Plant Cell Reports
Plant Cell Reports (1991) 9:575 578
9 Springer-Verlag1991
Arabidopsis thaliana: protocol for plant regeneration from protoplasts Hans Karesch, Roland Bilang, and Ingo Potrykus Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), ETH-Zentrum, CH-8092 Ztirich, Switzerland Received October 8, 1990/Revised version received October 30, 1990 - Communicated by H. L6rz
ABSTRACT
We report a protocol for plant regeneration from leaf protoplasts of Arabidopsis thaliana ecotype ZOrich. The protocol has been established in 1988 and has since been in routine use in our laboratory. Whereas recovery of proliferating protoplast-derived clones is routine, the success in plant regeneration from protoplast-derived clones is highly variable. In the hands of one of us (H.K.) average shoot regeneration frequency (% of calli regenerating at least one shoot) was ca. 60% and average plant regeneration frequency (% of calli yielding fertile plants in soil) was ca. 40%.
mation, e.g. for transient expression studies (Fromm and Walbot 1987), in gene targeting studies (Paszkowski et al. 1988) or in shotgun cloning approaches aimed at identifying DNA sequences that complement mutant phenotypes. We were therefore interested in developing appropriate protocols for culture and regeneration of protoplasts from Arabidopsis thaliana. A protocol on plant regeneration from protoplasts of Arabidopsis thaliana ecotype Columbia has been published by Damm and Willmitzer (1988). Although our protocol differs in several parameters from their procedure we also can confirm its basic suitability for another ecotype.
ABBREVIATIONS
2,4-D: 2,4-dichlorophenoxyacetic acid, IAA: indoleacetic acid, NAA: ~naphthaleneacetic acid, BAP: benzyiaminopurine, 2-iP: 2isopentenylaminopurine.
INTRODUCTION
Arabidopsis thaliana has attracted the interest of numerous laboratories working in the areas of plant molecular biology, plant development and genetic engineering. The properties which make this small crucifer so attractive have often been described, most recently in a review by EM. Meyerowitz (1989): small genome, little repetitive DNA, 5 chromosomes, more than 70 characterized genetic loci, more than 200 RFLP markers, rich mutant collection, short generation time, thousands of seeds per plant, small size allowing to grow many plants on little space, etc.. Further progress in the areas mentioned above depends strongly on routine and efficient techniques for transient or stable introduction of genes into Arabidopsis cells. Although stable integrative transformation is possible with several Agrobacterium-based protocols (Lloyd et al. 1986; Feldmann and Marks 1987; Valvekens et a1.1988;Schmidt and Willmitzer 1988), the method of direct gene transfer to protoplasts (Paszkowski et al. 1984; Shillito et al. 1985; Negrutiu et al. 1987) might offer certain advantages compared to Agrobacterium mediated transfor-
Offprint requests to: I. Potrykus
The work reported here (previously presented at the Second International Congress in Plant Molecular Biology, Jerusalem 1988; Karesch et al. 1988) and its application for gene transfer into Arabidopsis (Karesch and Potrykus 1988, Karesch et al. Plant Cell Reports, this issue) was part of the experimental work for a PhD thesis of Hans Karesch which he could not complete because of his sudden death on November 6, 1989. With Hans Karesch the scientific community lost a dedicated colleague with an outstanding interest and talent for plant cell culture.
MATERIAL & M ETHODS 1. Culture media MS: Murashige & Skoog, 1962, MS basal: MS salts and vitamins (1 mg/I thiamine HCI, 0.5 mg/I pyridoxin HCI, 0.5 mg/I nicotinic acid, 2 mg/I glycine, 100 mg/I m-inositol ), MS-O: MS basal + 3% (w/v) sucrose, pH 6.0 (before autoclaving), 0.8% agar (Difco Bacto), 1/2 MS-O: half concentration of MS basal, 1.5% sucrose, MS-PP: protoplast culture medial filtersterilized through 0.2 p.m, MS-PPh MSbasal, 0.4 M glucose, 1 mg/I 2,4-D, 0.15 mg/I kinetin, pH 5.8, MSPPIh MS basal, 0.2 M glucose, 0.05 mg/I 2,4-D, 2.0 mg/I NAA, 0.15 mg/I kinetin, 0.15 mg/I BAP, pH 5.8; for callus cultures: plus 0.8%
agar, pH 6.0 before autoclaving. MS-S (shoot-inducing): MS basal,
576 3% sucrose, 0.15 mg/I IAA, 5.0 mg/I 2-iP, 0.8% agar, pH 6.0 before autoclaving, MS-R (root-inducing): 1/2 MS-O, 1 mg/I NAA, 0.8% agar, pH 6.0 before autoclaving, MS-M: MS basal, 0.2 M glucose, 0.1 mg/I IAA, 1 mg/I kinetin, 0.8% Difco Bacto agar, pH 6.0 before autoclaving. MaCa solution: 0.4 M mannitol, 10 mM CaCIz, WS: (Menczel et al. 1981) 1000 ml contain 18.4 g CaCl=x 2 H20, 9.0 g NaCI, 1.0 g glucose, 0.8 g KCI, autoclaved.
2. Axenic plant cultures Plants ofArabidopsis thaliana ecotype Z0rich were collected at the Botanical Garden and confirmed by E. Landolt (ETH Zurich). Seeds were sterilized by vigorous shaking for 8 minutes in 10 ml of 7.2% (w/v) of Ca(OCI)2 plus Tween 80, collected on a sterile cotton filter (placed into a funnel) and carefully washed with sterile water. For germination 12-16 seeds were transferred with a painting brush onto 1/2 MS-O agar in plastic petri dishes and sealed with Parafilm. Following cold treatment at 4"C in the dark for 3 days, dishes were incubated for 7 days under 16 hours light (2800 lux),20-25"C. Night temperature was 16-20"C. Four seedlings each were carefully transferred with fine forceps onto 40 ml of 1/2 MS-O agar medium contained in 400 ml PLASTEM boxes (PE 474, Plastem AG, Schwarmburg, Switzerland). Good aeration was provided by a hole in the lid closed with a rubber sponge stopper (Cepharem stopper, Greiner, N0rttingen, FRG). Plants were raised in growth chambers with 16 h light (cool fluorescence, 4000 lux), 22"C. Night temperature was t6"C.
3. Protoplast isolation Protoplasts were isolated from leaves of 4 week old plants at the vegetative rosette stage. Leaves were wetted with and cut into fine slices in MaCa solution. Following washing in the same solution tissue slices were preincubated in MaCa solution for 1 h at 24"C in the dark. Thereafter MaCa was replaced with enzyme solution (10ml/ g leaf tissue) containing either 1% (w/v) Cellulysin + 0.1% Macerase, or 1% Cellulase Onozuka R10 + 0.25% Macerozyme R10, both dissolved in 0.4 M mannitol + 6.8 mM CaCI=, pH 5.5, 490 mOs/kg H20. Following 16 h of incubation (overnight) the digestion mix was shaken very gently and incubated for further 30 min. Protoplasts were separated from undigested tissue in stainless steel sieves of 100 and 50 I~m mesh. Following the addition of one volume of W5 solution to 2 volumes of enzyme/protoplast suspension the suspension was distributed to sterile capped plastic centrifuge tubes (Greiner, NCirttingen, FRG, 12 ml) and sedimented for 10 rain. at 80 g in a table centrifuge (Hettich Universial 1200). The sediment was resuspended in W5, sedimented and resuspended in a mixture of 1 vol. W5 + 2 vol. of 0.5 M mannitol. Following a final sedimentation the protoplasts were resuspended in 0.5 M mannitol (545 mOs) at a population density of 4x105/ml.
4. Protoplast culture Protoplasts developed best when embedded into a gel containing charged groups, e.g. alginate or pectinate. Preparation of thin layers of protoplasts in alginate or pectinate requires that calcium-free
protoplast suspensions are allowed to gel over a calcium-centaining basal layer. Therefore, protoplasts suspended at double plating density (4x105/ml) in 0.5 M mannitol were mixed with the same volume of either a solution of 2% (wN) Na-pectinate in 0.4 M mannitol (475 mOs,filtersterilized, Pektin N, no. 2-8913, Carl Roth, Karlsruhe, FRG) or with 2% Na-alginate in 0.4 M mannitol (467 rues, autoclaved, Sigma A 2158, low viscosity). One ml aliquots each were pipetted onto a basis layer of 5 ml solidified Difco Bacto agar containing either 100 mM CaCI= + 0.4 M mannitol for pectinate layers, or 10 mM CaCl= + 0.4 M mannitol for alginate layers, respectively. Following gellification for 30 min. the gellified surface was flooded with 2.5 ml of MaCa solution. Petri dishes were sealed with Parafilm and'incubated for 2 days at 4"C in the dark (without addition of any nutrients). Following this cold-starvation period protoplast gels were transferred with a spatula into fresh 6 cm Petri dishes containing 5 ml MaCa solution which was after a few minutes replaced by MS-PPI culture medium. Optimal gel/medium ratios were 0.4 (e.g. 0.5 ml protoplasts gel + 1.25 ml culture medium, gel rinsed once with MS-PPI before). The protoplast gels were incubated for 7 days in the dark at.22"C, followed by dim continous light at the same temperature. At day 10 from protoplast isolation 1.25 ml of MS-PPII was added to the conditioned MS-PPI medium yielding a gel/medium ratio of 0.2 (e.g. 0.5 ml gel + 2.5 ml medium). Thereafter MS-PPII was replaced in weekly intervals until week 9. When the yellow-green protoplast-derived clones had reached Ihe size of 1-2 mm they were liberated from the gel by chelating the calcium ions complexed with the polysaccharide chains of the gelling agent with potassium citrate: the culture medium was replaced by 2.5 ml of 20 mM K-citrate in 0.2 M glucose, pH 5.8,300 mOs, autoclaved. After 5 min. of incubation the citrate solution was replaced by 5 ml of fresh citrate solution, the Petri dish sealed with Parafilm and incubated on a gyratory shaker at 40 rpm for 30 minutes. Liberated clones were finally washed with MS-PPII.
5. Plant regeneration The protoplast-derived clones were individually transferred onto MSPPII agar (300 mOs, 20 ml per 9 c,nl petri dish). Dishes were sealed with Parafilm and incubated at 20"C in continuous dim light (ca. 700 lux). Within 3 weeks of culture clones reached a size of ca. 5 mm in diameter. At this stage the development of shoot meristems was induced by transfer to MS-S culture medium (190 mOs) and incubation at 20"C, 2000 lux continuous light. After 10-16 days of incubation shoot meristems could be identified as small dark-green areas. At this stage elongation and differentiation of shoots were supported by reduction of exogenous hormones and good aeration: calli with shoot primordia were transferred to 1/2 MS-O agar medium (0.8% Difco Bacto, 80 mOs, 40 ml per Plastem box, lid closed with Cepharem stopper). Incubation was at 20"C, 16 h 3000 lux, 16"C at darkness. After 2 weeks shoot rosettes could be excised from the calli for subsequent rooting. Remaining differentiating tissue pieces could be placed onto MS-M agar for a further multiplication cycle of shoots. Remaining undifferentiated callus could be placed back onto
577 1/2 MS-O for regeneration of further shoots. For rooting, the excised shoots were transferred to MS-R medium (in 9 cm petridishes) and incubated at 20"0, continuous dim light for 3 days. Root growth was promoted by subsequent transfer to hormone-free MS-O agar contained in Plastem boxes (with a hole in the lid, dosed with a Sepharem stopper) and incubation at 20"C, 16 h 3000 lux, 16"C during darkness. Rooted shoots were transferred to a mixture of 2 vol. potground + 1 vol perlite contained in small pots standing in shallow water in Plastem boxes with 3 holes in the lid, dosed with Cepharem stoppers. Culture was in the first two weeks still under sterile conditions at 20/16"C, 16 h 3000 lux per day. Adjustment to growth in a less humid atmosphere was achieved by stepwise opening the stoppers in the lid. The developing floral shoots were allowed to grow out of the Plastem boxes through the open holes in the lid. Flowering and fruit ripening took place in the open air at 20/ 16"C, 16 h 3000 lux per day. If watering was necessary this was done through the open holes.
Shoot Induction: (10-16 days). MS-S, 0.8% agar, 3% sucrose, 188 mOs; 20"C, continuous light; 2000 lux green shoot primordia at contact zone.
Shoot differentiation and elongation: (2 weeks). 1/2 MS-O, 0.8% agar, 1.5% sucrose, 80 rues; 20"C, continuous light, 2800 lux. Good aeration enhances shoot development and minimizes vitrification.
Excision and trimming of shoots/ root induction: (3 days). MS-R, 0.8% agar, 1.5% sucrose, 80 rues. 20"C, continuous dim light; 700 lux, development of root primordia.
Root eleongation: ( 10 days). 6. Flow-chart: Item protoplast to plant Preplasmolysis: (1 hour). MaCa-solution, 450 mOs/kg H20, 25"C, darkness.
1/2 MS-O, 0.8% agar; Plastem boxes with perforation; 20/16"C, 16 h day, 3000 lux. development of shoot resettes and branched root systems.
Transfer to soil and flowering: (7 weeks). Enzyme treatment: (16 hours). 1% Onozuka R10 + 0.25% Macerozyme R10, in MaCa, 490 mOs; 25"C, darkness
Potground + perlite (2+1), autoclaved, small pots in Plastem boxes with perforation; 7 days under sterile conditions; 20/16"C, 16 h day with 2800 lux.
Protoplast gels: (1 hour). 1% Na-pectinate in 0.4 M mannitol, or 1% Na-alginate in 0.4 M mannitol.
Cold-starvation: (36 hours). Protoplast gel over MaCa basis layer, under liquid MaCa; 4"C, darkness,
Protoplast culture: (7 days). MS-PPI, 530 mOs, 0.4 M glucose; 20"C, dark; first cell divisions at day 9.
Development of micro-colonies: (7 days). addition of same volume of MS-PPII, 400 rues, 0.3 M glucose; 20"C, continuous dim light, 700 lux. Development to green macrocolonles: (6 weeks). weekly replacement with MS-PPII; 300 rues, 0.2 M glucose; 20"C, continuous dim light, 700 lux.
Liberation from gel matrix: 20 mM K-citrate in 0.2 M glucose, 300 mOs.
Development of morphogenic callus: (3 weeks). MS-PPII, 0.8% agar, 0.2 M glucose, 300 mOs; 20"C, continuous dim light, 700 lux.
RESULTS & DISCUSSION The above (partially optimised) protocol has been developed on the basis of the published experience with protoplast and cell culture systems of Arabidopsis and Brassica (e.g. Damm and Wilmitzer 1988; Feldmann and Marks 1986; Gleba et al. 1978; Gleba and Hofmann 1978, 1980; Glimelius 1984; Gresshoff 1976; Huang and Yeoman 1984; Kao and Seguin-Swartz 1987; Negrutiu et a1.1975; Negrutiu and Jacobs 1978; Xuan and Menczel 1979, 1980) and describes a laboratory procedure which has been used in our group for two years with leaf protoplasts from Arabidopsis thaliana ecotype Z(Jrich. As with most other protoplast systems, the quantitative data vary between experiments, days, seasons and plants (Potrykus and Shillito 1986). Protoplast division frequency (ratio of cells entering first divisions) varied I>~tween0.1 and ca. 80% with an average of ca. 10%. The plating efficiency (ratio of cells yielding proliferating calli varied between ca. 0.01 and 5% with an average in the range of ca. 0.5%. Experiments from which no proliferating clones could be recovered were rare exceptions in a series of more than 200 experiments. When comparing plating efficiency, developmentof morphogenic calli, and shoot/plant regeneration from alginate- and pect~nate cultures the results clearly speak in favour of pectinate. As, however, alginate gave more consistent results over a longer period, we use this gelling agent in our routine procedures. Whereas recovery of protoplast-derived clones is more or less
578 predictable and achieved routinely by different experimentators, regeneration of fertile plants from protoplast-derived clones depended far stronger on the person involved. In the hands of one of us (H.K.) shoot and plant regeneration from protoplast-derived clones was reproducible in the range of ca. 40% and 60%, respectively, and could also be used for the regeneration of fertile transgenic plants after direct gene transfer to protoplasts (Karesch et al., this issue). The yield of fertile plants dropped significantly in experiments performed by other persons. Plant regeneration from protoplasts is, therefore, possible, even with relatively high efficiency, but the amount of necessary work and attention to obtain a high number of regenerants seems to depend on the general tissue culture experience of the experimentator and his/her readiness to further optimize the procedure. On the other hand, the procedure seems to be sufficient for studies of gene expression in transient systems (M. Schrott, unpublished results), as well as for large scale transformation experiments for gene targeting (R. B., unpublished results), where growth and selection of protoplast-derived calli can already answer the question of an experiment. As, however, plant regeneration from plant explants (leaf disk, root, petiole, stem, floral stem) is far less problematic, there should be a good reason if one decides to go the hard way via protoplasts. Acknowledgements We would like to thank Ortrun Mittelsten Scheid for very helpful discussions.
LITERATURE Datum B, Willmitzer L (1988) MoL Gen. Genet. 213: 15-20. Feldmann KA, Marks MD (1986) Plant Sci. 47: 63-69. Fromm M, Walbot V (1987) In: Plant Gene Research: Plant DNA Infectious Agents, Hohn T, Schell J. (eds), Springer, Wien, pp. 304-310. Glebe YY, Sidorov VA, Artemenko VS (1978) Fiziologiya Rastenii 25: 161-165. Glebe YY, Hofmann F (1978) Mol. Gen. Genet. 165: 257-264. Glebe YY, Hofmann F (1980) Planta 149:112-117. Glimelius K (1984) Physiol. Plant. 61 : 38-44. Gresshoff PM (1976) Arabidopsis Information Service 13:211-214. Kao HM, Seguin-Swartz G (1987) Plant Cell Tissue and Organ Culture 10: 79-90. Huang BC and Yeoman MM (1984) Plant Sci. Lett. 33: 353-363. Karesch H, Damm B, Potrykus I (1988) In: Abstracts, Second International Congress in Plant Molecular Biology, Jerusalem, Nov. 13-18, Abstr. No. 572. Karesch H, Petrykus I (1988) In: Abstracts, Second International Congress in Plant Molecular Biology, Jerusalem, Nov. 13-18, Abstr. No. 133. Karesch H, Bilang R, Mittelsten Scheid O. Potrykus I. Plant Cell Rep., this issue. Lloyd AM, Barnasen AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234: 464-466. Menczel L, Nagy F, Kiss Z, Maliga P (1981) Theer. Appl. Genet. 59: 191-195.
Meyerowitz EM (1989) Cell 56: 263-269. Mursshige T, Skoog F (1962) Physiol. Plant. 15: 473-497. Negrutlu I, Beeftink F, Jacobs M (1975) Plant SCl. Lett. 5: 293-304. Negrutlu I, Jacobs M (1978) Z. Pflanzenphysiol. 90: 423-430. Negrutlu I, Shillito RD, Potrykus I, Biasini G, Sala F (1987) Plant MoI. Biol. 8: 363-373. Paszkowski J, Shillito RD Saul MW, Mandak V, Hohn T, Hohn B, Potrykus I (1984) EMBO J. 3: 2712-2722. Peszkowski J, Baur M, Bogucki A, Potrykus I (1988) EMBO J. 7: 4021-4027. Potrykus I, ShilUto RD (1986) Methods Enzymol. 118: 550-578. Schmidt R, Willmitzer L (1988) Plant Cell Rep. 7: 583-586. Shillito RD, Saul MW, Paszkowski J, MOiler M, Potrykus I (1985) BioTechnol. 3:1099-1103. Valvekens D, Van Mentagu M, Van Lijsebettens M (1988) Proc. NatJ. Acad. Sci. USA 85: 5536-5540. Xuan LT, Menczel L (1979) Arabidopsis Information Service 16:82 83. Xuan LT, Menczel L (1980) Z. Pflanzenphysiol. 96: 77-80.
Plant Cell Reports (1991) 9:575 578
9 Springer-Verlag1991
Arabidopsis thaliana: protocol for plant regeneration from protoplasts Hans Karesch, Roland Bilang, and Ingo Potrykus Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), ETH-Zentrum, CH-8092 Ztirich, Switzerland Received October 8, 1990/Revised version received October 30, 1990 - Communicated by H. L6rz
ABSTRACT
We report a protocol for plant regeneration from leaf protoplasts of Arabidopsis thaliana ecotype ZOrich. The protocol has been established in 1988 and has since been in routine use in our laboratory. Whereas recovery of proliferating protoplast-derived clones is routine, the success in plant regeneration from protoplast-derived clones is highly variable. In the hands of one of us (H.K.) average shoot regeneration frequency (% of calli regenerating at least one shoot) was ca. 60% and average plant regeneration frequency (% of calli yielding fertile plants in soil) was ca. 40%.
mation, e.g. for transient expression studies (Fromm and Walbot 1987), in gene targeting studies (Paszkowski et al. 1988) or in shotgun cloning approaches aimed at identifying DNA sequences that complement mutant phenotypes. We were therefore interested in developing appropriate protocols for culture and regeneration of protoplasts from Arabidopsis thaliana. A protocol on plant regeneration from protoplasts of Arabidopsis thaliana ecotype Columbia has been published by Damm and Willmitzer (1988). Although our protocol differs in several parameters from their procedure we also can confirm its basic suitability for another ecotype.
ABBREVIATIONS
2,4-D: 2,4-dichlorophenoxyacetic acid, IAA: indoleacetic acid, NAA: ~naphthaleneacetic acid, BAP: benzyiaminopurine, 2-iP: 2isopentenylaminopurine.
INTRODUCTION
Arabidopsis thaliana has attracted the interest of numerous laboratories working in the areas of plant molecular biology, plant development and genetic engineering. The properties which make this small crucifer so attractive have often been described, most recently in a review by EM. Meyerowitz (1989): small genome, little repetitive DNA, 5 chromosomes, more than 70 characterized genetic loci, more than 200 RFLP markers, rich mutant collection, short generation time, thousands of seeds per plant, small size allowing to grow many plants on little space, etc.. Further progress in the areas mentioned above depends strongly on routine and efficient techniques for transient or stable introduction of genes into Arabidopsis cells. Although stable integrative transformation is possible with several Agrobacterium-based protocols (Lloyd et al. 1986; Feldmann and Marks 1987; Valvekens et a1.1988;Schmidt and Willmitzer 1988), the method of direct gene transfer to protoplasts (Paszkowski et al. 1984; Shillito et al. 1985; Negrutiu et al. 1987) might offer certain advantages compared to Agrobacterium mediated transfor-
Offprint requests to: I. Potrykus
The work reported here (previously presented at the Second International Congress in Plant Molecular Biology, Jerusalem 1988; Karesch et al. 1988) and its application for gene transfer into Arabidopsis (Karesch and Potrykus 1988, Karesch et al. Plant Cell Reports, this issue) was part of the experimental work for a PhD thesis of Hans Karesch which he could not complete because of his sudden death on November 6, 1989. With Hans Karesch the scientific community lost a dedicated colleague with an outstanding interest and talent for plant cell culture.
MATERIAL & M ETHODS 1. Culture media MS: Murashige & Skoog, 1962, MS basal: MS salts and vitamins (1 mg/I thiamine HCI, 0.5 mg/I pyridoxin HCI, 0.5 mg/I nicotinic acid, 2 mg/I glycine, 100 mg/I m-inositol ), MS-O: MS basal + 3% (w/v) sucrose, pH 6.0 (before autoclaving), 0.8% agar (Difco Bacto), 1/2 MS-O: half concentration of MS basal, 1.5% sucrose, MS-PP: protoplast culture medial filtersterilized through 0.2 p.m, MS-PPh MSbasal, 0.4 M glucose, 1 mg/I 2,4-D, 0.15 mg/I kinetin, pH 5.8, MSPPIh MS basal, 0.2 M glucose, 0.05 mg/I 2,4-D, 2.0 mg/I NAA, 0.15 mg/I kinetin, 0.15 mg/I BAP, pH 5.8; for callus cultures: plus 0.8%
agar, pH 6.0 before autoclaving. MS-S (shoot-inducing): MS basal,
576 3% sucrose, 0.15 mg/I IAA, 5.0 mg/I 2-iP, 0.8% agar, pH 6.0 before autoclaving, MS-R (root-inducing): 1/2 MS-O, 1 mg/I NAA, 0.8% agar, pH 6.0 before autoclaving, MS-M: MS basal, 0.2 M glucose, 0.1 mg/I IAA, 1 mg/I kinetin, 0.8% Difco Bacto agar, pH 6.0 before autoclaving. MaCa solution: 0.4 M mannitol, 10 mM CaCIz, WS: (Menczel et al. 1981) 1000 ml contain 18.4 g CaCl=x 2 H20, 9.0 g NaCI, 1.0 g glucose, 0.8 g KCI, autoclaved.
2. Axenic plant cultures Plants ofArabidopsis thaliana ecotype Z0rich were collected at the Botanical Garden and confirmed by E. Landolt (ETH Zurich). Seeds were sterilized by vigorous shaking for 8 minutes in 10 ml of 7.2% (w/v) of Ca(OCI)2 plus Tween 80, collected on a sterile cotton filter (placed into a funnel) and carefully washed with sterile water. For germination 12-16 seeds were transferred with a painting brush onto 1/2 MS-O agar in plastic petri dishes and sealed with Parafilm. Following cold treatment at 4"C in the dark for 3 days, dishes were incubated for 7 days under 16 hours light (2800 lux),20-25"C. Night temperature was 16-20"C. Four seedlings each were carefully transferred with fine forceps onto 40 ml of 1/2 MS-O agar medium contained in 400 ml PLASTEM boxes (PE 474, Plastem AG, Schwarmburg, Switzerland). Good aeration was provided by a hole in the lid closed with a rubber sponge stopper (Cepharem stopper, Greiner, N0rttingen, FRG). Plants were raised in growth chambers with 16 h light (cool fluorescence, 4000 lux), 22"C. Night temperature was t6"C.
3. Protoplast isolation Protoplasts were isolated from leaves of 4 week old plants at the vegetative rosette stage. Leaves were wetted with and cut into fine slices in MaCa solution. Following washing in the same solution tissue slices were preincubated in MaCa solution for 1 h at 24"C in the dark. Thereafter MaCa was replaced with enzyme solution (10ml/ g leaf tissue) containing either 1% (w/v) Cellulysin + 0.1% Macerase, or 1% Cellulase Onozuka R10 + 0.25% Macerozyme R10, both dissolved in 0.4 M mannitol + 6.8 mM CaCI=, pH 5.5, 490 mOs/kg H20. Following 16 h of incubation (overnight) the digestion mix was shaken very gently and incubated for further 30 min. Protoplasts were separated from undigested tissue in stainless steel sieves of 100 and 50 I~m mesh. Following the addition of one volume of W5 solution to 2 volumes of enzyme/protoplast suspension the suspension was distributed to sterile capped plastic centrifuge tubes (Greiner, NCirttingen, FRG, 12 ml) and sedimented for 10 rain. at 80 g in a table centrifuge (Hettich Universial 1200). The sediment was resuspended in W5, sedimented and resuspended in a mixture of 1 vol. W5 + 2 vol. of 0.5 M mannitol. Following a final sedimentation the protoplasts were resuspended in 0.5 M mannitol (545 mOs) at a population density of 4x105/ml.
4. Protoplast culture Protoplasts developed best when embedded into a gel containing charged groups, e.g. alginate or pectinate. Preparation of thin layers of protoplasts in alginate or pectinate requires that calcium-free
protoplast suspensions are allowed to gel over a calcium-centaining basal layer. Therefore, protoplasts suspended at double plating density (4x105/ml) in 0.5 M mannitol were mixed with the same volume of either a solution of 2% (wN) Na-pectinate in 0.4 M mannitol (475 mOs,filtersterilized, Pektin N, no. 2-8913, Carl Roth, Karlsruhe, FRG) or with 2% Na-alginate in 0.4 M mannitol (467 rues, autoclaved, Sigma A 2158, low viscosity). One ml aliquots each were pipetted onto a basis layer of 5 ml solidified Difco Bacto agar containing either 100 mM CaCI= + 0.4 M mannitol for pectinate layers, or 10 mM CaCl= + 0.4 M mannitol for alginate layers, respectively. Following gellification for 30 min. the gellified surface was flooded with 2.5 ml of MaCa solution. Petri dishes were sealed with Parafilm and'incubated for 2 days at 4"C in the dark (without addition of any nutrients). Following this cold-starvation period protoplast gels were transferred with a spatula into fresh 6 cm Petri dishes containing 5 ml MaCa solution which was after a few minutes replaced by MS-PPI culture medium. Optimal gel/medium ratios were 0.4 (e.g. 0.5 ml protoplasts gel + 1.25 ml culture medium, gel rinsed once with MS-PPI before). The protoplast gels were incubated for 7 days in the dark at.22"C, followed by dim continous light at the same temperature. At day 10 from protoplast isolation 1.25 ml of MS-PPII was added to the conditioned MS-PPI medium yielding a gel/medium ratio of 0.2 (e.g. 0.5 ml gel + 2.5 ml medium). Thereafter MS-PPII was replaced in weekly intervals until week 9. When the yellow-green protoplast-derived clones had reached Ihe size of 1-2 mm they were liberated from the gel by chelating the calcium ions complexed with the polysaccharide chains of the gelling agent with potassium citrate: the culture medium was replaced by 2.5 ml of 20 mM K-citrate in 0.2 M glucose, pH 5.8,300 mOs, autoclaved. After 5 min. of incubation the citrate solution was replaced by 5 ml of fresh citrate solution, the Petri dish sealed with Parafilm and incubated on a gyratory shaker at 40 rpm for 30 minutes. Liberated clones were finally washed with MS-PPII.
5. Plant regeneration The protoplast-derived clones were individually transferred onto MSPPII agar (300 mOs, 20 ml per 9 c,nl petri dish). Dishes were sealed with Parafilm and incubated at 20"C in continuous dim light (ca. 700 lux). Within 3 weeks of culture clones reached a size of ca. 5 mm in diameter. At this stage the development of shoot meristems was induced by transfer to MS-S culture medium (190 mOs) and incubation at 20"C, 2000 lux continuous light. After 10-16 days of incubation shoot meristems could be identified as small dark-green areas. At this stage elongation and differentiation of shoots were supported by reduction of exogenous hormones and good aeration: calli with shoot primordia were transferred to 1/2 MS-O agar medium (0.8% Difco Bacto, 80 mOs, 40 ml per Plastem box, lid closed with Cepharem stopper). Incubation was at 20"C, 16 h 3000 lux, 16"C at darkness. After 2 weeks shoot rosettes could be excised from the calli for subsequent rooting. Remaining differentiating tissue pieces could be placed onto MS-M agar for a further multiplication cycle of shoots. Remaining undifferentiated callus could be placed back onto
577 1/2 MS-O for regeneration of further shoots. For rooting, the excised shoots were transferred to MS-R medium (in 9 cm petridishes) and incubated at 20"0, continuous dim light for 3 days. Root growth was promoted by subsequent transfer to hormone-free MS-O agar contained in Plastem boxes (with a hole in the lid, dosed with a Sepharem stopper) and incubation at 20"C, 16 h 3000 lux, 16"C during darkness. Rooted shoots were transferred to a mixture of 2 vol. potground + 1 vol perlite contained in small pots standing in shallow water in Plastem boxes with 3 holes in the lid, dosed with Cepharem stoppers. Culture was in the first two weeks still under sterile conditions at 20/16"C, 16 h 3000 lux per day. Adjustment to growth in a less humid atmosphere was achieved by stepwise opening the stoppers in the lid. The developing floral shoots were allowed to grow out of the Plastem boxes through the open holes in the lid. Flowering and fruit ripening took place in the open air at 20/ 16"C, 16 h 3000 lux per day. If watering was necessary this was done through the open holes.
Shoot Induction: (10-16 days). MS-S, 0.8% agar, 3% sucrose, 188 mOs; 20"C, continuous light; 2000 lux green shoot primordia at contact zone.
Shoot differentiation and elongation: (2 weeks). 1/2 MS-O, 0.8% agar, 1.5% sucrose, 80 rues; 20"C, continuous light, 2800 lux. Good aeration enhances shoot development and minimizes vitrification.
Excision and trimming of shoots/ root induction: (3 days). MS-R, 0.8% agar, 1.5% sucrose, 80 rues. 20"C, continuous dim light; 700 lux, development of root primordia.
Root eleongation: ( 10 days). 6. Flow-chart: Item protoplast to plant Preplasmolysis: (1 hour). MaCa-solution, 450 mOs/kg H20, 25"C, darkness.
1/2 MS-O, 0.8% agar; Plastem boxes with perforation; 20/16"C, 16 h day, 3000 lux. development of shoot resettes and branched root systems.
Transfer to soil and flowering: (7 weeks). Enzyme treatment: (16 hours). 1% Onozuka R10 + 0.25% Macerozyme R10, in MaCa, 490 mOs; 25"C, darkness
Potground + perlite (2+1), autoclaved, small pots in Plastem boxes with perforation; 7 days under sterile conditions; 20/16"C, 16 h day with 2800 lux.
Protoplast gels: (1 hour). 1% Na-pectinate in 0.4 M mannitol, or 1% Na-alginate in 0.4 M mannitol.
Cold-starvation: (36 hours). Protoplast gel over MaCa basis layer, under liquid MaCa; 4"C, darkness,
Protoplast culture: (7 days). MS-PPI, 530 mOs, 0.4 M glucose; 20"C, dark; first cell divisions at day 9.
Development of micro-colonies: (7 days). addition of same volume of MS-PPII, 400 rues, 0.3 M glucose; 20"C, continuous dim light, 700 lux. Development to green macrocolonles: (6 weeks). weekly replacement with MS-PPII; 300 rues, 0.2 M glucose; 20"C, continuous dim light, 700 lux.
Liberation from gel matrix: 20 mM K-citrate in 0.2 M glucose, 300 mOs.
Development of morphogenic callus: (3 weeks). MS-PPII, 0.8% agar, 0.2 M glucose, 300 mOs; 20"C, continuous dim light, 700 lux.
RESULTS & DISCUSSION The above (partially optimised) protocol has been developed on the basis of the published experience with protoplast and cell culture systems of Arabidopsis and Brassica (e.g. Damm and Wilmitzer 1988; Feldmann and Marks 1986; Gleba et al. 1978; Gleba and Hofmann 1978, 1980; Glimelius 1984; Gresshoff 1976; Huang and Yeoman 1984; Kao and Seguin-Swartz 1987; Negrutiu et a1.1975; Negrutiu and Jacobs 1978; Xuan and Menczel 1979, 1980) and describes a laboratory procedure which has been used in our group for two years with leaf protoplasts from Arabidopsis thaliana ecotype Z(Jrich. As with most other protoplast systems, the quantitative data vary between experiments, days, seasons and plants (Potrykus and Shillito 1986). Protoplast division frequency (ratio of cells entering first divisions) varied I>~tween0.1 and ca. 80% with an average of ca. 10%. The plating efficiency (ratio of cells yielding proliferating calli varied between ca. 0.01 and 5% with an average in the range of ca. 0.5%. Experiments from which no proliferating clones could be recovered were rare exceptions in a series of more than 200 experiments. When comparing plating efficiency, developmentof morphogenic calli, and shoot/plant regeneration from alginate- and pect~nate cultures the results clearly speak in favour of pectinate. As, however, alginate gave more consistent results over a longer period, we use this gelling agent in our routine procedures. Whereas recovery of protoplast-derived clones is more or less
578 predictable and achieved routinely by different experimentators, regeneration of fertile plants from protoplast-derived clones depended far stronger on the person involved. In the hands of one of us (H.K.) shoot and plant regeneration from protoplast-derived clones was reproducible in the range of ca. 40% and 60%, respectively, and could also be used for the regeneration of fertile transgenic plants after direct gene transfer to protoplasts (Karesch et al., this issue). The yield of fertile plants dropped significantly in experiments performed by other persons. Plant regeneration from protoplasts is, therefore, possible, even with relatively high efficiency, but the amount of necessary work and attention to obtain a high number of regenerants seems to depend on the general tissue culture experience of the experimentator and his/her readiness to further optimize the procedure. On the other hand, the procedure seems to be sufficient for studies of gene expression in transient systems (M. Schrott, unpublished results), as well as for large scale transformation experiments for gene targeting (R. B., unpublished results), where growth and selection of protoplast-derived calli can already answer the question of an experiment. As, however, plant regeneration from plant explants (leaf disk, root, petiole, stem, floral stem) is far less problematic, there should be a good reason if one decides to go the hard way via protoplasts. Acknowledgements We would like to thank Ortrun Mittelsten Scheid for very helpful discussions.
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