- Email: [email protected]
Chemical Factors Controlling Morphogenesis of Petunia Cells Cultured in vitro
Biochem. Physiol. Pfhtnzen 170, S. 77 -84 (1!J76)
Chemical Factors Controlling Morphogenesis of Petunia Cells Cultured in vitro RAJBlR S. SANGW AN and HIROSHI HARADA Laboratoire de Physiologie Pluricellulaire, Hamean de I'Yvette, Bat. C, Gif-sUT Yvette, France Key Term Index: cell culture, chemical factors, morphogenesis, phytohormones; Petunia inflata, P. hybrida.
Summary Calluses of different types were obtained from in vitro cultured stem segments of Petunia inflata R. E. FRIES and Petunia hybrida L. (var. Cascade and Rose du Ciel) using different agarified media. A cell suspension from each type of callus was prepared in ,t liquid medium, the composition of which was the same as for the primary cultures (l\U: £lISa medium + 2,4-D 1 mg/l; MIl: MSb medium + 2,4-D 1 mg/l; £11111: £lISa medium + IAA 1 mg/l + BA 200 fLg/l + GA3 200 fLg/l). Each suspension culture was then transferred and grown in £lISa medium + 2,4-D 100 fLg/1 + kinetin 100 fLg/l (MIV). The morphogenetic potential of suspended cells depended upon the medium from which it had originally been derived. Cells derived from Ml gave somatic embryos, from £lIn gave roots whilst those from MIn gave undifferentiated calluses and shoot primordia. Most of the somatic embryos were morphologically similar to zygotic embryos and gave rise to new plants.
Introduction
EVer since STEWARD et al. (1958) demonstrated cellular totipotency, suspension cultures of plants cells are becoming increasingly important as experimental material for investigations on plant growth and metabolism. Some of the problems and potentials of the cell suspension culture technique have been discussed by CAREW and STABA (1965), STREET (1966), HALPERIN (1966), ROSSINI (1975) and JULLIEN (1975). Initial attempts to culture plants cells in liquid media necessitated the addition of coconut milk (STEWARD et al. 1964) or other organic supplement to the medium (TuLECKE 1961); but several cultures have now been observed to grow in chemically defined media of varying complexity (HALPERIN 1966). A large number of plants were obtained from single cell culture in tobacco (VASIL and HILDEBRANDT 1965a, 1965b), in carrot (HALPERIN 1966) and in Asparagus (JULLIEN 1975). Carrot tissues have been grown and induced to differentiate successfully by several workers but only STEWARD et al. (1970) and more particularly HALPERIN (1966) have identified and described two distinct forms of morphogenetic events in vitro: rhizogenesis and embryogenesis, respectively. More interesting investigations were conducted by BACKS-HuSEI\IANN and REINERT (1970) who employed isolated single cells from a suspension culture of carrot with subsequent study of their behaviour. Abbreviations used: BA, 6-benzyladenine; CM, coconut milk; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA 3, gibberellin A3; IAA, indole-3-acetic acid.
78
H. S. SAXGWAX and H. HARADA
The genus Petunia, like carrot, tobacco and Asparagus, has been widely studies from the tissue culture point of view. In m'tro bud formation has been observed from excised stem segments RAO et a1. (1973), and from protoplasts (DURAND et a1. 1972; BINDING 1974). On the other hand induction of androgenic plants in cultures of immature anthers was achieved in Petunia axillaris and Petunia hybrida (RAQUIN et a1. 1972 ; WAGNER and HESS 1974) and in culture of isolated pollen grains in Petunia hybrida (SANGWAN and NORREEL 1975). The present paper describes in detail the process of plant regeneration, through somatic embryogenesis or through root + bud formation, from separate cells of Petunia inflata or Petunia hybrida.
Material and Methods Growth conditions: Petunia inflata R. E. FRIES and Petunia hybrida L. (var. Cascade and Rose du Ciel) cultivated in a greenhouse under a temperature regime of 24°C (day) - 17 °C (night) and 16 hlight condition, formed the experimental material. Internodal stem segments (1 em in length) were excised from surface-sterilised plants and cultured on a medium comprising MUSASHIGE and SKOOG'S macroelements (1962), microelements of NITSCH (1968) and vitamin supplement (NITSCH and NITSCH 1965). This medium, hereafter referred to as MSa medium, contained 2 % sucrose and 0.6 % Difco-Bacto agar. Iron-EDTA was prepared as specified by MURASHIGE and SKOOG (1962) but using half their concentration. The MSb medium was the same as the MSa medium ex('ept that the NH4 NOa and KNO a were suppressed in MSb medium. The pH of the medium was adjusted to 5.6 with 0.1 N KOH and 0.1 N HCl before adding the agar and autoclaving (120°C, 20 min). All growth substances except CM and GAa, were added to the medium which was dispensed in 20 ml aliquots into culture tubes and autoclaved. CM and GAa were added sterilely after filtration through a Swinex Millipore filter (pore size 0.45 m,u). Cultures were grown at 28° ± 1 °C (day) and 21° ± 1 °C (night) and were illuminated by approximately 5000 lux for a 10 h period each day. For liquid medium, nitrogen was supplied as in MSa medium. The microelements, iron-EDTA and vitamins were the same as in the solid medium.
Isolation of the cells After a number of preliminary experiments, undifferentiated callus pieces (5 g) isolated from 4 week-old cultures which had been maintained on the following 3 different media were used: C
= MSa = MSb MIll = MSa
MI
:\m
+ 2,4-D (1 mg/I), + 2,4-D (1 mg/I), + IAA (1 mg/l) + BA (200,ug/l) + GAa (200 ,ug/l)
These callus pieces were put into culture flasks containing 100 ml of liquid media of the same compositions, and the culture flasks were agitated on a gyratory shaker at 60 rpm for 72 h at 25°C and with 18 hours of light per day. Single ('ells and small aggregates containing fewer than 5 cells were obtained by filtering the suspension through a sieve (150 ,um). This cell fraction was washed twice with the MIV medium (MSa + 2,4-D 100 fig/I + kinetin 100 pg/1) by centrifugation at 50 g for 5 min and then resuspended in MIV medium.
Cell suspension culture The separated cells were cultured in small Erlenmeyer flasks containing 10 ml of MIV which were inoculated with 5 x 104 cells per ml. The flasks were maintained at 25°C for 3 days in the dark (ENZ-
Chemical Factors Controlling Morphogenesis in Cell Cultures
79
1973) and then illuminated continuously with approximately 1000 lux. These cultures were agitated on a reciprocal shaker at 40 strokes per min at 25 ac. No study was made on the importance of light or temperature. 'IIANN-BECKER
Results
Callus formation Throughout the present investigation internodal stem segments of P. inflata and P. hybrida were used as starting material for callus formation. The explants showed a vigourous growth to produce a friable, soft, white callus with 2,4-D on both mineral media (MI and MIl), while with MIll a comparatively compact green callus was formed. Occasional limited nodular callus growth was observed on MSa and MSb medium without growth substances. Somatic embryogenesis Cell suspensions taken from MI-calluses gave rise to young somatic embryos after 4 weeks on MIV (Plate 1). The embryogenic suspension cultures Were composed of embryogenic clumps similar to those reported in carrot (SMITJI and STREET 1974), vacuolated free cells and small aggregates of such cells showing no evidence of division. The embryogenic clumps were composed of small cells rich in cytoplasm. When the suspension was transferred to liquid medium with low auxin concentration (MIV), it became studded with asynchronously developing embryos. Later, these embryos completed their development as free-floating structures (Figs. 8, 9). The somatic embryos were isolated or joined at the radicle ends in groups of two or more. Some of the isolatr'd embryos had a micro callus constituted of large cells with voluminous nuclei at the radicle end (Fig. 10). Embryos formation occurred within small calluses. They first appeared as little globular masses of meristematic cells which grew al1d then passed through heart and torpedo stages. Eventually cells of the hypocotyl underwent considerable expansion and elongation, and the embryos showed marked radical-plumule axis as well as the differentiation of vascular strands. At maturity the embryos usually possessed two cotyledons but occasionally pluricotyledonous embryos were also seen. When transferred to solid MSa medium without growth hormones al1d 0.5 % sucrose, most of the embryos gave rise to plantlets (Fig. 14) and then to plants which whert cultured in pots in a greenhouse, flowered normally. Feulgen-squashes from root-tips of regenerated plants revealed that they were all diploid (2n = 14). Rhizogenesis When the auxin-induced callus cells grown on the nitrogen lacking medium MIl were inoculated into low auxin liquid medium (MIV), a large number 9f roots (Fig. 11, 12), callusses with roots (Fig. 13) and abnormal embryos were formed after 3 weeks of culture. While studying the behaviour of this cell suspension, we found that disorganized cell clumps first developed. In the older clumps many areas in which a central tracheid-
80 like group of cells was surrounded by a ring of cambium-like cells, were observed. Generally the roots primordia originated from localized regions of non-vacuolated, densly cytoplasmic cells. There was no obvious developmental relationship between root primordia and tracheid-cambial associations present in the same clump. Callus and shoot development The cell fraction from the MIll induced calluses when innoculated into low auxin liquid medium (MIV), shOWing a few greenish multicellular structures. After 3 weeks of culture in the liquid medium, there was neither embryo development nor root formation, but only undifferentiated masses of green calluses were formed. Shoot development only took place by transferring the callus bearing bud primordia from liquid to solid medium of the same composition (MIV). After 2 weeks of culture on the solid medium, a large number of shoots developed from each callus but there was no root formation. We obtained root formation by transferring the shoots-bearing calluses to solid MSa medium with IAA (100 ",gil) and 1 % sucrose. Discussiou
The present experiments demonstrate that the ability of the suspended cells of P. inflata and P. hybrida to form somatic embryos, roots and shoots depends upon the physiological state of original calluses, influenced by different growth substances present in culture media. So it is not the last liquid medium which is responsible for the change in the morphogenetic responses but the primary media used to promote callus growth before isolation of the cells. Other workers also indicated some morphogenetical changes in various plant tissue cultures similar to those described here (HALPERIN 1966; TORREY 1966; KAO et al. 1970; KESSEL and CARR 1972; SMITH and STREET 1974). The calluses which were formed from stem explants cultured on the medium lacking nitrogen (MIl) usually consisted of a mass of large parenchymatous cells and a few compact multicellular units. When such callus is sieved and inoculated into low auxin medium with kinetin (MIV), multicellular clumps made of completely disorganized, loosely cohering cells, are formed. After a considerable production of parenchymatous or tracheid-like cells in such clumps, a relatively limited number of internal cells undergo the process of dedifferentiation and eumeristematic area appears. These internal cells give rise to a root primordium. The noteWorthy feature of rhizogenesis under these circumstances are a delayed formation of eumeristematic cells and an essentially endogenous origin of such cells. A similar observation was made by HALPERIN (1966) in carrot. On the other hand, stem explants grown on medium containing high amounts of reduced nitrogen and 2,4-D (MI) produced calluses and numerous multicellular structures containing relatively small cells. Apparently the high level of reduced nitrogen in the medium induces the cells of the callus to divide with a minimum expansion of daughter cells. When this material is sieved and inoculated into low auxin-kinetin medium (MIV), dedifferentiation and polarized growth proceed rapidly and within several days well-formed somatic proembryos are evident.
Chemical Factors Controlling Morphogenesis in Cell Cultures
81
In contrast, stem explants grown on medium containing IAAjBAj GA3 produced a compact green callus usually consisting of parenchymatous cells. When such a callus is sieved and inoculated into MIV medium, non-embryogenic masses without root and embryos are formed within several days. These experiments indicate that the cells from same explants and cultured in same liquid medium are capable of giving rise to root-bearing clumps, embryos, or to masses bearing bud primordia, depending upon the particular chemical environment in which they have been previously growll. Our results also have a bearing upon the determination of the factors required to induce somatic embryogenesis and subsequent embryo development. The role of auxins in inducing embryogenesis has been demonstrated by many authors. Although 2,4-D was generally used (HALPERIN and WETHERELL 1964; NAKAJIMA 1963; RAO and NARAYANASWAMI 1972; SANGWAN and HARADA 1975), sometimes IAA (KATO and TAKEUCJII 1963) or other auxins have been used (NORREEL and NITSCH 1968). The subsequent development of embryos into plantlets was accomplished by reducing the concentration of auxin (HALPERIN and WETHERELL 1964; HALPERIN 1966) or by omitting it from the medium (NAKAJIMA 1963). In the present investigations, 2,4-D along with reduced nitrogen was very effective in inducing embryogenesis, but for the development of embryos into plantlets, it was necessary to reduce the auxin concentration in the medium. Another important nutritive factor effecting somatic embryogenesis in vitro is the reduced nitrogen concentration and the form in which nitrogen is present in the nutrient medium (HALPERIN and WETHERELL 1965; NORREEL and NITSC;H 1968; TAZAWA and REINERT 1969; HALPERIN 1970). Reduced nitrogen is also required for the growth of isolated zygotic embryos cultured in vitro (MATSUBARA 1962; MONNIER 1973). We have shown that abnormal somatic embryogenesis occurs with low reduced nitrogen concentration or with medium lacking reduced nitrogen, and that high level of reduced nitrogen greatly enhances the rate of normal somatic embryos. TAZAWA and REINERT (1969) have also shown that the somatic embryos of carrot are readily produced in media containing relatively large amount of ammonium ions and nitrate, but embryos are never formed on a medium containing only nitrate in a low concentration. Although the presence of NH4 + in the medium is not necessary for embryo formation in vitro, it appears that a certain level of intercellular NH4 + is a prerequisite for this process. Since there is a positive correlation between embryo formation and the content in the cultures of both soluble and insoluble organic nitrogen, it is probable that NH4 + is important for embryo formation, being an essential substrate for the synthesis of organic nitrogen componds such as amino acids and proteins. We also found that cytokinin and gibberellin A3 when employed simultaneously, cause an inhibition of the appearance of embryogenic cells, but have little effect on the development of embryogenic cells to produce embroids. Callus maintained for several subcultures on media containing only 2,4-D soon undergoes a transformation involving an increase in the growth rate, a loss of chloro6 Bi ••hom. PhY8ioi. Pflanzen, Bd. 170
Ai
82
4$
R. S. SANGW AN and H.
,,;;; LA 4414libl I$ii;
*
H.~RAD.I
phyll and the iilability to form embryos. In carrot this type of callus was found to be entirely aneuploid (NEWCOMB and WET,HERELL 1970). Acknowledgement We are greatly indebted to Professor R. GORENFLOI' for his critical reading of the manuscrijit and to Doctor Brigitte SANGWAN-:'\ ORREEL for h~r help throughout the course of this research.
References BACKS-HuSEMAX, D., and REINERI', J., EmbryJbilduug durch isolierte Einzelzellen aus Gewebe kulturen von ])aucus carota. Protoplasma 70, 49 - 60 (1970). BINDING, H., Regeneration of haploid plants from protoplasts of Petunia hybrida L. Z. Pflanzenphysiol. 74, 327 -356 (1974). CAREW, D. P., and STRABA, E. J., Plant tissue culture: its fundamentals, application and relationship to medicinal plant stndies. Lloydia 28, 1-27 (1965). DeR,lxD, J., POTRYKUS, 1., aud DONN, G., Plantes issues de protoplastes de Petunia. Z. Pflanzenphysiol. 69, 26-34 (1973). ENZMANK-BECKER, G., Plating efficiency of protoplasts of tobacco in different light conditions. Z. Naturforschung 28c, 470-471 (1973). HALPERIN, W., Alternative morphogenetic events in cell suspension. Amer. J. Bot. ;'3, 443-453 (1966). Embryos from somatic plant cells. In: Control Mechanism in the Expression of Cellular Phenotypes, ed., H. A. Padykula, Symp. Int. Soc. Cell BioI. 9, 169-191. Academic Press, New York (1970). and WETHEIlELL, D. F., Adventive embryony in tissue cultures of wild carrot, Daucus ~arota,. Amer. J .. Bot. 51, 274-283 (1964). _ Ontogeny of adventive embryos of wild carrot. Science 147, 756-758 (1965). JULLIEN, M., Contribution a l'etude de rnltures ill vitro de cellules separees de tissns foliaires et de leurs potentialites organogenes ehez quelques plantes superieures, en particulier chez A.sparagus officinalis. These 3eme ryde, Univeriste de Paris VI (1975). KAO, K. N., MILLER, R. A., GA~IBORG, O. L., and HARVEY, B. L., Variations in chromosome number and structure in plant cells grown in suspension cultures. Can. J. Genet. Cytol. 12, 297 -301 (1970). KATO, II., and T.IKErcHI, M., Morphogenesis ill vitro starting from single cell of carrot root. Plant and Cell Physiol. 4, 243- 245 (1963). KESSEL, R. H. J., and CARll, A. H., The effect of dissolved oxygen concentration on growth and differentiation of carrot (Daucus carota) tissue. J. Exp. Bot. 23, 996-1007 (1972). MATSUBARA, S., Studies on a growth promoting substance "embryo factor" necessary for the culture of young embryos of Datura tatum ill ritro. Bot. Mag. Tokyo 75, 12 -18 (1962). MONNIER, M., Croissance et developpement des embryons globulaires de Capsella bursa-pastoris cultives in vitro dans un milieu it base d'une nouvelle solution minerale. Soc. bot. Fr., Memoires, ColI. Morphologie (Gif), 179 -194 (1973). MURASHIGE, T., and SKOOG, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantar. 15, 473-497 (1962). NEWCOMB, W., and WETHERELL, D. F., The effects of 2, 4, 6-trichlorophenoxyacetic acid on embryogenesis in wild carrot tissue cultures. Bot. Gaz. 131, 242-245 (1970). NAKAJIMAQ, T., On the plant tissue culture with special reference to embryogenesis. In: Cell Differentiation and Somatic Mutation, Gamma Fiels Symp. n° 2, Tokyo, 25-33 (1963). NITSCH, C., Induction in vitro de la floraison chez nne plante de jours courts, Plumbago indica L. These d'Universite, Masson Ed. (1968).
Chemical Fattors Controlling Morphogenesis in Cell Cultures
83
NITSCH, J. P.,
G*
--......,. 84:
-,,,.,
R.
...... ,,'
,~.,
~
....,.......
S. S.~NGW.~:'!
,.
and
......~-,
'-
_. . - ..
~.
~
....,
H. HARADA
Explanation of Plates Plate 1 Cell suspension cultures of Petunia hybrida (var. Rose du Ciel).
Fig. 1. Single cell on the first day of culture, (x 528). Fig. 2 to 6. Dividing cells and micro calluses after 2 (Fig. 2), 4 (Fig. 3), 10 (Fig. 4), 15 (Fig. 5) and 18 days (Fig. 6) in culture, (x 528). Fig. 7. Embryogenic calluses originated from cell suspension culture, ( x 1,5). Plate 2 Somatic embryos, roots and planllet issued from cell suspension culture of Petunia. Fig. 8 and 9. Somatic embryos at globular, heart (Fig. 8) and torpedo shaped (Fig. 9) stages, (x 18)
and torpedo shaped (Fig. 9) stages, ( X 18). Fig. 10. Somatic embryos with microcalluses at the radicle end, (x 18). Fig. 11 to 13. Rhizogenesis. Single root (Fig. 11), associated roots (Fig. 12) and root-bearing callus (Fig. 13), ( x 26). Fig. 14. Young plant after 40 days in culture, (x 0.8).
BPP 170
2
Plate 1
5
Sangwan and Harada VEB GUSTAV FISCHER VERLAG lENA
6
Pill/e :2
BPP 170
Sangwan and Harada VEB GUSTAV FISCHER VERLAG JENA
Chemical Factors Controlling Morphogenesis of Petunia Cells Cultured in vitro RAJBlR S. SANGW AN and HIROSHI HARADA Laboratoire de Physiologie Pluricellulaire, Hamean de I'Yvette, Bat. C, Gif-sUT Yvette, France Key Term Index: cell culture, chemical factors, morphogenesis, phytohormones; Petunia inflata, P. hybrida.
Summary Calluses of different types were obtained from in vitro cultured stem segments of Petunia inflata R. E. FRIES and Petunia hybrida L. (var. Cascade and Rose du Ciel) using different agarified media. A cell suspension from each type of callus was prepared in ,t liquid medium, the composition of which was the same as for the primary cultures (l\U: £lISa medium + 2,4-D 1 mg/l; MIl: MSb medium + 2,4-D 1 mg/l; £11111: £lISa medium + IAA 1 mg/l + BA 200 fLg/l + GA3 200 fLg/l). Each suspension culture was then transferred and grown in £lISa medium + 2,4-D 100 fLg/1 + kinetin 100 fLg/l (MIV). The morphogenetic potential of suspended cells depended upon the medium from which it had originally been derived. Cells derived from Ml gave somatic embryos, from £lIn gave roots whilst those from MIn gave undifferentiated calluses and shoot primordia. Most of the somatic embryos were morphologically similar to zygotic embryos and gave rise to new plants.
Introduction
EVer since STEWARD et al. (1958) demonstrated cellular totipotency, suspension cultures of plants cells are becoming increasingly important as experimental material for investigations on plant growth and metabolism. Some of the problems and potentials of the cell suspension culture technique have been discussed by CAREW and STABA (1965), STREET (1966), HALPERIN (1966), ROSSINI (1975) and JULLIEN (1975). Initial attempts to culture plants cells in liquid media necessitated the addition of coconut milk (STEWARD et al. 1964) or other organic supplement to the medium (TuLECKE 1961); but several cultures have now been observed to grow in chemically defined media of varying complexity (HALPERIN 1966). A large number of plants were obtained from single cell culture in tobacco (VASIL and HILDEBRANDT 1965a, 1965b), in carrot (HALPERIN 1966) and in Asparagus (JULLIEN 1975). Carrot tissues have been grown and induced to differentiate successfully by several workers but only STEWARD et al. (1970) and more particularly HALPERIN (1966) have identified and described two distinct forms of morphogenetic events in vitro: rhizogenesis and embryogenesis, respectively. More interesting investigations were conducted by BACKS-HuSEI\IANN and REINERT (1970) who employed isolated single cells from a suspension culture of carrot with subsequent study of their behaviour. Abbreviations used: BA, 6-benzyladenine; CM, coconut milk; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA 3, gibberellin A3; IAA, indole-3-acetic acid.
78
H. S. SAXGWAX and H. HARADA
The genus Petunia, like carrot, tobacco and Asparagus, has been widely studies from the tissue culture point of view. In m'tro bud formation has been observed from excised stem segments RAO et a1. (1973), and from protoplasts (DURAND et a1. 1972; BINDING 1974). On the other hand induction of androgenic plants in cultures of immature anthers was achieved in Petunia axillaris and Petunia hybrida (RAQUIN et a1. 1972 ; WAGNER and HESS 1974) and in culture of isolated pollen grains in Petunia hybrida (SANGWAN and NORREEL 1975). The present paper describes in detail the process of plant regeneration, through somatic embryogenesis or through root + bud formation, from separate cells of Petunia inflata or Petunia hybrida.
Material and Methods Growth conditions: Petunia inflata R. E. FRIES and Petunia hybrida L. (var. Cascade and Rose du Ciel) cultivated in a greenhouse under a temperature regime of 24°C (day) - 17 °C (night) and 16 hlight condition, formed the experimental material. Internodal stem segments (1 em in length) were excised from surface-sterilised plants and cultured on a medium comprising MUSASHIGE and SKOOG'S macroelements (1962), microelements of NITSCH (1968) and vitamin supplement (NITSCH and NITSCH 1965). This medium, hereafter referred to as MSa medium, contained 2 % sucrose and 0.6 % Difco-Bacto agar. Iron-EDTA was prepared as specified by MURASHIGE and SKOOG (1962) but using half their concentration. The MSb medium was the same as the MSa medium ex('ept that the NH4 NOa and KNO a were suppressed in MSb medium. The pH of the medium was adjusted to 5.6 with 0.1 N KOH and 0.1 N HCl before adding the agar and autoclaving (120°C, 20 min). All growth substances except CM and GAa, were added to the medium which was dispensed in 20 ml aliquots into culture tubes and autoclaved. CM and GAa were added sterilely after filtration through a Swinex Millipore filter (pore size 0.45 m,u). Cultures were grown at 28° ± 1 °C (day) and 21° ± 1 °C (night) and were illuminated by approximately 5000 lux for a 10 h period each day. For liquid medium, nitrogen was supplied as in MSa medium. The microelements, iron-EDTA and vitamins were the same as in the solid medium.
Isolation of the cells After a number of preliminary experiments, undifferentiated callus pieces (5 g) isolated from 4 week-old cultures which had been maintained on the following 3 different media were used: C
= MSa = MSb MIll = MSa
MI
:\m
+ 2,4-D (1 mg/I), + 2,4-D (1 mg/I), + IAA (1 mg/l) + BA (200,ug/l) + GAa (200 ,ug/l)
These callus pieces were put into culture flasks containing 100 ml of liquid media of the same compositions, and the culture flasks were agitated on a gyratory shaker at 60 rpm for 72 h at 25°C and with 18 hours of light per day. Single ('ells and small aggregates containing fewer than 5 cells were obtained by filtering the suspension through a sieve (150 ,um). This cell fraction was washed twice with the MIV medium (MSa + 2,4-D 100 fig/I + kinetin 100 pg/1) by centrifugation at 50 g for 5 min and then resuspended in MIV medium.
Cell suspension culture The separated cells were cultured in small Erlenmeyer flasks containing 10 ml of MIV which were inoculated with 5 x 104 cells per ml. The flasks were maintained at 25°C for 3 days in the dark (ENZ-
Chemical Factors Controlling Morphogenesis in Cell Cultures
79
1973) and then illuminated continuously with approximately 1000 lux. These cultures were agitated on a reciprocal shaker at 40 strokes per min at 25 ac. No study was made on the importance of light or temperature. 'IIANN-BECKER
Results
Callus formation Throughout the present investigation internodal stem segments of P. inflata and P. hybrida were used as starting material for callus formation. The explants showed a vigourous growth to produce a friable, soft, white callus with 2,4-D on both mineral media (MI and MIl), while with MIll a comparatively compact green callus was formed. Occasional limited nodular callus growth was observed on MSa and MSb medium without growth substances. Somatic embryogenesis Cell suspensions taken from MI-calluses gave rise to young somatic embryos after 4 weeks on MIV (Plate 1). The embryogenic suspension cultures Were composed of embryogenic clumps similar to those reported in carrot (SMITJI and STREET 1974), vacuolated free cells and small aggregates of such cells showing no evidence of division. The embryogenic clumps were composed of small cells rich in cytoplasm. When the suspension was transferred to liquid medium with low auxin concentration (MIV), it became studded with asynchronously developing embryos. Later, these embryos completed their development as free-floating structures (Figs. 8, 9). The somatic embryos were isolated or joined at the radicle ends in groups of two or more. Some of the isolatr'd embryos had a micro callus constituted of large cells with voluminous nuclei at the radicle end (Fig. 10). Embryos formation occurred within small calluses. They first appeared as little globular masses of meristematic cells which grew al1d then passed through heart and torpedo stages. Eventually cells of the hypocotyl underwent considerable expansion and elongation, and the embryos showed marked radical-plumule axis as well as the differentiation of vascular strands. At maturity the embryos usually possessed two cotyledons but occasionally pluricotyledonous embryos were also seen. When transferred to solid MSa medium without growth hormones al1d 0.5 % sucrose, most of the embryos gave rise to plantlets (Fig. 14) and then to plants which whert cultured in pots in a greenhouse, flowered normally. Feulgen-squashes from root-tips of regenerated plants revealed that they were all diploid (2n = 14). Rhizogenesis When the auxin-induced callus cells grown on the nitrogen lacking medium MIl were inoculated into low auxin liquid medium (MIV), a large number 9f roots (Fig. 11, 12), callusses with roots (Fig. 13) and abnormal embryos were formed after 3 weeks of culture. While studying the behaviour of this cell suspension, we found that disorganized cell clumps first developed. In the older clumps many areas in which a central tracheid-
80 like group of cells was surrounded by a ring of cambium-like cells, were observed. Generally the roots primordia originated from localized regions of non-vacuolated, densly cytoplasmic cells. There was no obvious developmental relationship between root primordia and tracheid-cambial associations present in the same clump. Callus and shoot development The cell fraction from the MIll induced calluses when innoculated into low auxin liquid medium (MIV), shOWing a few greenish multicellular structures. After 3 weeks of culture in the liquid medium, there was neither embryo development nor root formation, but only undifferentiated masses of green calluses were formed. Shoot development only took place by transferring the callus bearing bud primordia from liquid to solid medium of the same composition (MIV). After 2 weeks of culture on the solid medium, a large number of shoots developed from each callus but there was no root formation. We obtained root formation by transferring the shoots-bearing calluses to solid MSa medium with IAA (100 ",gil) and 1 % sucrose. Discussiou
The present experiments demonstrate that the ability of the suspended cells of P. inflata and P. hybrida to form somatic embryos, roots and shoots depends upon the physiological state of original calluses, influenced by different growth substances present in culture media. So it is not the last liquid medium which is responsible for the change in the morphogenetic responses but the primary media used to promote callus growth before isolation of the cells. Other workers also indicated some morphogenetical changes in various plant tissue cultures similar to those described here (HALPERIN 1966; TORREY 1966; KAO et al. 1970; KESSEL and CARR 1972; SMITH and STREET 1974). The calluses which were formed from stem explants cultured on the medium lacking nitrogen (MIl) usually consisted of a mass of large parenchymatous cells and a few compact multicellular units. When such callus is sieved and inoculated into low auxin medium with kinetin (MIV), multicellular clumps made of completely disorganized, loosely cohering cells, are formed. After a considerable production of parenchymatous or tracheid-like cells in such clumps, a relatively limited number of internal cells undergo the process of dedifferentiation and eumeristematic area appears. These internal cells give rise to a root primordium. The noteWorthy feature of rhizogenesis under these circumstances are a delayed formation of eumeristematic cells and an essentially endogenous origin of such cells. A similar observation was made by HALPERIN (1966) in carrot. On the other hand, stem explants grown on medium containing high amounts of reduced nitrogen and 2,4-D (MI) produced calluses and numerous multicellular structures containing relatively small cells. Apparently the high level of reduced nitrogen in the medium induces the cells of the callus to divide with a minimum expansion of daughter cells. When this material is sieved and inoculated into low auxin-kinetin medium (MIV), dedifferentiation and polarized growth proceed rapidly and within several days well-formed somatic proembryos are evident.
Chemical Factors Controlling Morphogenesis in Cell Cultures
81
In contrast, stem explants grown on medium containing IAAjBAj GA3 produced a compact green callus usually consisting of parenchymatous cells. When such a callus is sieved and inoculated into MIV medium, non-embryogenic masses without root and embryos are formed within several days. These experiments indicate that the cells from same explants and cultured in same liquid medium are capable of giving rise to root-bearing clumps, embryos, or to masses bearing bud primordia, depending upon the particular chemical environment in which they have been previously growll. Our results also have a bearing upon the determination of the factors required to induce somatic embryogenesis and subsequent embryo development. The role of auxins in inducing embryogenesis has been demonstrated by many authors. Although 2,4-D was generally used (HALPERIN and WETHERELL 1964; NAKAJIMA 1963; RAO and NARAYANASWAMI 1972; SANGWAN and HARADA 1975), sometimes IAA (KATO and TAKEUCJII 1963) or other auxins have been used (NORREEL and NITSCH 1968). The subsequent development of embryos into plantlets was accomplished by reducing the concentration of auxin (HALPERIN and WETHERELL 1964; HALPERIN 1966) or by omitting it from the medium (NAKAJIMA 1963). In the present investigations, 2,4-D along with reduced nitrogen was very effective in inducing embryogenesis, but for the development of embryos into plantlets, it was necessary to reduce the auxin concentration in the medium. Another important nutritive factor effecting somatic embryogenesis in vitro is the reduced nitrogen concentration and the form in which nitrogen is present in the nutrient medium (HALPERIN and WETHERELL 1965; NORREEL and NITSC;H 1968; TAZAWA and REINERT 1969; HALPERIN 1970). Reduced nitrogen is also required for the growth of isolated zygotic embryos cultured in vitro (MATSUBARA 1962; MONNIER 1973). We have shown that abnormal somatic embryogenesis occurs with low reduced nitrogen concentration or with medium lacking reduced nitrogen, and that high level of reduced nitrogen greatly enhances the rate of normal somatic embryos. TAZAWA and REINERT (1969) have also shown that the somatic embryos of carrot are readily produced in media containing relatively large amount of ammonium ions and nitrate, but embryos are never formed on a medium containing only nitrate in a low concentration. Although the presence of NH4 + in the medium is not necessary for embryo formation in vitro, it appears that a certain level of intercellular NH4 + is a prerequisite for this process. Since there is a positive correlation between embryo formation and the content in the cultures of both soluble and insoluble organic nitrogen, it is probable that NH4 + is important for embryo formation, being an essential substrate for the synthesis of organic nitrogen componds such as amino acids and proteins. We also found that cytokinin and gibberellin A3 when employed simultaneously, cause an inhibition of the appearance of embryogenic cells, but have little effect on the development of embryogenic cells to produce embroids. Callus maintained for several subcultures on media containing only 2,4-D soon undergoes a transformation involving an increase in the growth rate, a loss of chloro6 Bi ••hom. PhY8ioi. Pflanzen, Bd. 170
Ai
82
4$
R. S. SANGW AN and H.
,,;;; LA 4414libl I$ii;
*
H.~RAD.I
phyll and the iilability to form embryos. In carrot this type of callus was found to be entirely aneuploid (NEWCOMB and WET,HERELL 1970). Acknowledgement We are greatly indebted to Professor R. GORENFLOI' for his critical reading of the manuscrijit and to Doctor Brigitte SANGWAN-:'\ ORREEL for h~r help throughout the course of this research.
References BACKS-HuSEMAX, D., and REINERI', J., EmbryJbilduug durch isolierte Einzelzellen aus Gewebe kulturen von ])aucus carota. Protoplasma 70, 49 - 60 (1970). BINDING, H., Regeneration of haploid plants from protoplasts of Petunia hybrida L. Z. Pflanzenphysiol. 74, 327 -356 (1974). CAREW, D. P., and STRABA, E. J., Plant tissue culture: its fundamentals, application and relationship to medicinal plant stndies. Lloydia 28, 1-27 (1965). DeR,lxD, J., POTRYKUS, 1., aud DONN, G., Plantes issues de protoplastes de Petunia. Z. Pflanzenphysiol. 69, 26-34 (1973). ENZMANK-BECKER, G., Plating efficiency of protoplasts of tobacco in different light conditions. Z. Naturforschung 28c, 470-471 (1973). HALPERIN, W., Alternative morphogenetic events in cell suspension. Amer. J. Bot. ;'3, 443-453 (1966). Embryos from somatic plant cells. In: Control Mechanism in the Expression of Cellular Phenotypes, ed., H. A. Padykula, Symp. Int. Soc. Cell BioI. 9, 169-191. Academic Press, New York (1970). and WETHEIlELL, D. F., Adventive embryony in tissue cultures of wild carrot, Daucus ~arota,. Amer. J .. Bot. 51, 274-283 (1964). _ Ontogeny of adventive embryos of wild carrot. Science 147, 756-758 (1965). JULLIEN, M., Contribution a l'etude de rnltures ill vitro de cellules separees de tissns foliaires et de leurs potentialites organogenes ehez quelques plantes superieures, en particulier chez A.sparagus officinalis. These 3eme ryde, Univeriste de Paris VI (1975). KAO, K. N., MILLER, R. A., GA~IBORG, O. L., and HARVEY, B. L., Variations in chromosome number and structure in plant cells grown in suspension cultures. Can. J. Genet. Cytol. 12, 297 -301 (1970). KATO, II., and T.IKErcHI, M., Morphogenesis ill vitro starting from single cell of carrot root. Plant and Cell Physiol. 4, 243- 245 (1963). KESSEL, R. H. J., and CARll, A. H., The effect of dissolved oxygen concentration on growth and differentiation of carrot (Daucus carota) tissue. J. Exp. Bot. 23, 996-1007 (1972). MATSUBARA, S., Studies on a growth promoting substance "embryo factor" necessary for the culture of young embryos of Datura tatum ill ritro. Bot. Mag. Tokyo 75, 12 -18 (1962). MONNIER, M., Croissance et developpement des embryons globulaires de Capsella bursa-pastoris cultives in vitro dans un milieu it base d'une nouvelle solution minerale. Soc. bot. Fr., Memoires, ColI. Morphologie (Gif), 179 -194 (1973). MURASHIGE, T., and SKOOG, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantar. 15, 473-497 (1962). NEWCOMB, W., and WETHERELL, D. F., The effects of 2, 4, 6-trichlorophenoxyacetic acid on embryogenesis in wild carrot tissue cultures. Bot. Gaz. 131, 242-245 (1970). NAKAJIMAQ, T., On the plant tissue culture with special reference to embryogenesis. In: Cell Differentiation and Somatic Mutation, Gamma Fiels Symp. n° 2, Tokyo, 25-33 (1963). NITSCH, C., Induction in vitro de la floraison chez nne plante de jours courts, Plumbago indica L. These d'Universite, Masson Ed. (1968).
Chemical Fattors Controlling Morphogenesis in Cell Cultures
83
NITSCH, J. P.,
G*
--......,. 84:
-,,,.,
R.
...... ,,'
,~.,
~
....,.......
S. S.~NGW.~:'!
,.
and
......~-,
'-
_. . - ..
~.
~
....,
H. HARADA
Explanation of Plates Plate 1 Cell suspension cultures of Petunia hybrida (var. Rose du Ciel).
Fig. 1. Single cell on the first day of culture, (x 528). Fig. 2 to 6. Dividing cells and micro calluses after 2 (Fig. 2), 4 (Fig. 3), 10 (Fig. 4), 15 (Fig. 5) and 18 days (Fig. 6) in culture, (x 528). Fig. 7. Embryogenic calluses originated from cell suspension culture, ( x 1,5). Plate 2 Somatic embryos, roots and planllet issued from cell suspension culture of Petunia. Fig. 8 and 9. Somatic embryos at globular, heart (Fig. 8) and torpedo shaped (Fig. 9) stages, (x 18)
and torpedo shaped (Fig. 9) stages, ( X 18). Fig. 10. Somatic embryos with microcalluses at the radicle end, (x 18). Fig. 11 to 13. Rhizogenesis. Single root (Fig. 11), associated roots (Fig. 12) and root-bearing callus (Fig. 13), ( x 26). Fig. 14. Young plant after 40 days in culture, (x 0.8).
BPP 170
2
Plate 1
5
Sangwan and Harada VEB GUSTAV FISCHER VERLAG lENA
6
Pill/e :2
BPP 170
Sangwan and Harada VEB GUSTAV FISCHER VERLAG JENA