 mg 1-1. During this time the cotyledon fragments roughly doubled in size and became green. The first cell divisions were observed after 3 days in culture and comprised a row of spongy parenchyma cells between vascular bundles (Figs. 1 and 2) and parenchymatous cells around the midrib vein (Fig. 3). The divisions soon proceeded in other mesophyll cells and after 2-4 weeks the inner tissue of the cotyledon blades was transformed into numerous meristematic regions (Fig. 4). A yellowish callus, partly containing anthocyanin, was visible on the edge of the blades. This callus was used for further subcultures. Growth of callus tissue and organogenesis The callus tissue grew poorly when subcultured on the same medium in which cell division was induced, but its growth was very much improved by lowering the 2,4-D concentration to 1 mg 1-1 and by increasing the KIN concentration to 1 mg 1-1. Ana-
Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
In vitro plant regeneration in buckwheat
39
Figs. 1-3: Transverse sections through cotyledon explants grown for 3 days on the medium for cell division (2,4-D 5, KIN 0.1 mg 1-1). Fig. 1: Cell division in a row of spongy parenchyma cells between the vascular bundles (30x). - Fig. 2: Detail of dividing cells between two neighbouring bundles (126x). - Fig. 3: Divided cells around the midrib vein (60 x ). Fig. 4: Meristematic nodules in cotyledon explants grown for 15 days on the medium for callus maintenance (2,4-D 1, KIN 1 mg 1- 1)(65x). tomical observation showed that the callus was composed mainly of meristematic nodules, developing into root initials, with very little parenchymatous tissue between them. Numerous roots were elongated when the 2,4-D concentration was further decreased to 0.1 mg 1-\ or when the auxin was omitted (Fig. 5), but these calluses survived 5-6 transfers and then became necrotic. The calluses kept on 2,4-D+KIN media did never produce shoots. It was found that shoot formation could only be induced when the primary cotyledon explants were removed after 3 - 5 days from the medium suitable for cell division, and transferred to another medium, with a high cytokinin to auxin ratio. Meristematic nodules developed then into typical shoot meristems. By the end of the first, or during the second passage, the calluses in up to 40% of explants gave rise to a large number of buds. Growth of the buds apparently improved callus growth and this in turn led to the formation of many more buds. However, the buds failed to develop if the tissue was transferred to the inductive medium later than at 4 weeks. The first tissue with buds was obtained in May 1978 and it is still maintained in culture (Fig. 6), with an unchanged capacity for shoot formation. The same experiment was repeated several times with numerous fresh explants and the results obtained were always similar.
Z. Pjlanzenphysiol. Bd. 104. S. 37-42.1981.
40
VEROSLAVA SREJOVIC
and MIRJANA
NESKOVIC
Fig. 5: Calluses with roots at the end of 2nd subculture (2,4-D 1, KIN 0.1 mg 1-') (1 x). - Fig. 6: Calluses with numerous buds and shoots at the end of 9th subculture (IAA 10- 6 M, BAP 10- 5 M) (Ix). - Fig. 7: A shoot with adventitious roots, developed after 10 days on the medium without hormones (1.3 x). - Fig. 8: Flowering plant in soil (0.35 x).
The combination of hormones which was used for shoot induction consisted of IAA 10- 6 M and BAP 10- 5 M. Substitution of IAA with 2,4-D or NAA, as well as the substitution of BAP by KIN, all in equimolar concentrations, produced a considerabZ. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
In vitro plant regeneration in buckwheat
41
ly lower number of shoots, the tissue grew slowly, and had to be abandoned, usually after 2 passages. In the IAA + BAP medium the number of bud initials per callus was rarely less than 50. Regeneration of whole plants
Shoots which differentiated on IAA + BAP medium did never produce roots on the same medium. Well developed shoots were isolated, cleaned of callus pieces and adventitious roots developed on. their basal parts (Fig. 7). The rooting was best on BM + lEA (1 mg 1-1), but roots also developed when all hormones were omitted. Plantlets which grew well on agar were carefully taken out, washed from the medium and grown first in peat, moistened with mineral solution. When the plants became vigorous, they were transferred into pots with soil, where they continued growing to a height of 30 cm or more (Fig. 8). A cluster of normal, white flowers developed in almost all regenerated plants. Flowering was always preceded by, or occurred simultaneously, with rooting. Although the large scale propagation of buckwheat plants was not the primary object of the present study, it is clear from the results obtained that it can be achieved with high frequency. The transfer of plants into the soil was not accompanied by great difficulties, provided the plants were protected in a humid atmosphere. Discussion Buckwheat tissue culture, described in the present paper, was initiated in cotyledon explants, composed of differentiated, but not fully grown cells. The cells, which were most responsive to the cell division stimulus, were those lying between the vascular bundles, in continuity with the remaining cambium. In this respect buckwheat cotyledons differ from some other leaf explants, in which cell division and subsequent organogenesis originate in epidermal or subepidermal cell layers (Tran Thanh Van and Drira, 1971; Venverloo, 1976). The sequential use of hormones in the present work has enabled us to distinguish between the two phases in organ development: the initial renewal of cell division and the subsequent phase of organ determination. This sequential change of hormones may not be indispensible for the complete regeneration of plants. A very limited number of shoots developed when the first medium was omitted. It seems, therefore, that the high 2,4-D content in the beginning influenced the abundance of bud formation, probably by increasing the number of dedifferentiated cells. These cells become subsequently determined to form root or shoot initials, in response to the specific auxin to cytokinin ratio. The number of species in which plantlets can be regenerated from explants in vitro is increasing rapidly. Nevertheless, there are still many recalcitrant plants or tissues, which do not respond to the usual hormonal stimuli. If. in such a species root and Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
42
VEROSLAVA SREJOVIC and MIRJANA NESKovrc
shoot formation are alternative events, like in buckwheat, one has probably to consider sequential changes in hormone complex and to find out the correct time to apply them. It is clear that a better understanding of the primary events in cell determination (Hicks, 1980) is the key for further progress in this field. References CHEN, H. and A. W. GALSTON: Growth and development of Pelargonium pith cells in vitro. II. Initiation of organized development. Physiol. Plant. 20, 533-539 (1967). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). HICKS, G. S.: Patterns of organ development in plant tissue culture and the problem of organ determination. Bot. Rev. 46,1-23 (1980). MURASHIGE, T.: Manipulation of organ initiation in plant tissue cultures. Bot. Bull. Acad. Sin. 19,1-24(1977). - The impact of plant tissue culture on agriculture. In: T. A. THORPE (Ed.): Frontiers of Plant Tissue Culture 1978, pp. 15-26. University of Calgary, Calgary, 1978. NARAYANASWAMY, S.: Regeneration of plants from tissue cultures. In: J. REINERT and Y. P. S. BAJAJ (Eds.): Plant Cell, Tissue and Organ Culture, pp. 179-248. Springer-Verlag, Berlin, 1977. SKOOG, F. and C. O. MILLER: Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exptl. BioI. 11, 118 -131 (1957). STEWARD, F. c., A. E. KENT, and M. O. MAPES: Growth and organization in cultured cells: Sequential and synergistic effects of growth regulating substances. In: Plant Growth Regulators. Ann. N. Y. Acad. Sci. 144, 326-334 (1967). TRAN THANH VAN, M. and A. DRIRA: Definition of a simple experimental system of directed organogenesis de novo: organ neoformation from epidermal tissues of Nautilocalyx lynchei. In: ColI. Intern. C. N. R. S. No. 193, 169-176 (1971). VENVERLOO, C. J.: The formation of adventitious organs. III. A comparison of root and shoot formation on Nautilocalyx explants. Z. Pflanzenphysiol. 80, 310-322 (1976). YAMANE, Y.: Induced differentiation of buckwheat plants from subcultured calluses in vitro. Jap. J. Genet. 49,139-146 (1974).
Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
Summary Fragments excised from cotyledons of imbibed seeds were initially cultivated on media with high auxin (2,4-D) and low cytokinin (kinetin) content. Cell division in spongy parenchyma cells was induced within 5 days and the fragments were then transferred to different media. Employing various types and concentrations of auxins and cytokinins, it was possible to direct further development and obtain undifferentiated calluses, or calluses bearing either roots or shoots. Whole plantlets were obtained when the shoots were stimulated to produce adventitious roots. Flowering was observed in all rooted plants. When transferred from sterile culture conditions to peat and soil, most plantlets were able to survive. It has been concluded that the regeneration of whole buckwheat plants is feasible from differentiated cotyledon cells, provided three different media were subsequently used, each one suitable for a specific phase of development.
Key words: Fagopyrum esculentum, cotyledon explants, organogenesis, plant regeneration in
vitro.
Introduction Vegetative propagation of many economic plants has so far been achieved through tissue culture methods (Murashige, 1978). Buckwheat is a plant of economic importance, as a food plant, due to its starch reserves in the seeds. Yamane (1974) showed that isolated hypocotyls and cotyledons of buckwheat could be induced to develop calluses, with the capacity for organogenesis and restoration of plantlets. With the exception of Yamane's paper, it seems that neither buckwhest, nor any other species of Polygonaceae has much been used for tissue culture up to now (Murashige, 1978; Narayanaswamy, 1977). While the general conditions for organ formation seem to be well established (Skoog and Miller, 1957; Murashige, 1977), there are differences between species in regard to the optimal hormone concentrations or balance. Many plant tissues require a proper sequence of hormones, in order to regenerate whole plants, either via embryogenesis (Steward et aI., 1967) or organogenesis (Chen and Galston, 1967). The purpose of the present investigation was to study the capacity of buckwheat cells to undergo the change in their differentiation program and to develop into Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
38
VEROSLAVA SREJOVIC and MIRJANA NESKOVIC
whole plantlets. It seems that buckwheat tissue provides a suitable system for studying the sequential role of hormones in organ redifferentiation.
Material and Methods Fruits of Fagopyrum esculentum Moench. cv. Pennquad, of a tetraploid line, were surface sterilized for 2 min with ethanol, the pericarp was removed and the peeled seeds further sterilized for 15 min in 5% calcium hypochlorite solution. After washing, they were left in sterile water for 3 h to imbibe. Then the endosperm was removed under sterile conditions, the embryo axis cut off and the folded cotyledons cut transversely into apical and basal parts, which were put into culture. Three segments were cultivated on 40 ml of agar media, in 100 ml Erlenmayer flasks. Each experiment was started with 12 flasks and was repeated at least three times. Basal medium (BM) for the cultivation of tissue contained the mineral solution Bs {Gam borg et aI., 1968),3% sucrose, 1% agar and (in mg I-I): thiamine 2, pyridoxine 1, nicotinic acid 1, minositol 100 and casein hydrolysate 2000. Auxins 2,4-dichlorophenoxyacetic acid (2,4-D), unaphtaleneacetic acid (NAA), indolyl-3-acetic acid (IAA) and indolyl-3-butyric acid (IBA), as well as cytokinins kinetin (KIN) and benzylaminopurine (BAP) were added in different concentrations, as specified in the text. All cultures were maintained in diffuse light of 4500 K «Tesla» fluorescent tubes, at light intensity of 1500 Ix. The day length was 16 h, and the temperature was regulated at 25 ± 1 0c. Callus pieces in the course of organogenesis were fixed in Carnoy fixative, embedded in paraffin and stained with gallocyanin, hematoxylin, safranin or light green.
Results Induction of cell division in cotyledon explants In mature seeds cotyledons have a large, thin, tightly folded blade, with the anatomy of a typical leaf. At the time of excision, vascular bundles were not fully differentiated and 2 - 3 layers of cambial cells were visible between xylem and phloem. Cotyledon explants were cultivated for the first 5 days on a medium suitable to induce cell division, and containing BM supplemented with 2,4-D 5 mg 1-1 and KIN 0.1 mg 1-1. During this time the cotyledon fragments roughly doubled in size and became green. The first cell divisions were observed after 3 days in culture and comprised a row of spongy parenchyma cells between vascular bundles (Figs. 1 and 2) and parenchymatous cells around the midrib vein (Fig. 3). The divisions soon proceeded in other mesophyll cells and after 2-4 weeks the inner tissue of the cotyledon blades was transformed into numerous meristematic regions (Fig. 4). A yellowish callus, partly containing anthocyanin, was visible on the edge of the blades. This callus was used for further subcultures. Growth of callus tissue and organogenesis The callus tissue grew poorly when subcultured on the same medium in which cell division was induced, but its growth was very much improved by lowering the 2,4-D concentration to 1 mg 1-1 and by increasing the KIN concentration to 1 mg 1-1. Ana-
Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
In vitro plant regeneration in buckwheat
39
Figs. 1-3: Transverse sections through cotyledon explants grown for 3 days on the medium for cell division (2,4-D 5, KIN 0.1 mg 1-1). Fig. 1: Cell division in a row of spongy parenchyma cells between the vascular bundles (30x). - Fig. 2: Detail of dividing cells between two neighbouring bundles (126x). - Fig. 3: Divided cells around the midrib vein (60 x ). Fig. 4: Meristematic nodules in cotyledon explants grown for 15 days on the medium for callus maintenance (2,4-D 1, KIN 1 mg 1- 1)(65x). tomical observation showed that the callus was composed mainly of meristematic nodules, developing into root initials, with very little parenchymatous tissue between them. Numerous roots were elongated when the 2,4-D concentration was further decreased to 0.1 mg 1-\ or when the auxin was omitted (Fig. 5), but these calluses survived 5-6 transfers and then became necrotic. The calluses kept on 2,4-D+KIN media did never produce shoots. It was found that shoot formation could only be induced when the primary cotyledon explants were removed after 3 - 5 days from the medium suitable for cell division, and transferred to another medium, with a high cytokinin to auxin ratio. Meristematic nodules developed then into typical shoot meristems. By the end of the first, or during the second passage, the calluses in up to 40% of explants gave rise to a large number of buds. Growth of the buds apparently improved callus growth and this in turn led to the formation of many more buds. However, the buds failed to develop if the tissue was transferred to the inductive medium later than at 4 weeks. The first tissue with buds was obtained in May 1978 and it is still maintained in culture (Fig. 6), with an unchanged capacity for shoot formation. The same experiment was repeated several times with numerous fresh explants and the results obtained were always similar.
Z. Pjlanzenphysiol. Bd. 104. S. 37-42.1981.
40
VEROSLAVA SREJOVIC
and MIRJANA
NESKOVIC
Fig. 5: Calluses with roots at the end of 2nd subculture (2,4-D 1, KIN 0.1 mg 1-') (1 x). - Fig. 6: Calluses with numerous buds and shoots at the end of 9th subculture (IAA 10- 6 M, BAP 10- 5 M) (Ix). - Fig. 7: A shoot with adventitious roots, developed after 10 days on the medium without hormones (1.3 x). - Fig. 8: Flowering plant in soil (0.35 x).
The combination of hormones which was used for shoot induction consisted of IAA 10- 6 M and BAP 10- 5 M. Substitution of IAA with 2,4-D or NAA, as well as the substitution of BAP by KIN, all in equimolar concentrations, produced a considerabZ. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
In vitro plant regeneration in buckwheat
41
ly lower number of shoots, the tissue grew slowly, and had to be abandoned, usually after 2 passages. In the IAA + BAP medium the number of bud initials per callus was rarely less than 50. Regeneration of whole plants
Shoots which differentiated on IAA + BAP medium did never produce roots on the same medium. Well developed shoots were isolated, cleaned of callus pieces and adventitious roots developed on. their basal parts (Fig. 7). The rooting was best on BM + lEA (1 mg 1-1), but roots also developed when all hormones were omitted. Plantlets which grew well on agar were carefully taken out, washed from the medium and grown first in peat, moistened with mineral solution. When the plants became vigorous, they were transferred into pots with soil, where they continued growing to a height of 30 cm or more (Fig. 8). A cluster of normal, white flowers developed in almost all regenerated plants. Flowering was always preceded by, or occurred simultaneously, with rooting. Although the large scale propagation of buckwheat plants was not the primary object of the present study, it is clear from the results obtained that it can be achieved with high frequency. The transfer of plants into the soil was not accompanied by great difficulties, provided the plants were protected in a humid atmosphere. Discussion Buckwheat tissue culture, described in the present paper, was initiated in cotyledon explants, composed of differentiated, but not fully grown cells. The cells, which were most responsive to the cell division stimulus, were those lying between the vascular bundles, in continuity with the remaining cambium. In this respect buckwheat cotyledons differ from some other leaf explants, in which cell division and subsequent organogenesis originate in epidermal or subepidermal cell layers (Tran Thanh Van and Drira, 1971; Venverloo, 1976). The sequential use of hormones in the present work has enabled us to distinguish between the two phases in organ development: the initial renewal of cell division and the subsequent phase of organ determination. This sequential change of hormones may not be indispensible for the complete regeneration of plants. A very limited number of shoots developed when the first medium was omitted. It seems, therefore, that the high 2,4-D content in the beginning influenced the abundance of bud formation, probably by increasing the number of dedifferentiated cells. These cells become subsequently determined to form root or shoot initials, in response to the specific auxin to cytokinin ratio. The number of species in which plantlets can be regenerated from explants in vitro is increasing rapidly. Nevertheless, there are still many recalcitrant plants or tissues, which do not respond to the usual hormonal stimuli. If. in such a species root and Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.
42
VEROSLAVA SREJOVIC and MIRJANA NESKovrc
shoot formation are alternative events, like in buckwheat, one has probably to consider sequential changes in hormone complex and to find out the correct time to apply them. It is clear that a better understanding of the primary events in cell determination (Hicks, 1980) is the key for further progress in this field. References CHEN, H. and A. W. GALSTON: Growth and development of Pelargonium pith cells in vitro. II. Initiation of organized development. Physiol. Plant. 20, 533-539 (1967). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). HICKS, G. S.: Patterns of organ development in plant tissue culture and the problem of organ determination. Bot. Rev. 46,1-23 (1980). MURASHIGE, T.: Manipulation of organ initiation in plant tissue cultures. Bot. Bull. Acad. Sin. 19,1-24(1977). - The impact of plant tissue culture on agriculture. In: T. A. THORPE (Ed.): Frontiers of Plant Tissue Culture 1978, pp. 15-26. University of Calgary, Calgary, 1978. NARAYANASWAMY, S.: Regeneration of plants from tissue cultures. In: J. REINERT and Y. P. S. BAJAJ (Eds.): Plant Cell, Tissue and Organ Culture, pp. 179-248. Springer-Verlag, Berlin, 1977. SKOOG, F. and C. O. MILLER: Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exptl. BioI. 11, 118 -131 (1957). STEWARD, F. c., A. E. KENT, and M. O. MAPES: Growth and organization in cultured cells: Sequential and synergistic effects of growth regulating substances. In: Plant Growth Regulators. Ann. N. Y. Acad. Sci. 144, 326-334 (1967). TRAN THANH VAN, M. and A. DRIRA: Definition of a simple experimental system of directed organogenesis de novo: organ neoformation from epidermal tissues of Nautilocalyx lynchei. In: ColI. Intern. C. N. R. S. No. 193, 169-176 (1971). VENVERLOO, C. J.: The formation of adventitious organs. III. A comparison of root and shoot formation on Nautilocalyx explants. Z. Pflanzenphysiol. 80, 310-322 (1976). YAMANE, Y.: Induced differentiation of buckwheat plants from subcultured calluses in vitro. Jap. J. Genet. 49,139-146 (1974).
Z. Pjlanzenphysiol. Bd. 104. S. 37-42. 1981.