PlantCell Reports
Plant Cell Reports (1983) 2:19-20
© Springer-Verlag 1983
Shoot Regeneration from Glycine canescens Tissue Cultures J. M. Widholm and S. Rick Agronomy Department, University of Illinois, Urbana, IL 61801, USA Received August 20, 1982/November 12, 1982
Abstract Shoots were reproducibly obtained from calli of both cotyledons and hypocotyls of Glycine canescens, a species related to soybean. A few shoots were also obtained from suspension cultures initiated from the callus tissues. Root formation was observed infrequently so no complete plants were recovered.
Abbreviations B5, basal medium described by Gamborg et al. (1968); BA, benzyladenine; 2,4-D, 2,4dichlorophenoxyacetic acid; IAA, 3-indoleacetic acid; IBA, 3-indolebutyric acid; KIN, kinetin; MS, basal medium described by Murashige and Skoog (1962); N, medium described by Nitsch (1972) with 1% sucrose; NAA, naphthaleneacetic acid.
Introduction Despite extensive effort, plant regeneration from tissue cultures of the important grain legume Glycine max has not been achieved. The induction of "embryo like" structures has been reported (Beversdorf and Bingham 1977; Oswald et al. 1977; Phillips and Collins 1981) but these fail to develop into viable plants. The Research Group of Soya Bean Tissue Culture (1976) reported regeneration of plants from hypocotyl callus, but the callus had to be placed on regeneration medium within I0 d after forming. This rapid loss of plant regeneration capability suggests that the plants arose from preformed buds. The rapid loss of morphogenic capacity precludes the use of such manipulative techniques as selection or genetic transformation before plant regeneration. Kameya and Widholm (1981) showed that Glycine canescens hypocotyl sections, with the cotyledonary node buds removed, could be induced to form shoots while those from G. max or six other wild relatives failed to do so. These results led us to initiate G. caneseens tissue cultures and further evaluate the plant regeneration from cultured cells.
Materials and Methods Glycine canescens seeds (kindly provided by T. Hymowitz) were surface sterilized for i0 min in
sodium hypochlorite (Chlorox diluted 10-fold with distilled water) with one drop of liquid detergent per i00 ml and rinsed twice for 5 min in autoclaved distilled water. The seed coats were nicked with a scalpel and the seeds placed on MS basal medium (0.8% agar) lacking hormones (20 ml in i0 cm plastic Petri dishes). Throughout this paper each medium is denoted by the abbreviation for the basal medium, B5, MS or N, followed by the hormone levels in mg/l in brackets. The MS medium with no hormones is denoted MS (0). Following incubation for seven d at 27-28°C under 16 h light (i000 lux) the cotyledons and hypocotyls were excised and placed on 0.8% agarsolidified B5 (0.5 IAA), B5 (2 IAA), B5 (0.5 NAA), B5 (2 NAA), B5 (0.5 2,4-D) and B5 (2 2,4-D). Shoot formation was induced by placing calli or suspension cultured cells on 0.8% agar-solidified medium in plastic petri dishes incubated under the conditions described above. Suspension cultures were initiated by placing calli, grown on B5 (0.5 NAA) for 4 transfers, into 50 ml of MS (0.4 2,4-D) in 125 ml Erlenmeyer flasks. The suspensions were incubated on a reciprocating shaker (80 cpm with a 7 cm stroke) at 27 to 28°C under continuous dim light and were transferred every 14 days. To induce shoot formation, 9 day old suspensions were collected under sterile conditions on Miracloth filters, rinsed with MS (0) liquid medium and then resuspended in MS (0) liquid medium. One ml aliquots were placed on agar-solidified MS basal with the following hormone combinations in mg/l: 5 BA, 0. i IAA and 5 BA, 0.2 IAA and 5 BA, 0.2 IAA and i0 BA, 0.5 IAA and 5 BA, 1 1 A A and 0.64 KIN, 1 IAA and 1 KIN, 1 IAA and 1 BA, 2 NAA and 1 BA. The shoots which formed were maintained by monthly transfer on the following media (0.8% agar) to attempt to induce root formation: MS(O), MS(0.01 IBA), M S ( 0 . 1 1 B A ) , these three media with 0.1% charcoal, or N(0).
Results and Discussion Callus formation from excised Glycine canescens cotyledons or hypocotyls was most rapid on B5(2 IAA), B5(2 NAA) and B5(2 2,4-D) media while continued callus growth was better when these hormones were present at 0.5 mg/l. The calli generally grew slowly as dark-brown soft masses. Although all the experiments reported here were carried out with calli grown in the B5-based media, later work showed that calli maintained on MS basal medium with 0.4 mg/l 2,4-D were lighter in color and grew faster.
20 When calli formed from hypocotyls or cotyledons on B5(0.5 NAA) were placed on MS (0.2 IAA and 5BA) shoots formed readily within 8 weeks. These cultures retained the ability to form shoots even after three transfers over a 19 week period on B5(0.5 NAA) or B5(0.5 IAA) (Figure I). Shoots formed when subcultured calli were placed on MS(0.2 IAA and i0 BA), MS(0.5 IAA and 5 BA) or MS(0.2 IAA and 5 BA) for 5 weeks and then transferred to MS (0.5 IAA and 5 BA), MS(0.2 IAA and 5 BA) or MS(0.5 BA). More than half of the calli produced from one to twenty shoots.
Figure 3. ,Shoot formed from C. canescens suspension cultured cells plated on MS (-- 5 BA) for 35 d.
Figure I. Shoots formed from G. canescens calli grown for three transfers on B~ (0.5 NAA) and then placed on MS (0.5 IAA and 5 BA) for 37 d. Whether the sequential transfer to different media is required was not studied fully. In most cases the origin of the tissue, i.e. hypocotyl or cotyledon, did not affect the ability of the cultures to form shoots. The shoots elongated on several media including MS (O), N (0) and MS (0.5 BA) (Figure 2).
Root formation was obtained very rarely in spite of many attempts with several media. Thus whole plants have not been recovered. These preliminary results show that G. canescens is apparently more amenable to tissue culture plant regeneration than is G. max where plant regeneration from tissue cultures has been rarely reported. During the experiments reported here, cultures were also initiated using similar methods from G. max (L.) Merr. var. Bragg, Dunn and Wayne, but shoot formation was never observed. The G. canescens system must be studied further to determine the optimal culture conditions, how long the cultured cells will continue to regenerate shoots using optimal conditions, how to root the shoots that do form and if protoplasts from leaves or cultured cells are totipotent. Studies with G. canescens may help us learn how to manipulate tissue cultures of the commercially important species, G. max.
Acknowledgments This research was supported by funds from the Illinois Agricultural Experiment Station, the American Soybean Association and the Illinois Soybean Program Operating Board. Technical assistance was provided by Maria Dimas.
References
Figure 2. Shoots formed from G. canescens calli grown on B5 (0.5 NAA) then MS (0.5 IAA and 5 BA) and finally MS (0.5 BA). A suspension culture composed of cell clumps up to 2 mm in diameter was initiated from hypocotyl derived calli grown on B5(0.5 NAA) for four transfers. Following three transfers at 14 d intervals in liquid MS (0.4 2,4-D) suspensions plated on MS(5 BA) agar-solidified medium still formed a few shoots after five weeks (Figure 3). Cultures maintained longer in liquid medium did not regenerate shoots.
Beversdorf WD, Bingham ET (1977) Crop Sci 17: 307311 Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50: 148-151 Kameya T, Widholm J (1981) Plant Sci Lett 21: 289294 Murashige T, Skoog F (1962) Physiol Plant 15: 473497 Nitsch JP (1972) Z Pflanzenzuchtg 6 7 : 3 - 1 8 Oswald TH, Smith AE, Phillips DV (1977) Physiol Plant 39:129-134 Phillips GC, Collins GB (1981) Plant Cell, Tissue and Organ Culture i: 123-129 Research Group of Soya Bean Tissue Culture, Institute of Crop Breeding, Kirin Academy of Agriculture (1976) Acta Botanica Sinica 1 8 : 2 5 8 - 2 6 2
Plant Cell Reports (1983) 2:19-20
© Springer-Verlag 1983
Shoot Regeneration from Glycine canescens Tissue Cultures J. M. Widholm and S. Rick Agronomy Department, University of Illinois, Urbana, IL 61801, USA Received August 20, 1982/November 12, 1982
Abstract Shoots were reproducibly obtained from calli of both cotyledons and hypocotyls of Glycine canescens, a species related to soybean. A few shoots were also obtained from suspension cultures initiated from the callus tissues. Root formation was observed infrequently so no complete plants were recovered.
Abbreviations B5, basal medium described by Gamborg et al. (1968); BA, benzyladenine; 2,4-D, 2,4dichlorophenoxyacetic acid; IAA, 3-indoleacetic acid; IBA, 3-indolebutyric acid; KIN, kinetin; MS, basal medium described by Murashige and Skoog (1962); N, medium described by Nitsch (1972) with 1% sucrose; NAA, naphthaleneacetic acid.
Introduction Despite extensive effort, plant regeneration from tissue cultures of the important grain legume Glycine max has not been achieved. The induction of "embryo like" structures has been reported (Beversdorf and Bingham 1977; Oswald et al. 1977; Phillips and Collins 1981) but these fail to develop into viable plants. The Research Group of Soya Bean Tissue Culture (1976) reported regeneration of plants from hypocotyl callus, but the callus had to be placed on regeneration medium within I0 d after forming. This rapid loss of plant regeneration capability suggests that the plants arose from preformed buds. The rapid loss of morphogenic capacity precludes the use of such manipulative techniques as selection or genetic transformation before plant regeneration. Kameya and Widholm (1981) showed that Glycine canescens hypocotyl sections, with the cotyledonary node buds removed, could be induced to form shoots while those from G. max or six other wild relatives failed to do so. These results led us to initiate G. caneseens tissue cultures and further evaluate the plant regeneration from cultured cells.
Materials and Methods Glycine canescens seeds (kindly provided by T. Hymowitz) were surface sterilized for i0 min in
sodium hypochlorite (Chlorox diluted 10-fold with distilled water) with one drop of liquid detergent per i00 ml and rinsed twice for 5 min in autoclaved distilled water. The seed coats were nicked with a scalpel and the seeds placed on MS basal medium (0.8% agar) lacking hormones (20 ml in i0 cm plastic Petri dishes). Throughout this paper each medium is denoted by the abbreviation for the basal medium, B5, MS or N, followed by the hormone levels in mg/l in brackets. The MS medium with no hormones is denoted MS (0). Following incubation for seven d at 27-28°C under 16 h light (i000 lux) the cotyledons and hypocotyls were excised and placed on 0.8% agarsolidified B5 (0.5 IAA), B5 (2 IAA), B5 (0.5 NAA), B5 (2 NAA), B5 (0.5 2,4-D) and B5 (2 2,4-D). Shoot formation was induced by placing calli or suspension cultured cells on 0.8% agar-solidified medium in plastic petri dishes incubated under the conditions described above. Suspension cultures were initiated by placing calli, grown on B5 (0.5 NAA) for 4 transfers, into 50 ml of MS (0.4 2,4-D) in 125 ml Erlenmeyer flasks. The suspensions were incubated on a reciprocating shaker (80 cpm with a 7 cm stroke) at 27 to 28°C under continuous dim light and were transferred every 14 days. To induce shoot formation, 9 day old suspensions were collected under sterile conditions on Miracloth filters, rinsed with MS (0) liquid medium and then resuspended in MS (0) liquid medium. One ml aliquots were placed on agar-solidified MS basal with the following hormone combinations in mg/l: 5 BA, 0. i IAA and 5 BA, 0.2 IAA and 5 BA, 0.2 IAA and i0 BA, 0.5 IAA and 5 BA, 1 1 A A and 0.64 KIN, 1 IAA and 1 KIN, 1 IAA and 1 BA, 2 NAA and 1 BA. The shoots which formed were maintained by monthly transfer on the following media (0.8% agar) to attempt to induce root formation: MS(O), MS(0.01 IBA), M S ( 0 . 1 1 B A ) , these three media with 0.1% charcoal, or N(0).
Results and Discussion Callus formation from excised Glycine canescens cotyledons or hypocotyls was most rapid on B5(2 IAA), B5(2 NAA) and B5(2 2,4-D) media while continued callus growth was better when these hormones were present at 0.5 mg/l. The calli generally grew slowly as dark-brown soft masses. Although all the experiments reported here were carried out with calli grown in the B5-based media, later work showed that calli maintained on MS basal medium with 0.4 mg/l 2,4-D were lighter in color and grew faster.
20 When calli formed from hypocotyls or cotyledons on B5(0.5 NAA) were placed on MS (0.2 IAA and 5BA) shoots formed readily within 8 weeks. These cultures retained the ability to form shoots even after three transfers over a 19 week period on B5(0.5 NAA) or B5(0.5 IAA) (Figure I). Shoots formed when subcultured calli were placed on MS(0.2 IAA and i0 BA), MS(0.5 IAA and 5 BA) or MS(0.2 IAA and 5 BA) for 5 weeks and then transferred to MS (0.5 IAA and 5 BA), MS(0.2 IAA and 5 BA) or MS(0.5 BA). More than half of the calli produced from one to twenty shoots.
Figure 3. ,Shoot formed from C. canescens suspension cultured cells plated on MS (-- 5 BA) for 35 d.
Figure I. Shoots formed from G. canescens calli grown for three transfers on B~ (0.5 NAA) and then placed on MS (0.5 IAA and 5 BA) for 37 d. Whether the sequential transfer to different media is required was not studied fully. In most cases the origin of the tissue, i.e. hypocotyl or cotyledon, did not affect the ability of the cultures to form shoots. The shoots elongated on several media including MS (O), N (0) and MS (0.5 BA) (Figure 2).
Root formation was obtained very rarely in spite of many attempts with several media. Thus whole plants have not been recovered. These preliminary results show that G. canescens is apparently more amenable to tissue culture plant regeneration than is G. max where plant regeneration from tissue cultures has been rarely reported. During the experiments reported here, cultures were also initiated using similar methods from G. max (L.) Merr. var. Bragg, Dunn and Wayne, but shoot formation was never observed. The G. canescens system must be studied further to determine the optimal culture conditions, how long the cultured cells will continue to regenerate shoots using optimal conditions, how to root the shoots that do form and if protoplasts from leaves or cultured cells are totipotent. Studies with G. canescens may help us learn how to manipulate tissue cultures of the commercially important species, G. max.
Acknowledgments This research was supported by funds from the Illinois Agricultural Experiment Station, the American Soybean Association and the Illinois Soybean Program Operating Board. Technical assistance was provided by Maria Dimas.
References
Figure 2. Shoots formed from G. canescens calli grown on B5 (0.5 NAA) then MS (0.5 IAA and 5 BA) and finally MS (0.5 BA). A suspension culture composed of cell clumps up to 2 mm in diameter was initiated from hypocotyl derived calli grown on B5(0.5 NAA) for four transfers. Following three transfers at 14 d intervals in liquid MS (0.4 2,4-D) suspensions plated on MS(5 BA) agar-solidified medium still formed a few shoots after five weeks (Figure 3). Cultures maintained longer in liquid medium did not regenerate shoots.
Beversdorf WD, Bingham ET (1977) Crop Sci 17: 307311 Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50: 148-151 Kameya T, Widholm J (1981) Plant Sci Lett 21: 289294 Murashige T, Skoog F (1962) Physiol Plant 15: 473497 Nitsch JP (1972) Z Pflanzenzuchtg 6 7 : 3 - 1 8 Oswald TH, Smith AE, Phillips DV (1977) Physiol Plant 39:129-134 Phillips GC, Collins GB (1981) Plant Cell, Tissue and Organ Culture i: 123-129 Research Group of Soya Bean Tissue Culture, Institute of Crop Breeding, Kirin Academy of Agriculture (1976) Acta Botanica Sinica 1 8 : 2 5 8 - 2 6 2
