- Email: [email protected]
Organogenesis from callus cultures of the legume, Stylosanthes hamata
Plant Science Letters, 7 (1976) 39--42 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
39
O R G A N O G E N E S I S F R O M CALLUS C U L T U R E S OF THE LEGUME, S T Y L O S A N T H E S H A M A TA
W.R. SCOWCROFT and J.A. ADAMSON Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra, A.C. T. (Australia) (Received February 27th, 1976) (Accepted March 17th, 1976)
SUMMARY
Callus cultures of the perennial pasture legume, Stylosanthes hamata can be readily induced to form shoots and roots. Sequential transfer from culture medium without growth regulators to one with kinetin led to regeneration of entire plantlets. Long-term embryogenic potential was maintained.
INTRODUCTION Cell cultures of legume species have proven refractory to plant regeneration, despite the considerable use of legumes in tissue culture studies. Limited differentiation o f legume callus includes the formation of shoot primordia on callus on Pisum sativum stems [1] and r o o t formation from b o t h P. sativum [2] and Vigna unguiculata [3] callus. Although complete plant regeneration has been reported from callus ofMedicago sativa [4] and from shoot apices o f P. sativum [ 5], the callus phase b e t w e e n explant and plantlet regeneration was transitory. There remained the desideratum of long term embryogenic potential o f legume cell cultures. In the course o f our studies on nitrogen fixation in c o w p e a rhizobia [6--8] a legume was sought which could be cultured in vitro in an undifferentiated state and from which plants could be readily regenerated. This report concerns the cell culture and subsequent regeneration of the tropical legume Stylosanthes hamata. This species is an erect, smooth-stemmed herbaceous perennial which has been developed as a pasture species for northern Australia. It is noduiated b y the non-specific " c o w p e a group" o f rhizobia.
METHODS Seed was scarified with sandpaper, surface-sterilized with 0.2% HgC12 (30 min) and washed 6 times with sterile, distilled water. The seed was germinated
40
in petri dishes on agar medium [9] with 9-10- 6 M 2,4-dichlorophenoxyacetic acid and 2.3-10 -v M kinetin. Subsequent experiments were performed on a modified medium (CS5) in which the combined nitrogen sources of Schenk and Hildebrandt [9] were replaced by (in raM) NH4 NO3 (15.5), NH4 H2 PO4 (4.0), KNO3 (24.8) and which contained the same growth regulators as above. Callus and liquid cultures (50 ml per 250 ml flask at 110 rpm on a gyratory shaker) were grown in the dark at 29°C. To induce plantlet regeneration, callus was transferred to 20 ml CS5 medium with altered auxin and/or cytokinin concentrations in 50 ml plastic containers and cultured in a 16 h light (1500 lux)/8 h dark regime at 25°C. RESULTS AND DISCUSSION
Seed germinated within 8 days and within 3 weeks callus proliferated from both the radicle and from the cotyledon (Fig. 1). Callus from both the cotyledon and radicle was excised and each treated separately. In subsequent regeneration experiments the response of callus derived from the cotyledon was identical with that from the radicle. The callus was friable with a relatively rapid growth rate and could be maintained in an undifferentiated state for at least 15 months.
Fig.l. Callus formation from the cotyledons (A) and the radicle (B) o f S. hamata
The formation of shoots and roots was variable depending on the type and concentration of growth regulators used. Roots were formed on CS5 medium and on CS5 without growth regulators; extensive root formation often occurred from callus cultures growing in the dark on standard CS5 medium. The number of roots could be enhanced by incorporation 6 "10 -7 M indole acetic acid into the medium. However, this was not necessarily
41
favourable since shoots formed subsequently on callus with a large n u m b e r of small roots developed less robust plants than that with a few well developed roots. Shoot development from callus was most p r o n o u n c e d with 1.4-10 -s M kinetin. S h o o t primordia could be discerned within 3--4 weeks and these gave rise to 10--20 distinct shootlets/culture within 6 weeks (Fig. 2). However, conditions which favoured shoot formation tended to suppress subsequent r o o t initiation.
Fig.2. Shoot formation from callus of $. hamata. Fig.3. Entire plantlet formation from callus of S. hamata. To achieve consistent regeneration of entire plantlets, undifferentiated callus was initially cultured on CS5 w i t h o u t growth regulators until roots, 1--2 cm long had developed. Callus with roots was then transferred t o medium with 1 . 4 . 1 0 -5 M kinetin to induce shoot formation. Entire plantlets continued to develop on this medium (Fig. 3), b u t the rate of plantlet growth could be enhanced if callus with shoots and roots was transferred back to CS5 medium w i t h o u t growth regulators. In this way 5--10 plantlets per container can be differentiated which are sufficiently robust to survive transfer to greenhouse pots. Such plantlets yield fully fertile, normal plants (Fig. 4). Callus o f S. h a m a t a can be maintained as undifferentiated callus on CS5 w i t h o u t losing its embryogenic potential. Using the transfer sequence described above, plantlets were readily regenerated from 15-month-old
42
Fig.4. Flowering part of regenerated plant, S. hamata; total plant height 25 cm.
callus cultures. In liquid suspension culture the cellsaggregate. These cell aggregates give rise t o s hoot pr i m or di a w hen transferred t o CS5 agar m e d i u m containing 1 . 4 . 1 0 -s M kinetin. In plant cell culture genetics it is desirable t hat genetic m odi fi cat i on at t h e cellular level be evaluated in m a t ur e plants, particularly if t he consequences of such manipulation be o f value t o plant improvement. The ability t o regenerate a legume readily m a y be o f value in heterospecific gene transfer experiments which a t t e m p t t o develop non-legume crops which are less d e p e n d e n t on applied nitrogen fertilizer. ACKNOWLEDGEMENTS
W e wish to thank Dr. J.E. Begg for originallysuggesting 8. hamata and supplying seed and Dr. J.D. Pagan for discussion. REFERENCES
1 A.C. Hildebrandt, J.C. Wilmar, H. Johns and A.J. Riker, Am. J. Bot., 50 (1963) 248. 2 J.G. Torrey, Physiol. Plant., 20 (1967) 265. 3 M.R. Davey, E. Bush and J.B. Power, Plant Sci. Lett., 3 (1974) 127. 4 J.W. Saunders and E.T. Bingham, Crop Sci., 12 (19.72) 804. 5 0 . L . Gamborg, F. Constabel and J.P. Shyluk, Physiol. Plant., 30 (1974) 125. 6 W.R. Scowcroft and A.H. Gibson, Nature, 253 (1975) 351. 7 A.H. Gibson, J.J. Child, J.D. Pagan and W.R. Scowcroft, Planta, 128 (1976) 233. 8 J.D. Pagan, J.J. Child, W.R. Scowcroft and A.H. Gibson, Nature, 256 (1975) 406. 9 R.V. Schenk and A.C. Hildebrandt, Can. J. Bot., 50 (1972) 199.
39
O R G A N O G E N E S I S F R O M CALLUS C U L T U R E S OF THE LEGUME, S T Y L O S A N T H E S H A M A TA
W.R. SCOWCROFT and J.A. ADAMSON Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra, A.C. T. (Australia) (Received February 27th, 1976) (Accepted March 17th, 1976)
SUMMARY
Callus cultures of the perennial pasture legume, Stylosanthes hamata can be readily induced to form shoots and roots. Sequential transfer from culture medium without growth regulators to one with kinetin led to regeneration of entire plantlets. Long-term embryogenic potential was maintained.
INTRODUCTION Cell cultures of legume species have proven refractory to plant regeneration, despite the considerable use of legumes in tissue culture studies. Limited differentiation o f legume callus includes the formation of shoot primordia on callus on Pisum sativum stems [1] and r o o t formation from b o t h P. sativum [2] and Vigna unguiculata [3] callus. Although complete plant regeneration has been reported from callus ofMedicago sativa [4] and from shoot apices o f P. sativum [ 5], the callus phase b e t w e e n explant and plantlet regeneration was transitory. There remained the desideratum of long term embryogenic potential o f legume cell cultures. In the course o f our studies on nitrogen fixation in c o w p e a rhizobia [6--8] a legume was sought which could be cultured in vitro in an undifferentiated state and from which plants could be readily regenerated. This report concerns the cell culture and subsequent regeneration of the tropical legume Stylosanthes hamata. This species is an erect, smooth-stemmed herbaceous perennial which has been developed as a pasture species for northern Australia. It is noduiated b y the non-specific " c o w p e a group" o f rhizobia.
METHODS Seed was scarified with sandpaper, surface-sterilized with 0.2% HgC12 (30 min) and washed 6 times with sterile, distilled water. The seed was germinated
40
in petri dishes on agar medium [9] with 9-10- 6 M 2,4-dichlorophenoxyacetic acid and 2.3-10 -v M kinetin. Subsequent experiments were performed on a modified medium (CS5) in which the combined nitrogen sources of Schenk and Hildebrandt [9] were replaced by (in raM) NH4 NO3 (15.5), NH4 H2 PO4 (4.0), KNO3 (24.8) and which contained the same growth regulators as above. Callus and liquid cultures (50 ml per 250 ml flask at 110 rpm on a gyratory shaker) were grown in the dark at 29°C. To induce plantlet regeneration, callus was transferred to 20 ml CS5 medium with altered auxin and/or cytokinin concentrations in 50 ml plastic containers and cultured in a 16 h light (1500 lux)/8 h dark regime at 25°C. RESULTS AND DISCUSSION
Seed germinated within 8 days and within 3 weeks callus proliferated from both the radicle and from the cotyledon (Fig. 1). Callus from both the cotyledon and radicle was excised and each treated separately. In subsequent regeneration experiments the response of callus derived from the cotyledon was identical with that from the radicle. The callus was friable with a relatively rapid growth rate and could be maintained in an undifferentiated state for at least 15 months.
Fig.l. Callus formation from the cotyledons (A) and the radicle (B) o f S. hamata
The formation of shoots and roots was variable depending on the type and concentration of growth regulators used. Roots were formed on CS5 medium and on CS5 without growth regulators; extensive root formation often occurred from callus cultures growing in the dark on standard CS5 medium. The number of roots could be enhanced by incorporation 6 "10 -7 M indole acetic acid into the medium. However, this was not necessarily
41
favourable since shoots formed subsequently on callus with a large n u m b e r of small roots developed less robust plants than that with a few well developed roots. Shoot development from callus was most p r o n o u n c e d with 1.4-10 -s M kinetin. S h o o t primordia could be discerned within 3--4 weeks and these gave rise to 10--20 distinct shootlets/culture within 6 weeks (Fig. 2). However, conditions which favoured shoot formation tended to suppress subsequent r o o t initiation.
Fig.2. Shoot formation from callus of $. hamata. Fig.3. Entire plantlet formation from callus of S. hamata. To achieve consistent regeneration of entire plantlets, undifferentiated callus was initially cultured on CS5 w i t h o u t growth regulators until roots, 1--2 cm long had developed. Callus with roots was then transferred t o medium with 1 . 4 . 1 0 -5 M kinetin to induce shoot formation. Entire plantlets continued to develop on this medium (Fig. 3), b u t the rate of plantlet growth could be enhanced if callus with shoots and roots was transferred back to CS5 medium w i t h o u t growth regulators. In this way 5--10 plantlets per container can be differentiated which are sufficiently robust to survive transfer to greenhouse pots. Such plantlets yield fully fertile, normal plants (Fig. 4). Callus o f S. h a m a t a can be maintained as undifferentiated callus on CS5 w i t h o u t losing its embryogenic potential. Using the transfer sequence described above, plantlets were readily regenerated from 15-month-old
42
Fig.4. Flowering part of regenerated plant, S. hamata; total plant height 25 cm.
callus cultures. In liquid suspension culture the cellsaggregate. These cell aggregates give rise t o s hoot pr i m or di a w hen transferred t o CS5 agar m e d i u m containing 1 . 4 . 1 0 -s M kinetin. In plant cell culture genetics it is desirable t hat genetic m odi fi cat i on at t h e cellular level be evaluated in m a t ur e plants, particularly if t he consequences of such manipulation be o f value t o plant improvement. The ability t o regenerate a legume readily m a y be o f value in heterospecific gene transfer experiments which a t t e m p t t o develop non-legume crops which are less d e p e n d e n t on applied nitrogen fertilizer. ACKNOWLEDGEMENTS
W e wish to thank Dr. J.E. Begg for originallysuggesting 8. hamata and supplying seed and Dr. J.D. Pagan for discussion. REFERENCES
1 A.C. Hildebrandt, J.C. Wilmar, H. Johns and A.J. Riker, Am. J. Bot., 50 (1963) 248. 2 J.G. Torrey, Physiol. Plant., 20 (1967) 265. 3 M.R. Davey, E. Bush and J.B. Power, Plant Sci. Lett., 3 (1974) 127. 4 J.W. Saunders and E.T. Bingham, Crop Sci., 12 (19.72) 804. 5 0 . L . Gamborg, F. Constabel and J.P. Shyluk, Physiol. Plant., 30 (1974) 125. 6 W.R. Scowcroft and A.H. Gibson, Nature, 253 (1975) 351. 7 A.H. Gibson, J.J. Child, J.D. Pagan and W.R. Scowcroft, Planta, 128 (1976) 233. 8 J.D. Pagan, J.J. Child, W.R. Scowcroft and A.H. Gibson, Nature, 256 (1975) 406. 9 R.V. Schenk and A.C. Hildebrandt, Can. J. Bot., 50 (1972) 199.