Plant Cell Reports

Plant Cell Reports (t988) 7:379-384

© Springer-Verlag 1988

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts H. M. Zhang, H. Yang, E. L. Rech, T. J. Golds, A. S. Davis, B. J. Mulligan, E. C. Cocking, and M. R. Davey Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham, NG7 2RD, UK Received August 10, 1988 - Communicated by I. Potrykus

ABSTRACT Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering. ABBREVIATIONS NPTII, neomycin phosphotransferase; dodecyl sulphate.

SDS, sodium

rice tissues following DNA uptake into rice (Taipei 309) protoplasts (Yang et al. 1988b). We have now combined these optimum conditions for transformation using electroporation with those for plant regeneration to produce transgenic rice plants resistant to kanamycin. MATERIALS AND METHODS Protoplast

isolation

Protoplasts were isolated using a routine procedure (Abdullah et al. 1986) from an established (more than 10 months old) cell suspension of Oryza sativa L. v Taipei 309. The suspension was initiated from leaf base callus and designated line LB3. Some preparations of freshly isolated protoplasts were heat shocked at 45°C for 5 min, followed by 10 sec on ice (Thompson et al. 1986b) prior to plasmid uptake by electroporation.

INTRODUCTION

Plasmid constructs

A major constraint in the production of transgenic plants in most of the cereals is the inability to induce plant regeneration from protoplasts treated with DNA. To date, plant regeneration from transformed protoplast-derived tissues has been achieved only in Zea mays (Rhodes et al. 1988).

E. coli HBIO] was transformed hy the calcium chloride procedure (Mandel and Higa 1970) with pCaMVNEO (Fromm et al. 1986) carrying a chimaeric gene consisting of the CaMV 35S promoter, the neomycin phosphotransferase (NpTII) gene from Tn5 and the nos polyadenylation region, pHP23 was provided in the same E. coli strain. This plasmid was constructed by fusing the 35S promoter of CaMV to the 5' end of the EcoRV fragment of pABDI. The EcoRV fragment carried the NPTII gene and the 19S promoter from gene VI of CaMV (Raszkowski et al. 1986). After construct verification (Holmes and Quigley 1981), large scale plasmid isolation from E. coli was performed using the alkaline lysis method (Birnboim and Doly 1979). Plasmid was sterilised by ethanol precipitation and dissolved in sterile TE buffer (Maniatis et al. 1982) at 1.0 mg/ml.

The experimental approach used most extensively involves DNA uptake into protoplasts by treatment with polyethylene glycol or electroporation. This technique has been used to study transient gene expression (Fromm et al. 1985; Ou-Lee et al. 1986; Werr and L~rz 1986; Hauptmann et al. 1987; Junker et al., 1987) and has also resulted in the production of stably transformed tissues of gramineous species, including Lolium multiflorum (Potrykus et al. 1985b}, Oryza sativa (Uchimiya et al. 1986; Yang et al. 1988a,b), Panicum ma×imum (Hauptmann et al. 1988), Saccharum spp. (Chen et al. 1987), Triticum monococcum (LSrz et al. 1985; Hauptmann et al. 1988), and Zea mays (Fromm et al. 1986). Our studies have established a reproducible procedure for the regeneration of fertile plants from rice protoplasts (Oryza sativa L.v Taipei 309) with a regeneration efficiency adequate for assessments of transgenic plant production (Abdullah et al. 1986; 1988). Recently, we have reported that electroporation is the most efficient procedure for the production of kanamycin resistant

Offpr~t requests to." M. R. Davey

Electroporation

of protoplasts

Protoplasts were resuspended at 2.5 x I06/mi in electroporation medium which was modified from a published formulation (Fromm et al. 1986) and contained 0.8 g/l NaCl, 0.02 g/l KCI, 0.02 g/l KH2PO 4 and 100 g/l glucose, pH 7.1. One ml volumes of protoplast suspension were mixed with 20 ~g of pCaMVNEO in 20 ~i of TE buffer. Four hundred ~i samples of protoplast/plasmid mixture were transferred to the chamber of an electroporator (DIA-LOG, G.m.b.H., 4 D~sseldorf 13, West Germany). The plexiglass chamber, which had two parallel

380 stainless steel electrodes 1.0 cm apart, was sterilized by immersion in 80% (v/v) ethanol (30 min) prior to use. Three successive exponential pulses of an initial voltage of 2000V were applied to the samples, at 10 sec. intervals, by discharging a 40 nF capacitor across the electrodes of the chamber. The time decay constant {defined as the time taken for the pulse to decay to 63% of the initial voltage) was t~RC, where R was the resistance of the sample in the chamber and C the capacitance. The resistance of the sample (measured using a BI83-LCR meter; Thorn, EMI Instruments Ltd., Dover, U.K.) was 1,7002, resulting in a time constant of 68 ~sec. After electroporation, protoplasts were incubated on ice for 10 min, followed by 20 min at 22°C. Protoplasts were also electroporated in the absence of plasmid. Protoplast culture and selection of kanamycin resistant colonies Protoplasts were cultured in 1.5 ml volumes of KPR medium (Thompson et al. 1986a) made semi-solid with 1.2% w/v agarose (Sea Plaque, ~MC Corporation, Rockland, ME04841, USA) at 6.0 x 10J/ml in 3.0 cm diameter Petri dishes. The latter were sealed with Nescofilm (Nippon Shoji Kaisha Ltd., Osaka, Japan). Cultures were incubated in the dark at 27°C. After 7 days, the agarose medium in each dish was cut into 4 sectors, the latter transferred to 5.0 cm diameter Petri dishes, and the sectors bathed in 3.0 ml of KPR liquid medium. At day 14 of culture, the number of dividing protoplasts was determined and expressed as a percentage of the number of protoplasts originally plated. At this time, half of the cultures were maintained in KPR medium to ensure that the plasmid-treated protoplasts were capable of cell colony formation. In the remaining cultures, the liquid medium in each dish was replaced by 3.0 ml of KPR medium containing 100 bg/ml of kanamycin. The kanamycin supplemented medium was itself replaced every 7 days over a period of 28 days with medium containing the same concentration of antibiotic. The number of colonies which had reached a diameter of 1.0 mm or more in antibiotic-free KPR medium and in the kanamycin selection plates was recorded 42 days after protoplast plating. Samples of protoplasts which had been heat shocked and electroporated in the absence of plasmid were also cultured to colonies without kanamycin selection to ensure a supply of non-transformed callus for biochemical analysis. Plant regeneration

from kanamycin resistant

tissues

Individual cell colonies from the kanamycin selection plates were placed, using fine forceps, onto the surface of hormone-free N6 regeneration medium (Chu et al. 1975) containing 8% w/v sucrose and 0.4% w/v agarose (Sigma, type I) in 100 mm square 5 x 5 compartment dishes (Sterilin Ltd., Feltham, Middlesex, UK). Four colonies were cultured on 2.0 ml aliquots of medium in each compartment. Four hundred colonies were transferred to N6 medium with 50 ~g/ml of kanamycin, and the same number to N6 medium lacking antibiotic. Cultures were maintained at 27°C in the dark for 2 to 3 weeks before transfer to the light {continuous daylight fluorescent illumination; 2500 lux). Plantlets were transferred to agar-solidified (0.8% w/v; Sigma) hormone-free Murashige and Skoog (1962) based medium and maintained under the same conditions to stimulate shoot and root development.

Fifty randomly selected protoplast-derived colonies which failed to regenerate on N6 medium were transferred to AA medium (Abdullah et al. 1986) (27°C; dark) and this material used for biochemical analysis. Regenerated plants were potted, when 10-15 cm tall, in a mixture of equal volumes of John Innes No. 3 potting compost and Levington M3 compost (Fisons Horticultural Division, Ipswich, UK). Plants were sprayed with distilled water after potting and covered with a plastic bag. They were maintained for 6 weeks in a growth room (day and night temperatures of 28°C and 25°C respectively; 10 h day/14 h night cycle), during which time they were hardened off by gradual removal of the plastic bags. Subsequently, plants were transferred to the glasshouse and grown under natural daylight with day and night temperature maxima of 30°C and 18°C respectively. A similar plant regeneration procedure was used for tissues derived from heat shocked protoplasts which had been electroporated in the absence of plasmid. Some colonies from such protoplasts were also grown on AA medium in the absence of kanamycin to provide non-transformed callus for analysis. DNA isolation and hybridisation Total genomic DNA was isolated from rice callus and leaves of regenerated plants using a published technique (Dellaporta et al. 1983). Ten ~g of DNA of each sample were digested with BamHI as described by the manufacturer (Northumbria Biochemicals Ltd., Cramlington, UK) and the fragments electrophoresed (25V; 16h) in a 0.8% w/v agarose gel. One pg of pCaMVNEO DNA restricted with BamHI was also run on the gel. DNA fragments were blotted to nylon membrane (Hybond-N; Amersham International, Amersham, UK) and hybridised to the 1.0 Kb BamHI fragment of pCaMVNEO containing the structural sequence of the NPTII gene. The 1.0 Kb BamHI fragment was labelled using a Multiprime DNA Labelling System (RPN.1601Y; Amersham International). After hybridisation, the membrane was washed twice with a solution of 2 x S S P E (Maniatis et al. 1982) and 0.1% w/v SDS (10 min each wash) at 22°C, followed by 2 washes (30 min each) at 65°C. The membrane was given 2 additional washes (30 min each) in 0.3 x SSPE with 0.1% SDS at 65°C, dried, and exposed to X-ray film (Fuji RX) using an intensifying screen. Neomycin phosphotransferase

(NPTII) assay

Two hundred mg of leaf material or 0.5g of callus were ground with 50 ~i of extraction buffer containing 62.5 mM Tris-HC1 (pH 6.8), 40 mM dithiothreitol, 1.0 mM 1,10-phenanthroline and 5.5 mM phenylmethyl-sulphonyl fluoride. Aprotinin (Sigma) was added to a final concentration of 100 bg/ml. Extracts were centrifuged (12000 rpm; 5 min). Protein concentrations were measured (Bradford 1976). Fifty bg of protein of each sample were electrophoresed in a 10% w/v polyacrylamide gel for 18 h (80V) at 4°C. Subsequent stages of the procedure were as reported (Schreier et al. 1985). RESULTS Protoplasts electroporated with pCaMVNEO entered division within 4 days of plating. Heat shock treatment of freshly isolated protoplasts prior to electroporation increased the percentage of protoplasts dividing by day 14. This stimulatory effect of heat shock on protoplast division was

381 Table 1.

Colony production and transformation frequencies following electroporation of rice protoplasts with

pCaMVNEO.

Treatment

Experiment

% of protoplasts dividing at day 14

Number o~ colonies from 1.0 x 10 protoplasts 42 days after electroporation

Kanamycinfree medium

-HS;

+HS;

EP+pCaMVNEO

EP+pCaMVNEO

-HS;

EP-pCaMVNEO

+HS;

EP-CaMVNEO

1 2 3

O.2 0.8 0.7

56 151 129

4 5 6

4.0 5.0 7.0

1023 1152 1249

7 8

0.9 1.2

137 163

9 10

7.0 8.0

1197 1264

Mean relative transformation frequency (%)

Mean absolute transformation frequency (x 10 -~)

Medium with 100 ~g/ml kanamycin

9 28 17

16

2.0

212 265 369

25

28

0 0

0

0

0 0

0

0

EP, electroporati~n; HS, heat shock. Experiments I-6 each involved 8.0 x 106 protoplasts; experiments 7-10 each involved 7.5 x 10 protoplasts. Relative transformation frequency = number of colonies on medium with kanamycin expressed as a percentage of the number of colonies on kanamycin-free medium. Absolute transformation frequency = number of colonies on medium with kanamycin compared to the number of protoplasts plated. reflected in the increased number of kanamycin resistant colonies which developed following exposure of protoplast-derived cells to the antibiotic (Table I). The relative and absolute£ transformation frequencies were 25Z and 28 x 10 respectively for hea~ shocked protoplasts compared to 16Z and 2.0 x 10 -J respectively for protoplasts not subjected to this treatment prior to electroporation with plasmid. Protoplasts electreporated in the absence of plasmid failed to grow on kanamycin-containing medium (Table I; compare Figs. 1A and IB). These kanamycin resistant rice colonies continued to grow following transfer from the selection plates to either N6 regeneration medium containing 50 #g/ml of kanamycin or to N6 regeneration medium without this antibiotic (Fig. IC). On N6 regeneration medium containing kanamycin, only 2 plantlets were regenerated from 400 resistant colonies and these plantlets failed to survive in culture. In contrast, about 30Z of the kanamycin resistant protoplast-derived colonies maintained in the dark at 27°C on N6 medium without kanamycin produced one or more somatic embryos within 2 to 3 weeks. About 10% of the somatic embryos (Fig. ID) germinated and produced coleoptiles which became green (Fig. IE) following transfer of the cultures to light. Further shoot and root development followed transfer of regenerated plants to hormone-free Murashige and Skoog based medium (Fig. IF). Of 400 kanamycin resistant colonies transferred to N6 medium without kanamycin, 6 colonies produced 12 green plants and 2 colonies produced 2 albino plants. These albino plants failed to survive in culture after reaching about 10 cm in height. Green plants were transferred to

the glasshouse (Fig. IG), where their development was comparable to that of rice (Taipei 309) plants regenerated from non-transformed protoplast-derived tissues and to seed-derived plants. Six of the regenerated plants and 9 kanamycin resistant colonies chosen at random were analysed for the presence of the NPTII gene and its expression. Genomic DNA isolated from the kanamycin resistant colonies and the regenerated plants hybridised to the internal 1.0 Kb BamHI fragment of pCaMVNEO containing the NPTII gene (Fig. 2A), confirming the presence of the foreign DNA in the kanamycin resistant colonies and the regenerated rice plants. In contrast, DNA from rice callus and from a rice plant regenerated from heat shocked protoplasts, electroporated in the absence of plasmid, failed to hybridise to the labelled probe. All nine of the kanamycin resistant colonies expressed NPTII activity, but only 2 of the regenerated plants derived from individual kanamycin resistant colonies gave a positive enzyme assay. A typical assay is shown in Fig. 2B. Extracts from rice callus and from a rice plant regenerated from protoplasts, electroporated in the absence of plasmid, did not exhibit NPTII activity. In addition to using pCaMVNEO, we have also obtained transgenic rice plants at a relative frequency of 25Z by electroporation of rice protoplasts with pHP23. Two plants were regenerated carrying the NPTII gene, but these failed to express NPTII enzyme activity (data not shown).

382 FIGURE l .

Sbages in the regeneration of a tranagenic rice plant.

A. Kamamycin resistant colonies developing from protoplasts heat shocked prior to eleetroporation ~ith pCaHVNEO. Culture photographed 42 days after plating and after exposure of protoplasts to lO0 pg/ml of kanamycin for 28 days (Bar = 1.O em)° B. A control culture showing the absence of kanamycin resistant colonies. Protoplaste ~ere heat shocked but electroporated in the absence of plasmid. The selection conditions were the same as in Fig. 1A. (Bar = 1.O cm). C. Four nodular kanamycin resistant tissues, in one of the compartments of a 5 x 5 well dish, after 7 days on N6 regeneration medium lacking the antibiobic. (Bar = 1.O cm)° D. A germinating somatic embryo (se) on rice callus after 2 weeks on N6 medium. (Bar = 1.0 mm). E. Young rice plants with coleoptiles (c) and roots (r) which have developed by somatic embryogeneais. The plants are still attached to the protoplast-derived callus, lheir coleoptiles have turned green following transfer of the cultures to the light. (Bar = 2.0 mm). F. A green regenerated rice plant undergoing shoot and root development on hormone-free Murashige and Skoog medium. (Bar = l.O cm). G. A rice plant derived from one of the kanamycin resistant colonies shown in Fig. IA. (Bar = 1.O cm). FIGURE 2. Confirmation of transformation by DNA hybridisation and NPTII enzyme activity.

A Kb

A. Southern blot analysis of genomie DNA from protoplast-derived kanamycin resistant colonies and regenerated rice plants. DNA digested with BamHI and 32 probed with the P-labelled 1.O Kb BamHI fragment of pCaMVNEO containing the structural sequence of the NPTII gene.

1.90

1.33

0.98 0.83

2

1

5

6

7

8

9

Hybridisation of the probe to DNA from tissues and plants derived from protoplasts electroporated with pCaHVNEO confirmed the presence of the NPTII gene in this material. (DNA from 7 other tissues and 2 regenerated plants derived from protoplasta electroporated with pCaMVNEO also hybridised to the probe).

B

1

10

Lane 1. ~ DNA cut with EcoRV/Hind IIl. Lane 2. 1.0 pg of pCaMVNEO digested with BamHI. Lane 3. Control rice callus from protoplasts electroporated without plasmid. Lanes 4 and 5. Kanamycin resistant tissues from protoplasts electropcrated with pCaHVNEOo Lane 6. Rice plant from prstoplasts electroparated ~ithout plasmid. Lanes 7 to i0. Rice plants regenerated from kanamycin resistant colonies.

2

3

4

5

6

7

8

B. Detection of NPTII activity in plant extracts. Autoradiograph showing the presence of phosphorylated kanamycin resulting from NPTII activity in extracts from kanamycin resiatanb tissues (lanes 2 and 3), and t~o plants regenerated from kanamycin resistant tissues (lanes 5 and 6). NPTII activity was absent in callus (lane i) and a plant regenerated from rice protoplasts electroporated in the absence of pCaMVNEO (lane &). Two plants containing the NPTII gene failed to show NPTII activity (lanes 7 and 8). (NPTII activity was also confirmed in 7 other kanamyein resistant tissues).

383 DISCUSSION It has been proposed that the high natural levels of resistance to kanamycin exhibited by cultured cells of several monocotyledons may preclude the use of this antibiotic for selecting transformed protoplast-derived cells (Vasil 1987). Our results show that resistance to this antibiotic can, in fact, be used to sglect transformed rice cells, negating the necessity to screen other antibiotics such as hygromycin B (Blochlinger and Diggelmann 1984) and the associated plasmid constructs, as agents to select transformed rice cells. However, as the natural kanamycin resistance of non-transformed rice protoplasts increases with age (Yang et al. 1988b), the time of application and concentration of antibiotic employed for selection are critical. Our previous experiments (Yang et al. 1988b) demonstrated that growth of non-transformed rice protoplasts was inhibited by exposure of the cells to 100 ~g/ml of kanamycin at day 14 of culture. Therefore, this concentration of kanamycin was used to select transformed protoplast-derived colonies in the present experiments. The period for which the transformed colonies were exposed to the antibiotic was also important, since inclusion of kanamycin in the regeneration medium at 50 ~g/ml reduced the number of plants produced. Indeed, those plants which did .develop in the presence of the antibiotic failed to survive in vitro. The absolute transformation frequency in rice protoplasts, not subjected to heat shock prior to electroporation compares favourably with that of protoplasts of other monocotyledons (Potrykus et al. 1985b; L~rz et al. 1985; Fromm et al. 1986; Hauptmann et al. 1988) and dicotyledons (Paszkowski et al. 1984; 1986; BalJzs et al. 1985; Hain et al. 1985; Meyer et al. 1985; Potrykus et al. 1985a;_~uerche_~t al. 1987) where frequencies of 1.0 x 10 - to 10 v have been reported with E. coli based vectors carrying chimaeric genes. Heat shock treatment of the freshly isolated protoplasts not only stimulated protoplast division as previously reported for rice (Thompson et al. 1986b), but also increased significantly the throughput of transformed colonies. The maximum relative transformation frequency of 25% obtained in the present experiments was significantly higher than those of 2% for tobacco (Shillito et al. 1985) and 5% reported for maize (Rhodes et al. 1988) following electroporation mediated uptake of plasmids carrying a similar chimaeric gene. Although exposure of electroporated maize protoplasts to nurse ceils maximised the throughput of transformed colonies (Rhodes et al. 1988), this approach was of no benefit in the present experiments with rice. In fact, the use of cells of Taipei 309 as a nurse culture reduced the number of transformed colonies selected (data not shown). The nine kanamycin resistant tissues chosen randomly for analysis and the six regenerated rice plants all contained an apparently unmodified form of the NPTII gene. Interestingly, previous DNA hybridisation analysis of rice (Yang et al. 1988b) and maize (Fromm et al. 1986) colonies resulting from the electroporation of protoplasts with the same plasmid as used in the present experiments (pCaMVNEO), demonstrated the presence of bands additional to the 1.0 Kb fragment which hybridised to the NPTII gene probe. Transgenic maize plants also contained re-arranged NPTII gene sequences (Rhodes et al. 1988). Once selected on kanamycin

containing medium, transformed rice colonies expressed NPTII activity even after growth on N6 or AA medium in the absence of the antibiotic, confirming the constitutive expression of the inserted foreign gene in plant cells. Similarly, transformed maize tissues grown for 10 months on kanamycin-free medium still expressed NPTII activity (Rhodes et al. 1988). Although it is not clear why 4 of the transgenic rice plants failed to express NPTII activity, it is conceivable that this may have been a gene dosage effect similar to that reported for the NPTII gene in tobacco (Potrykus et al. 1985a). Under glasshouse conditions, the transgenic rice plants produced in this study were similar morphologically to non-transformed plants derived from protoplasts of Taipei 309 using the same plant regeneration procedure and to seed-derived plants. The fact that electroporation is the preferred DNA delivery method for the production of transgenic rice plants from protoplasts is of interest, since this treatment is known to stimulate DNA synthesis in protoplasts (Rech et al. 1988) and also to promote plant regeneration from protoplast-derived tissues (Ochatt et al. 1988; Chand et al. 1988). The ability to produce transgenic rice plants from protoplasts now provides the opportunity to assess the feasibility of introducing non-selectable genes by co-transformation (Schocher et al. 1988), into this economically important cereal. ACKNOWLEDGEMENTS This work was supported, in part, by the Rockefeller Foundation and Overseas Development Administration. The authors acknowledge support for the workers involved in this study: HY (Royal Fellow administered by the Royal Society of London), HMZ (Fellowship from the Chinese Government), ELR (Empresa Brasileira de Pesquisa Agropeeuaria), TJG (SERC CASE Studentship) and ASD (University of Nottingham Studentship). Thanks are extended to Drs. M. Fromm and V. Walbot (Department of Biological Sciences, Stanford University, USA) for pCaMVNEO, Dr. J. Paszkowski (Swiss Federal Institute of Technology, Zurich) for pHP23, and Mr. B.V. Case for photographic assistance. REFERENCES Abdullah R, Cocking EC, Thompson JA (1986) Bio/Technology 4 : 1 0 8 7 - 1 0 9 0 Abdullah R, Thompson JA, Kush GS, Cocking EC (1988) Theor. Appl. Genet. (submitted) BalJzs E, Bouzoubaa S, Guilley H, Jonard G, Paszkowski J, Richards K (1985) Gene 4 0 : 3 4 3 - 3 4 8 Birnboim HC, Doly J (1979) Nucleic Acids Res. 7: 1513-1523 Blochlinger K, Diggelmann H (1984) Molec. and Cell Biol. 4:2929-2931 Bradford HM (1976) Anal. Biochem. 7 2 : 2 4 8 - 2 5 4 Chand PK, Ochatt SJ~ Rech EL, Power JB, Davey MR (1988) J. Exp. Bot. (in press) Chen WH, Gartland KMA, Davey MR, Sotak RR, Gartland JS, Mulligan BJ, Power JB, Cocking EC (1987) Plant Cell Reports 6:297-301 Chu CC, Wang CC, Sun CS (1975) Scientia Sinica 18: 659-668 Dellaporta SL, Wood J, Hicks JB (1983) Plant Mol. Biol. Reporter I: 19-21 From m M, Taylor LP, Walbot V (1985) Proc. Natl. Acad. Sci. USA 8 2 : 5 8 2 4 - 5 8 2 8 Fromm ME, Taylor LP, Walbot V (1986) Nature 319: 791-793

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