Plant Cell Reports
Plant Cell Reports (1986) 5:101 103
© Springer-Verlag 1986
A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus T. L. Barsby, S. A. Yarrow, and J. F. Shepard 1 Allelix I n c 6850 Goreway Drive, Mississauga, Ontario L4V 1P l, Canada Received January 17, 1986 / Revised version received January 31, 1986 - Communicated by F. Constabel
ABSTRACT
Protoplasts of several spring and winter varieties of Brassica napus were isolated from hypocotyl tissue. Proto---6-~Fasts divided and formed cell colonies at high frequency, without browning when cultured in modified Shepards' medium. This high efficiency of proliferation was sustained through to plant regeneration with all varieties cultured. This has been attributed to the incorporation of a reservoir medium, the presence of 2,4-D in the proliferation medium, and the presence of kinetin in conjunction with lowering of the sucrose concentration in the regeneration medium. ABBREVIAT IONS NAA = 1-naphthaleneacetic acid;
BAP = 6-benzylaminopurine; IAA = 3-indoleacetic acid; 2,4-D = 2,4-dichlorophenoxyacetic acid. INTRODUCTION
In order that cell technologies (eg., somatic hybridisation, transformation, etc.) may present a major contribution to conventional breeding techniques of agriculturally important crop species, high regeneration frequencies from protoplasts are essential. Recently, much attention has been directed to the Brassicaceae, to address this problem. Regeneration from protoplasts of a number of tissue sources has been reported, namely from leaves (Bidney et a l . , 1983, Guo and Schieder, 1983, Li and Kohlenbac-ElS,---1982, Newell et a l . , 1984), stem embryos (Kohlenbach et a l . , -T9E~-), cotyledons (Vatsya and Bhaskaran, Tg82~--Lu et a l . , 1982), roots (Xu et a l . , 1982) and hypocotyl-~-T~limelius, 1984, Chuong et a l . , 1985). The advantages of using hypocotyT~ ~ v e been described (Glimelius, 1984), but the production of brown phenolics that i n h i b i t protoplast development s t i l l remains problematical unless very careful timing of cell transfers is employed. Simplification of the culture procedure has been achieved (Chuong et a l . , 1985) but at a cost of efficiency. This report describes an alternative procedure for e f f i c i e n t regeneration of plants from hypocotyl protoplasts of Brassica napus. MATERIALS AND METHODS
Plant Material Conditions established by this study are suitable for a number of spring and winter types of B. napus, including Regent, Westar, Olga, Jet Neuf, S ~ t ~ 1 Present address: R. R_ No. 1, TerraCotta, Ontario LOP 1NO, Canada Offprint requests to: T. L Barsby
Seeds were s u r f a c e - s t e r i l i s e d by immersion in a 10% solution of commercial hypochlorite ('Javex'; containing 6.0% sodium hypochlorite by volume) for i0 min and then rinsed for 5 min in s t e r i l e d i s t i l l e d deionized water. Batches of seed for which this s t e r i l i z a t i o n procedure was i n e f f e c t i v e were immersed in a solution of 0.05% mercuric chloride and 0.1% sodium dodecyl sulfate for i0 min and rinsed 6 times in s t e r i l e water. S t e r i l i s e d seeds were placed (20/10cm petri plate) on hormone-free MS basal media (Murashige and Skoog, 1962) c o n t a i n i n g lOg/l sucrose, and s o l i d i f i e d with 4g/l agarose (Sigma Type i ) or with 2g/l Gellan Gum (Kelco KA940). Seeds were germinated in the dark at 25°C. Protoplast Isolation Hypocotyls were removed from dark-grown seedlings a f t e r 3-5 days; on occasion, hypocotyls as old as 17 days gave rise to viable protoplasts and eventually to plants. Excised hypocotyls were chopped into 2-5mm transverse segments and immediately placed into an enzyme solution, containing 1.0% Cellulase R-IO, 0.1% Macerozyme R-IO (Yakult Honsha Co., Ltd., Tokyo, Japan), 1.0% polyvinylpyrrolidone (PVP-IO, Sigma) 0.35M sucrose, in 5mM 2[N-morpholino] ethanesulphonic acid (MES) buffer, pH 5.6. Up to 200 hypocotyls (3.0-3.5g) were added to 50ml of enzyme solution, and the mixture incubated for 16h (overnight) with gentle agitation (50rpm) at 25°C. Protoplasts were collected as described by Shepard and Totten (1977). The digestion mixture was f i l t e r e d through two layers of cheesecloth and collected by centrifugation (750rpm, 10 min) prior to washing once in a solution containing sucrose and MES, in the proportions specified for the enzyme solution. Following a second centrifugation, a band comprising mostly protoplasts concentrated at the surface. Protoplast Culture P r o t o p l a s t s were suspended at a d e n s i t y of lO0,O00/ml in the cell layer medium of Shepard (1980), modified by adding 0.06% agarose in place of 0.4% washed agar, and substituting the hormone concentrations of Medium A of Glimelius (1984) (O.img/l NAA, O.4mg/l BAP, 1.0mg/l 2,4-D). The suspension was plated in p l a s t i c quadrant plates in contact with a reservoir medium (Shepard, 1980) containing O.img/l NAA and l.Omg/l 2,4-D. Plated protoplasts were incubated at 25°C in the dark.
102 Eight days after plating, the suspension of dividing cells was transferred to a lOcm petri plate and diluted with an equal volume of a medium similar to the reservoir, but modified by the replacement of the hormones specified with 1.0mg/l 2,4-D and O.lmg/l Kinetin. The agarose concentration was also altered to 0.06%. All subsequent procedures were carried out at 25°C with a 16h photoperiod of 4000 lux.
in v i t r o step and rooting shoots d i r e c t l y in peat, a~ e ~ i b e d . Only occasional loss of p l a n t l e t s occurred, due to fungal contamination. Reducing the photoperiod to lOh at this stage helped to prevent premature flowering. The jars in which the peat p e l l e t s were placed, were opened for 24h p r i o r to transfer of the plants to potting compost, to gradually acclimatize the plants to the lower humidity.
Callus Proliferation and Plant Regeneration Cell colonies were transferred four to fourteen days after d i l u t i o n , by which time they had reached O.5-1.0mm in size. Transfer was effected either by pipetting the suspension of colonies onto the surface of the proliferation medium and carefully drawing off excess dilution medium, or by individual colony transfer. The preferred proliferation medium was MS-based with 1.0mg/l 2,4-D, O.img/l kinetin and 1.0% sucrose.
Published protocols for regeneration of plants from B. napus protoplasts (Li and Kohlenbach, 1982., Glime~u~84., Chuong et a l . , 1985) were also investigated. None was as'-ef}'i%ient at a l l stages from protoplast to plant using the c u l t i v a r s l i s t e d , in our laboratory.
C a l l i of l-2mm in diameter were transferred to a s i m i l a r basal medium containing 2.0mg/l k i n e t i n , 2.0mg/l zeatin riboside, O.Img/l IAA with 0.2% sucrose to induce d i f f e r e n t i a t i o n . Temperature and l i g h t regimes were as described above for diluted colonies. Colonies with primordial shoots were transferred to B5 basal medium (Gamborg et a l . , 1968) containing 0.2% sucrose and O.03mg/l -~-A3~'-to induce grow-out of the shoots. Shoots with v i s i b l y differentiated meristems were transferred to s t e r i l e ' J i f f y 7' ( J i f f y Products (N.B.) Ltd., Shippegan, Canada) peat pellets to induce root formation. These were placed within s t e r i l e jars, to maintain a r e l a t i v e l y high humidity, under 10h photoperiod. When root development was evident, established plants were transferred to 'Metromix' potting compost (W.R. Grace & Co. of Canada Ltd.). RESULTS
Protoplast Isolation and Cell Division Three day old hypocotyls gave the most reproducible y i e l d s (3xlOb protoplasts per 100 dark-grown hypocotyls) and the most consistently high plating efficiencies (50-70%). Plant Regeneration Following d i l u t i o n (I-2 weeks), macroscopic colonies developed from approximately 90% of the dividing protoplasts. Compact, 2-5mm diameter colonies were transferred to regeneration medium. Selection of c a l l i at the correct stage for transfer was c r i t i c a l - calli less than 2mm in diameter or of loose appearance turned brown and died within 2-3 days of transfer, whereas compact c a l l i of 2-5mm began to form shoot primordia after 10 d a y s . MS-medium s o l i d i f i e d with 0.6% agarose Type I and containing 0.2% sucrose, 2.0mg/l kinetin, 2.0mg/l zeatin and O.img/l IAA gave high rates of shoot primordia formation, achieving up to 70% for 'Regent' With other cultivars, regeneration rates were lower, but not less than 10% in any case. Shoot primordia were a bright green colour, in comparison to the callus tissue which remained cream in colour. Those c a l l i that did develop a green colour, invariably failed to produce shoot primordia. The production of shoots was often associated with h a i r - l i k e structures on the callus.
After 2-3 weeks the primordia developed into recognizable shoots, and these developed further following transfer to the grow-out medium. Early problems with the induction of adventitious roots on regenerated shoots were resolved by eliminating the
DISCUSSION This report presents a concisely defined method for regenerating plants from protoplasts of B. napus. I t is acknowledged that the empirical nature oTmany plant regeneration studies allows at most, the development of techniques which apply repeatedly to a p a r t i c u l a r set of circumstances. In our hands, the reported technique is r e l i a b l e and e f f i c i e n t , and s u f f i c i e n t l y reproducible to be used in the production of somatic hybrids and cybrids. The main advantage of the present method, over s i m i l a r published reports, is that the high e f f i c i e n c y of early division is sustained through to the recovery of plants. No browning is observed in early culture. Recovery of colonies by incorporating three simple steps ( d i l u t i o n , p r o l i f e r a t i o n and regeneration), is far greater than by u t i l i s i n g simpler in concept 'one-step' methods, where the m a j o r i t y of p r o t o p l a s t s are not recovered to regeneration media. We a t t r i b u t e the improved e f f i c i e n c y mainly to i ) the use of a reservoir medium, i i ) early d i l u t i o n of colonies and the presence of 2,4-D in the p r o l i f e r a t i o n medium, i i i ) the presence of kinetin and the lowering of sucrose to 0.2% in the regeneration and shoot grow-out media, iv) the use of peat as a rooting medium. Protoplast regeneration is fundamental to a number of suggested endeavours with somatic cells. Careful monitoring of, and subtle improvements to systems, always w i l l be required i f these a c t i v i t i e s are to be successful. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of N. Maclean, W. Shurben and A. Walker. This research was funded in part by the National Research Council Canada, Ottawa, Ontario, Canada, under the Program for Industry/Laboratory Projects technology transfer. A r r a n g e m e n t No. CA910-4-OO10/B-31. REFERENCES
Bidney DL, Shepard JF, Kaleikau E (1983) Protoplasma 117:89-92 Chuong PV, Pauls KP, Beversdorf WD (1985) Plant Cell Reports 4:4-6 Gamborg OL, M i l l e r RA, Ojima K (1968) Exptl Cell Res 50:151-158 Glimelius K (1984) Physiol Plant 61:38-44 Guo BJ, Schieder 0 (1983) Z Pflanzenphysiol 110:375-377 Kohlenbach HW, Wenzel G, Hoffman F (1982) Z Pflanzenphysiol 105:131-142 Li L, Kohlenbach HW (1982) Plant Cell Reports 1:209-211 Lu DY, Pental D, Cocking EC (1982) Z Pflanzenphysiol 107:59-63
103 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Newell CA, Rhoads ML, Bidney DL (1984) Can J Genet Cytol 26:752-761 Shepard JF, (1980) In: Rubenstein I, Gengenbach B, Phillips RL, Green CE (eds) Genetic Improvement of Crops: Emergent Techniques, University of Minnesota Press, Minneapolis. pp 185-219
Shepard JF, Torten RE (1977) Plant Physiol 60:313-316 Vatsya B, Bhaskaran S (1982) Protoplasma 113:161-163 Xu ZH, Davey MR, Cocking EC (1982) Plant Sci Lett 24:117-121
Plant Cell Reports (1986) 5:101 103
© Springer-Verlag 1986
A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus T. L. Barsby, S. A. Yarrow, and J. F. Shepard 1 Allelix I n c 6850 Goreway Drive, Mississauga, Ontario L4V 1P l, Canada Received January 17, 1986 / Revised version received January 31, 1986 - Communicated by F. Constabel
ABSTRACT
Protoplasts of several spring and winter varieties of Brassica napus were isolated from hypocotyl tissue. Proto---6-~Fasts divided and formed cell colonies at high frequency, without browning when cultured in modified Shepards' medium. This high efficiency of proliferation was sustained through to plant regeneration with all varieties cultured. This has been attributed to the incorporation of a reservoir medium, the presence of 2,4-D in the proliferation medium, and the presence of kinetin in conjunction with lowering of the sucrose concentration in the regeneration medium. ABBREVIAT IONS NAA = 1-naphthaleneacetic acid;
BAP = 6-benzylaminopurine; IAA = 3-indoleacetic acid; 2,4-D = 2,4-dichlorophenoxyacetic acid. INTRODUCTION
In order that cell technologies (eg., somatic hybridisation, transformation, etc.) may present a major contribution to conventional breeding techniques of agriculturally important crop species, high regeneration frequencies from protoplasts are essential. Recently, much attention has been directed to the Brassicaceae, to address this problem. Regeneration from protoplasts of a number of tissue sources has been reported, namely from leaves (Bidney et a l . , 1983, Guo and Schieder, 1983, Li and Kohlenbac-ElS,---1982, Newell et a l . , 1984), stem embryos (Kohlenbach et a l . , -T9E~-), cotyledons (Vatsya and Bhaskaran, Tg82~--Lu et a l . , 1982), roots (Xu et a l . , 1982) and hypocotyl-~-T~limelius, 1984, Chuong et a l . , 1985). The advantages of using hypocotyT~ ~ v e been described (Glimelius, 1984), but the production of brown phenolics that i n h i b i t protoplast development s t i l l remains problematical unless very careful timing of cell transfers is employed. Simplification of the culture procedure has been achieved (Chuong et a l . , 1985) but at a cost of efficiency. This report describes an alternative procedure for e f f i c i e n t regeneration of plants from hypocotyl protoplasts of Brassica napus. MATERIALS AND METHODS
Plant Material Conditions established by this study are suitable for a number of spring and winter types of B. napus, including Regent, Westar, Olga, Jet Neuf, S ~ t ~ 1 Present address: R. R_ No. 1, TerraCotta, Ontario LOP 1NO, Canada Offprint requests to: T. L Barsby
Seeds were s u r f a c e - s t e r i l i s e d by immersion in a 10% solution of commercial hypochlorite ('Javex'; containing 6.0% sodium hypochlorite by volume) for i0 min and then rinsed for 5 min in s t e r i l e d i s t i l l e d deionized water. Batches of seed for which this s t e r i l i z a t i o n procedure was i n e f f e c t i v e were immersed in a solution of 0.05% mercuric chloride and 0.1% sodium dodecyl sulfate for i0 min and rinsed 6 times in s t e r i l e water. S t e r i l i s e d seeds were placed (20/10cm petri plate) on hormone-free MS basal media (Murashige and Skoog, 1962) c o n t a i n i n g lOg/l sucrose, and s o l i d i f i e d with 4g/l agarose (Sigma Type i ) or with 2g/l Gellan Gum (Kelco KA940). Seeds were germinated in the dark at 25°C. Protoplast Isolation Hypocotyls were removed from dark-grown seedlings a f t e r 3-5 days; on occasion, hypocotyls as old as 17 days gave rise to viable protoplasts and eventually to plants. Excised hypocotyls were chopped into 2-5mm transverse segments and immediately placed into an enzyme solution, containing 1.0% Cellulase R-IO, 0.1% Macerozyme R-IO (Yakult Honsha Co., Ltd., Tokyo, Japan), 1.0% polyvinylpyrrolidone (PVP-IO, Sigma) 0.35M sucrose, in 5mM 2[N-morpholino] ethanesulphonic acid (MES) buffer, pH 5.6. Up to 200 hypocotyls (3.0-3.5g) were added to 50ml of enzyme solution, and the mixture incubated for 16h (overnight) with gentle agitation (50rpm) at 25°C. Protoplasts were collected as described by Shepard and Totten (1977). The digestion mixture was f i l t e r e d through two layers of cheesecloth and collected by centrifugation (750rpm, 10 min) prior to washing once in a solution containing sucrose and MES, in the proportions specified for the enzyme solution. Following a second centrifugation, a band comprising mostly protoplasts concentrated at the surface. Protoplast Culture P r o t o p l a s t s were suspended at a d e n s i t y of lO0,O00/ml in the cell layer medium of Shepard (1980), modified by adding 0.06% agarose in place of 0.4% washed agar, and substituting the hormone concentrations of Medium A of Glimelius (1984) (O.img/l NAA, O.4mg/l BAP, 1.0mg/l 2,4-D). The suspension was plated in p l a s t i c quadrant plates in contact with a reservoir medium (Shepard, 1980) containing O.img/l NAA and l.Omg/l 2,4-D. Plated protoplasts were incubated at 25°C in the dark.
102 Eight days after plating, the suspension of dividing cells was transferred to a lOcm petri plate and diluted with an equal volume of a medium similar to the reservoir, but modified by the replacement of the hormones specified with 1.0mg/l 2,4-D and O.lmg/l Kinetin. The agarose concentration was also altered to 0.06%. All subsequent procedures were carried out at 25°C with a 16h photoperiod of 4000 lux.
in v i t r o step and rooting shoots d i r e c t l y in peat, a~ e ~ i b e d . Only occasional loss of p l a n t l e t s occurred, due to fungal contamination. Reducing the photoperiod to lOh at this stage helped to prevent premature flowering. The jars in which the peat p e l l e t s were placed, were opened for 24h p r i o r to transfer of the plants to potting compost, to gradually acclimatize the plants to the lower humidity.
Callus Proliferation and Plant Regeneration Cell colonies were transferred four to fourteen days after d i l u t i o n , by which time they had reached O.5-1.0mm in size. Transfer was effected either by pipetting the suspension of colonies onto the surface of the proliferation medium and carefully drawing off excess dilution medium, or by individual colony transfer. The preferred proliferation medium was MS-based with 1.0mg/l 2,4-D, O.img/l kinetin and 1.0% sucrose.
Published protocols for regeneration of plants from B. napus protoplasts (Li and Kohlenbach, 1982., Glime~u~84., Chuong et a l . , 1985) were also investigated. None was as'-ef}'i%ient at a l l stages from protoplast to plant using the c u l t i v a r s l i s t e d , in our laboratory.
C a l l i of l-2mm in diameter were transferred to a s i m i l a r basal medium containing 2.0mg/l k i n e t i n , 2.0mg/l zeatin riboside, O.Img/l IAA with 0.2% sucrose to induce d i f f e r e n t i a t i o n . Temperature and l i g h t regimes were as described above for diluted colonies. Colonies with primordial shoots were transferred to B5 basal medium (Gamborg et a l . , 1968) containing 0.2% sucrose and O.03mg/l -~-A3~'-to induce grow-out of the shoots. Shoots with v i s i b l y differentiated meristems were transferred to s t e r i l e ' J i f f y 7' ( J i f f y Products (N.B.) Ltd., Shippegan, Canada) peat pellets to induce root formation. These were placed within s t e r i l e jars, to maintain a r e l a t i v e l y high humidity, under 10h photoperiod. When root development was evident, established plants were transferred to 'Metromix' potting compost (W.R. Grace & Co. of Canada Ltd.). RESULTS
Protoplast Isolation and Cell Division Three day old hypocotyls gave the most reproducible y i e l d s (3xlOb protoplasts per 100 dark-grown hypocotyls) and the most consistently high plating efficiencies (50-70%). Plant Regeneration Following d i l u t i o n (I-2 weeks), macroscopic colonies developed from approximately 90% of the dividing protoplasts. Compact, 2-5mm diameter colonies were transferred to regeneration medium. Selection of c a l l i at the correct stage for transfer was c r i t i c a l - calli less than 2mm in diameter or of loose appearance turned brown and died within 2-3 days of transfer, whereas compact c a l l i of 2-5mm began to form shoot primordia after 10 d a y s . MS-medium s o l i d i f i e d with 0.6% agarose Type I and containing 0.2% sucrose, 2.0mg/l kinetin, 2.0mg/l zeatin and O.img/l IAA gave high rates of shoot primordia formation, achieving up to 70% for 'Regent' With other cultivars, regeneration rates were lower, but not less than 10% in any case. Shoot primordia were a bright green colour, in comparison to the callus tissue which remained cream in colour. Those c a l l i that did develop a green colour, invariably failed to produce shoot primordia. The production of shoots was often associated with h a i r - l i k e structures on the callus.
After 2-3 weeks the primordia developed into recognizable shoots, and these developed further following transfer to the grow-out medium. Early problems with the induction of adventitious roots on regenerated shoots were resolved by eliminating the
DISCUSSION This report presents a concisely defined method for regenerating plants from protoplasts of B. napus. I t is acknowledged that the empirical nature oTmany plant regeneration studies allows at most, the development of techniques which apply repeatedly to a p a r t i c u l a r set of circumstances. In our hands, the reported technique is r e l i a b l e and e f f i c i e n t , and s u f f i c i e n t l y reproducible to be used in the production of somatic hybrids and cybrids. The main advantage of the present method, over s i m i l a r published reports, is that the high e f f i c i e n c y of early division is sustained through to the recovery of plants. No browning is observed in early culture. Recovery of colonies by incorporating three simple steps ( d i l u t i o n , p r o l i f e r a t i o n and regeneration), is far greater than by u t i l i s i n g simpler in concept 'one-step' methods, where the m a j o r i t y of p r o t o p l a s t s are not recovered to regeneration media. We a t t r i b u t e the improved e f f i c i e n c y mainly to i ) the use of a reservoir medium, i i ) early d i l u t i o n of colonies and the presence of 2,4-D in the p r o l i f e r a t i o n medium, i i i ) the presence of kinetin and the lowering of sucrose to 0.2% in the regeneration and shoot grow-out media, iv) the use of peat as a rooting medium. Protoplast regeneration is fundamental to a number of suggested endeavours with somatic cells. Careful monitoring of, and subtle improvements to systems, always w i l l be required i f these a c t i v i t i e s are to be successful. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of N. Maclean, W. Shurben and A. Walker. This research was funded in part by the National Research Council Canada, Ottawa, Ontario, Canada, under the Program for Industry/Laboratory Projects technology transfer. A r r a n g e m e n t No. CA910-4-OO10/B-31. REFERENCES
Bidney DL, Shepard JF, Kaleikau E (1983) Protoplasma 117:89-92 Chuong PV, Pauls KP, Beversdorf WD (1985) Plant Cell Reports 4:4-6 Gamborg OL, M i l l e r RA, Ojima K (1968) Exptl Cell Res 50:151-158 Glimelius K (1984) Physiol Plant 61:38-44 Guo BJ, Schieder 0 (1983) Z Pflanzenphysiol 110:375-377 Kohlenbach HW, Wenzel G, Hoffman F (1982) Z Pflanzenphysiol 105:131-142 Li L, Kohlenbach HW (1982) Plant Cell Reports 1:209-211 Lu DY, Pental D, Cocking EC (1982) Z Pflanzenphysiol 107:59-63
103 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Newell CA, Rhoads ML, Bidney DL (1984) Can J Genet Cytol 26:752-761 Shepard JF, (1980) In: Rubenstein I, Gengenbach B, Phillips RL, Green CE (eds) Genetic Improvement of Crops: Emergent Techniques, University of Minnesota Press, Minneapolis. pp 185-219
Shepard JF, Torten RE (1977) Plant Physiol 60:313-316 Vatsya B, Bhaskaran S (1982) Protoplasma 113:161-163 Xu ZH, Davey MR, Cocking EC (1982) Plant Sci Lett 24:117-121
