Plant Cell Reports
Plant Cell Reports (1982) 1 : 261-263
© Springer-Verlag 1982
Propagation of Fern (Nephrolepis) Through Tissue Culture M. A. Padhya and A. R. Mehta Department of Botany, The Maharaja Sayajirao University of Baroda, Baroda-390002, India Received July 16, 1982/September 20, 1982
Abstract Rapid clonal propagation in three stolon bearing species of Nephrolepis was achieved by tissue culture methods. Explants from young stolons were grown on B5 (Gamborg et a l . 1968) medium supplemented with indolebutyric acid where they produced several l e a f primordia. These l e a f primordia were isolated and grown into entire p l a n t l e t s . An overall m u l t i p l i c a t i o n factor of 30-40 was attained within three months.
medium with 2% sucrose (basal medium-BM) and BM - supplemented with various growth regulators such as napthalenacetic acid (NAA), 2, 4-di chlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), kinetin (K), 6-benzyladenine (BAP) i n d i v i dually as per the requirements of each experiment. Three concentrations (0.5, 1.0, 2.0 mg/l) of each of these growth regulators were tested. The medium was adjusted to pH 5.7 before s o l i d i f y i n g with 0.6% agar. All cultures were incubated in continuous fluorescent l i g h t (1000Lux) at 25+_2~C.
Introduction Ferns have been c u l t i v a t e d for t h e i r ornamental foliage. Their demand as indoor plants has been increasing. Excised l e a f primordia of Osmunda cinnamomea (Steeves, 1966), Adiantum pedatum and other ferns (Wetmore, 1970) have been' grown to adult p l a n t l e t s in axenic cultures. The conventional method employed for propagation of ferns has been by a runnerset procedure. But ferns derived through t h i s method produced a single growth center from where only a few fronds developed at a time. In contrast, the ferns derived through tissue culture techniques produced several growth centers and from each growth center numerous fronds were produced, thus giving an a t t r a c t i v e look to the plants (Murashige, 1977). In the present i n v e s t i g a t i o n , an attempt has been made to standardise tissue culture techniques for rapid m u l t i p l i c a t i o n and propagation in three species of Nephrolepis. Materials and Methods Stolon segments from three species of Nephrol~pis: ~. c o r d i f o l i a P r e s l . , N. exaltata Schott, and N. acuta Presl., a l l growing in the University Botanical Gardens, Baroda, were collected. They were surface s t e r i l i s e d with mercuric chloride (0.1%), washed with s t e r i l e d i s t i l l e d wateG cut into 1 to 2 cm pieces and inoculated on various culture media. The culture media included B5 (Gamborg et al. 1968)
Results and Discussion Stolon segments of all three Nephrolepis species cultured on basal medium (BM) and BM supplemented with NAA, K, or BAP failed to express any morphogenic response, while segments cultured on BMwith 2, 4-D (2 mg/l) showed white friable callus formation from their cut ends. All stolon segments of three species of Nephrolepis responded to IBA ().0 mg/l) treatment. These segments showed greenish proliferations along their margin. In fact, these were the meristematlc growth centers from which 18-20 leaf primordia emerged by the third week. The leaf primordia situated on the outer side were old and developed into full-fledged leaves at the end of four weeks (Fig. 1). On the other hand, each of the younger leaf primordia situated near the center, on isolation and transfer to fresh medium after three weeks developed multiple shoots. The very young shoot primordia could thus be effectively used as propagules for rapid multiplication. Similar differential responses of young and old leaf primordia have been observed earlier in Osmunda cinnamomea by Steeves (1961) and also by Haight and Kuehnert (1971). I f the shoot primordia were transferred to freshly made medium after four weeks, instead of three, root formation was induced resulting into complete plantlet regeneration (Fig. 2). Formation of roots by regenerated shoots (after 4 weeks) on transfer to fresh medium of the same composition can possibly
0721-7714/82/0001/0261/$ 01.00
262 be explained e i t h e r by depletion of some essent i a l metabolites in the spent medium or i t s extreme hardness due to desiccation. I f the t r a n s f e r of shoot primordia was f u r t h e r delayed to f i v e or s i x weeks, stolons were formed (Fig. 3). Such stolons also provided ideal explant source for m u l t i p l e shoot primordia, which in turn could be separated for f u r t h e r m u l t i p l i c a t i o n . The o l a n t l e t s were successf u l l y transferred to pots (Fig. 4).
in these stolon segments or preformed shoot apices. All the Nephrolepis plants propagated through tissue culture techniques exhibited u n i f o r m i t y in t h e i r q u a l i t y . Moreover, larqe-scale propagation of these ferns in short time was possible. Both these factors would contribute to commercial f e a s i b i l i t y of t h i s aporoach by fern growers.
Stolon seqments of NeDhrolenis c o r d i f o l i a qrown on BM+IBA (1.0 mq/l) Incubation: at 25+2% in continuous l i g h t (1000 L u x ) Fiq. I. Production of numerous leaves - 4 week old Fig. 2. Plantlets produced from isolated l e a f primordium - 6 week old Fig. 3. Stolon formation from p l a n t l e t s - 8 week old Fig. 4. Transferred p l a n t l e t s to pots A l l three specices of Nephrolepis studied responded to IBA (1,0 mg/l) treatment and showed m u l t i ple shoot formation. Wardlaw (1966) had demonstrated a s i m i l a r response from excised Dryopteris rhizomes. Nn the other hand, Peterson (1969) reported bud formation from Ophioglossum roots with cytokinins. In Nephrolepis species, no exogenous cytokinins were found necessary to induce l e a f primordia. This might be due to the high endogenous level of cytokinins
References Gamborg OL, ~ l i l l e r RA, Ojima K (1968) Expt Cell Res 50:151-58 Haight TH, Kuehnert CC (1971) Can. J. Bot.49:1941-45 Murashige T (1977) In: Barz W, Reinhard E, Zenk ~IH (eds) Plant tissue culture and i t s biotechnoloflical a p p l i c a t i o n , Springer-Verlag, Berlin Heidelberg, New York po 392-402
263 Peterson RL (1969) Can. J. Bot 47:1285-87 Steeves TA (1961) Phytomorphology 11:346-59 Steeves TA (1966) In: Cutter EC (ed) Trends in plant morphogenesis, Longmans London 200-219 Wardlaw CW (1966): In: Beerman et al. (eds)
Cell d i f f e r e n t i a t i o n and morphogenesis, North-Holland publishing company, Amsterdam 96-119 Netmore RH (1970) In: Hillman N (ed) Papers in Plant Physiology 525-43
Plant Cell Reports (1982) 1 : 261-263
© Springer-Verlag 1982
Propagation of Fern (Nephrolepis) Through Tissue Culture M. A. Padhya and A. R. Mehta Department of Botany, The Maharaja Sayajirao University of Baroda, Baroda-390002, India Received July 16, 1982/September 20, 1982
Abstract Rapid clonal propagation in three stolon bearing species of Nephrolepis was achieved by tissue culture methods. Explants from young stolons were grown on B5 (Gamborg et a l . 1968) medium supplemented with indolebutyric acid where they produced several l e a f primordia. These l e a f primordia were isolated and grown into entire p l a n t l e t s . An overall m u l t i p l i c a t i o n factor of 30-40 was attained within three months.
medium with 2% sucrose (basal medium-BM) and BM - supplemented with various growth regulators such as napthalenacetic acid (NAA), 2, 4-di chlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), kinetin (K), 6-benzyladenine (BAP) i n d i v i dually as per the requirements of each experiment. Three concentrations (0.5, 1.0, 2.0 mg/l) of each of these growth regulators were tested. The medium was adjusted to pH 5.7 before s o l i d i f y i n g with 0.6% agar. All cultures were incubated in continuous fluorescent l i g h t (1000Lux) at 25+_2~C.
Introduction Ferns have been c u l t i v a t e d for t h e i r ornamental foliage. Their demand as indoor plants has been increasing. Excised l e a f primordia of Osmunda cinnamomea (Steeves, 1966), Adiantum pedatum and other ferns (Wetmore, 1970) have been' grown to adult p l a n t l e t s in axenic cultures. The conventional method employed for propagation of ferns has been by a runnerset procedure. But ferns derived through t h i s method produced a single growth center from where only a few fronds developed at a time. In contrast, the ferns derived through tissue culture techniques produced several growth centers and from each growth center numerous fronds were produced, thus giving an a t t r a c t i v e look to the plants (Murashige, 1977). In the present i n v e s t i g a t i o n , an attempt has been made to standardise tissue culture techniques for rapid m u l t i p l i c a t i o n and propagation in three species of Nephrolepis. Materials and Methods Stolon segments from three species of Nephrol~pis: ~. c o r d i f o l i a P r e s l . , N. exaltata Schott, and N. acuta Presl., a l l growing in the University Botanical Gardens, Baroda, were collected. They were surface s t e r i l i s e d with mercuric chloride (0.1%), washed with s t e r i l e d i s t i l l e d wateG cut into 1 to 2 cm pieces and inoculated on various culture media. The culture media included B5 (Gamborg et al. 1968)
Results and Discussion Stolon segments of all three Nephrolepis species cultured on basal medium (BM) and BM supplemented with NAA, K, or BAP failed to express any morphogenic response, while segments cultured on BMwith 2, 4-D (2 mg/l) showed white friable callus formation from their cut ends. All stolon segments of three species of Nephrolepis responded to IBA ().0 mg/l) treatment. These segments showed greenish proliferations along their margin. In fact, these were the meristematlc growth centers from which 18-20 leaf primordia emerged by the third week. The leaf primordia situated on the outer side were old and developed into full-fledged leaves at the end of four weeks (Fig. 1). On the other hand, each of the younger leaf primordia situated near the center, on isolation and transfer to fresh medium after three weeks developed multiple shoots. The very young shoot primordia could thus be effectively used as propagules for rapid multiplication. Similar differential responses of young and old leaf primordia have been observed earlier in Osmunda cinnamomea by Steeves (1961) and also by Haight and Kuehnert (1971). I f the shoot primordia were transferred to freshly made medium after four weeks, instead of three, root formation was induced resulting into complete plantlet regeneration (Fig. 2). Formation of roots by regenerated shoots (after 4 weeks) on transfer to fresh medium of the same composition can possibly
0721-7714/82/0001/0261/$ 01.00
262 be explained e i t h e r by depletion of some essent i a l metabolites in the spent medium or i t s extreme hardness due to desiccation. I f the t r a n s f e r of shoot primordia was f u r t h e r delayed to f i v e or s i x weeks, stolons were formed (Fig. 3). Such stolons also provided ideal explant source for m u l t i p l e shoot primordia, which in turn could be separated for f u r t h e r m u l t i p l i c a t i o n . The o l a n t l e t s were successf u l l y transferred to pots (Fig. 4).
in these stolon segments or preformed shoot apices. All the Nephrolepis plants propagated through tissue culture techniques exhibited u n i f o r m i t y in t h e i r q u a l i t y . Moreover, larqe-scale propagation of these ferns in short time was possible. Both these factors would contribute to commercial f e a s i b i l i t y of t h i s aporoach by fern growers.
Stolon seqments of NeDhrolenis c o r d i f o l i a qrown on BM+IBA (1.0 mq/l) Incubation: at 25+2% in continuous l i g h t (1000 L u x ) Fiq. I. Production of numerous leaves - 4 week old Fig. 2. Plantlets produced from isolated l e a f primordium - 6 week old Fig. 3. Stolon formation from p l a n t l e t s - 8 week old Fig. 4. Transferred p l a n t l e t s to pots A l l three specices of Nephrolepis studied responded to IBA (1,0 mg/l) treatment and showed m u l t i ple shoot formation. Wardlaw (1966) had demonstrated a s i m i l a r response from excised Dryopteris rhizomes. Nn the other hand, Peterson (1969) reported bud formation from Ophioglossum roots with cytokinins. In Nephrolepis species, no exogenous cytokinins were found necessary to induce l e a f primordia. This might be due to the high endogenous level of cytokinins
References Gamborg OL, ~ l i l l e r RA, Ojima K (1968) Expt Cell Res 50:151-58 Haight TH, Kuehnert CC (1971) Can. J. Bot.49:1941-45 Murashige T (1977) In: Barz W, Reinhard E, Zenk ~IH (eds) Plant tissue culture and i t s biotechnoloflical a p p l i c a t i o n , Springer-Verlag, Berlin Heidelberg, New York po 392-402
263 Peterson RL (1969) Can. J. Bot 47:1285-87 Steeves TA (1961) Phytomorphology 11:346-59 Steeves TA (1966) In: Cutter EC (ed) Trends in plant morphogenesis, Longmans London 200-219 Wardlaw CW (1966): In: Beerman et al. (eds)
Cell d i f f e r e n t i a t i o n and morphogenesis, North-Holland publishing company, Amsterdam 96-119 Netmore RH (1970) In: Hillman N (ed) Papers in Plant Physiology 525-43
