CHAPTER 13

Protoplast Electrofusion and Regeneration in Potato

Jianping Cheng and James A. Saunders

1. Introduction Protoplast fusion and subsequent in vitro plant regeneration, leading

to somatic hybridization, offer opportunities for transferring entire genomes from one plant into another, regardless of the interspecific crossing barriers. In contrast to techniques for plant transformation that are aimed at single-gene transfer, protoplast fusion is needed when poly­genic traits are concerned, as is frequently encountered in the genetics of higher plants. Several Solanaceous species, including potato, have been used with greater success than other higher plant species in somatic hybridization because they are more responsive to the protoplast regen­eration process. There are two commonly used procedures to induce cell fusion, namely polyethylene glycol (PEG)-induced protoplast fusion and protoplast electrofusion. These procedures have been the subject of sev­eral reviews indicating that electrofusion is generally more efficient (1—5). Electrofusion is superior to PEG-induced protoplast fusion in the following aspects:

1. Simplicity of the fusion process; 2. Less toxicity and less physical damage to the protoplasts; 3. Large fusion volume allowing more protoplasts to be treated; and 4. Fine control of the fusion process with the availability of commercial

electrofusion equipment.

From Methods in Molecular Biology, Vol 55 Plant Cell Electroporation and Electrofusion Protocols Edited by J A Nickoloff Humana Press Inc , Totowa, NJ

181

182 Cheng and Saunders

The following procedures for protoplast preparation, electrofiision, and regeneration have been used successfully in several potato species, including Solarium phureja, S. chacoense, and dihaploid and tetraploid S. tuberosum, and they are likely also to be suitable for many other potato species with minor modifications.

2. Materials 2.1, Plants for Protoplast Isolation

Use in vitro grown plants initiated from nodal cuttings of greenhouse-or field-grown plants or from seeds for protoplast preparation. Nodal cuttings with 1 or 2 axillary buds are surface-sterilized by immersion in 20% (v/v) commercial bleach for 10 min (seeds are disinfected in 50% [v/v] commercial bleach for 10 min), then rinsed in sterilized distilled water three times. Bleached ends of the cuttings are excised prior to inserting the lower end of the tissue into the propagation medium (A, Table 1) contained in 25 x 150-mm glass culture tubes or other dispos­able plastic culture vessels. Place disinfected seeds on the surface of the medium. The cultures are grown at 25 °C under cool white fluorescent light (approx 30 jiE/m- /̂s) with 12 h/d photoperiod. Subculture the plants at monthly intervals.

2.2. Solutions and Media 1. Rinse solution: 0.3MKC1, pH 5.7. 2. Flotation solution: 20% sucrose, pH 5.7. 3. Electrofiision solution: O.SMmanitol. Sterilize all solutions by autoclav-

ing (see Note 1). 4. Media: See Table 1.

3. Methods 3.1. Protoplast Isolation

1. Isolate protoplasts from young plants (3-wk-old cultures). Excise the upper halves (stems together with leaves) of the plants and immerse in 25 mL of pretreatment solution (G, Table 1) in a sterile 100 x 15-mm Petri dish. Perform this soaking pretreatment at 25°C in the dark for 48 h.

2. Following the preincubation, mince the plant material in a sterile 100 x 15-mm Petri dish containing 1 mL of filter-sterilized cell-wall-digesting solu­tion (B, Table 1) (approx 1 g of plant material/dish) with a pair of surgical scalpels. Add filter-sterilized digesting solution (12 mL) to the minced plant material, seal the dish with parafilm, and wrap in aluminum foil.

Electrofusion in Potato 183

Digestion proceeds in the dark at 25 °C with gentle shaking (60 rpm) overnight.

3. After completion of digestion, add an equal volume of the rinse solution to the protoplast suspension to lower the density of the digesting solution. Sieve the diluted digested material through a screen (approx 50-80 ^m pore diameter) into a 15-mL sterile centrifuge tube and centrifuge at 50g for 5 mm.

4. Carefully remove the supernatant, and resuspend the pellet in 10 mL of the flotation solution {see Note 2). Carefully layer approx 1 mL of rinse solu­tion on the top of the flotation solution. Centrifuge the tube at 50g for 10 min. Intact protoplasts will float to the interface between the flotation solu­tion and the rinse solution.

5. Collect the protoplasts with a sterilized glass pipet from the sucrose inter­face, and transfer to a new 15-mL centrifuge tube. Dilute the protoplast suspension with 12 mL of rinse solution, and pellet the protoplasts by cen-trifiigation at 50g for 5 min. It is important not to remove too much (no more than 2 mL) of the suspension liquid while collecting protoplasts from the interface, because too much sucrose solution in the rinse will prevent the protoplasts from pelleting.

6. Remove the supernatant and resuspend the protoplast pellet m 1 mL of 0.571/ manmtol for electrofusion. Determine the protoplast density by counting cells under a microscope using a hemocytometer and adjust the protoplast density to 1 x 10̂ protoplasts/mL for electrofusion {see Note 3).

3.2. Electrofusion 1. Perform electrofusion usmg a square-wave electrofusion apparatus. Cell

and protoplast electrofusion can be accomplished using some exponen­tial discharge machines. However, most of these types of machines are designed primarily for electroporation and cannot the function to align cells before the fusion pulse. The use of the square-wave pulse generator also allows successful fusion and electroporation over a much broader range of conditions than the exponential pulse generators (6). A square-wave electroporator that has worked well in our hands is the Electro Cell Manipulator, Model 200 by BTX, Inc. (San Diego, CA). Set align­ment voltage meter at 40—80 V/cm field strength and with an alignment duration of 20 s. Set the field strength of the pulse at 1.250 kV/cm with a pulse duration of 60 ins and 1 or 2 consecutive pulses. The second pulse may increase the fusion frequency, but it also decreases the percentage of viable cells. It may be advisable to try one pulse initially to determine if the protoplast population in use can withstand the second pulse.

184 Cheng and Saunders

Table 1 Chemical Composition in 1 L Solution

for Potato Protoplast Preparation, Culture, and Regeneration Media"

Component B D

NH4NO3 KNO3 KH2PO4 MgS04 • 7H2O

1650 — — — — 1650 — 1900 950 950 950 950 1900 950 170 85 85 85 85 270 85 370 185 185 185 185 370 185

CaCl2-2H20 440 660 660 660 660 440 440 Na2EDTA 37.3 18.65 18 65 18.65 18.65 37 3 — FeS04-7H20 27 8 13.9 13.9 13 9 13 9 27 8 — MnS04-4H20 22 3 11.15 11.15 11.15 1115 22.3 — ZnS04 7H2O 10.6 5.3 5 3 5 3 5 3 10 6 — H3BO3 6.2 3 1 3 1 3 1 3 1 6 2 — KI 0.83 0 415 0 415 0.415 0 415 0 83 — Na2Mo04 2H2O 0 25 0.125 0 125 0 125 0 125 0 25 — C0CI2 6H2O 0 025 0.013 0.013 0 013 0 013 0 025 — CuS04-5H2O 0 025 0.013 0 013 0 013 0.013 0.025 — Glycine 2 0 2 0 2.0 2.0 2 0 2 0 2 0 Nicotinic acid 0 5 0.5 0 5 0 5 0.5 0.5 0 5 Pyridoxine 0 5 0.5 0 5 0 5 0 5 0.5 0 5 Thiamine 1 0 10.0 10 0 10.0 10 0 10 10 Inositol 100 0 100 0 100 0 100.0 100 0 100 0 100 0 Casein hydrolysate 250 0 — 250 0 250 0 250.0 250 0 —

Glutamine — — 100 0 100 0 100 0 100 0 — Adenine sulfate — — — — — 100 0 — Sucrose 20 g — 10 g 10 g 10 g 10 g — Glucose — 18 g 30 g 30 g 30 g — — Mannitol — 73 g — — — — — Sorbitol — — 50 g 50 g 50 g 30 g — PVP-10* — 5g — — — — — MES — I g — — — — — lAA — — — — — 0 01 — NAA — — 1.25 1.25 1.25 — 1.25 2,4-D — — 0 25 0 25 0 25 — — Zeatin — — 10 10 10 30 — BAP — — — — — — 0 5 GA3 — — — — — 05 — Agar 7g — — — — — — Phytagel — — — — 2g 2g — LGT agarose — — — 8g — — — pH 5.7 5.7 5 7 5 7 5 7 5 7 57

Continued on facing page

Electrofusion in Potato 185

2. Gently mix protoplasts in a 1:1 ratio from each fusion partner in a 15-mL centrifuge tube. Transfer 400 ^L of protoplast mixture into a 2-mm gap electroporation cuvet (e.g., BTX catalog # 620) and cover with a cuvet lid. Insert the cuvet into the safety chamber at the position for electric dis­charge, and press the discharge button to start electrofiision (see Note 4).

3. After electrofusion, the protoplasts, still contained in the electroporation cuvet, are protected from any physical disturbance in a laminar hood for 30 min to allow protoplast membranes to recover from the electrically induced damage.

4. After the recovery period, gently transfer the protoplasts with a sterile glass pipet into a 15-mL centrifuge tube and centrifuge at 50g for 5 mm.

5. Resuspend the protoplast pellet m 0.5 mL of liquid culture medium (C, Table 1).

3.3. Protoplast Culture and Regeneration 1. Culture the electrofusion-treated protoplasts in a semisolid medium by

very gently mixing 0.5 mL of protoplasts suspended in the liquid culture medium after electrofusion with 0.5 mL of 0.8% (w/v) low-gelling-tem­perature agarose-embedding medium (D, Table 1) in a 30 x 10-mm Petri dish. The agarose-embedding medium is maintained at 45 °C in a water bath before being used to prevent gelling (see Notes 5 and 6).

2. Incubate the Petri dishes containing protoplasts in a growth chamber at 25°C m the dark. When protoplast-calli (p-calli) reach approx 1 mm m diameter, they are transferred together with the agarose block onto the top of p-callus growth medium (E, Table 1) in a 100 x 20-mm Petri dish and kept in a growth chamber at 25 °C in the dark.

3. P-calli remain on p-callus growth medium without subculture until they are approx 3 mm in diameter. At this size, transfer p-calli onto the shoot-regeneration medium (F, Table 1) in a 100 x 20-mm Petri dish, and grow the callus under an average light intensity of 20 |a.E/m /̂s in a 16 h/d photo-period. Subculture monthly until shoots regenerate.

"All media are filter-stenlized, except media A and G are autoclaved To make a filter-steril-ized medium contaimng a gelling agent (agarose or phytagel), the gelling agent is dissolved separately in distilled water m double concentration, pH adjusted to 5.7, and autoclaved, the other components of the medium are dissolved in another container in distilled water in double concentration, pH adjusted to 5 7, and filter-sterilized, mix the two separately prepared parts 1 to 1 under aseptic conditions when the autoclaved gelling solution cools to 70°C All units are in milligrams, unless specified

^Abbreviations PVP-10. Polyvinylpyrrolidone, mol wt 10,000; MES 2-(Af-Morpholino) ethanesulfonic acid, NAA Naphthaleneacetic acid; lAA Indole-3-acetic acid, 2,4-D 2,4-Dichlorophenoxyacetic acid, BAP' 6-Benzylaminopurine, GA Gibberellic acid, LGT agarose low-gelling-temperature agarose

186 Cheng and Saunders

4. Cut off regenerated shoots from p-calli when they reach approx 20 mm m length, and plant them into the propagating medium (A, Table 1) in culture tubes. Maintain culture conditions as described earlier for nodal cuttings. These shoots regenerate roots readily in the propagating medium. Supplement the propagating medium with 0.05 mg/L naph-thaleneacetic acid (NAA) and 0.05 mg/L gibberellic acid (GA3) if dif­ficulties in rooting arise.

5. Maintain regenerated plants m vitro under controlled conditions prior to transfer to a greenhouse. To transfer regenerated plants from in vitro cul­ture to a greenhouse, the plants are carefully lifted from agar medium and planted into a garden soil mix in small Jiffy paper garden pots. It is easier to lift the plants from agar medium with their roots intact when the roots are <3 cm in length. Keep the potted plants in a growth chamber for 2 wk under a plastic cover to prevent excessive evaporation. Remove the plastic cover after 2 wk and maintain the plants in the growth chamber without a plastic cover for an additional 2 wk. Transplant the plants and soil from the paper pots into regular garden pots, and transfer to a greenhouse. Shading may be provided during the first 2 wk in a greenhouse.

4. Notes 1. The maintenance of aseptic conditions is vital throughout the entire proce­

dure. To this end, we routinely perform all the procedures in a laminar flow hood, except when the samples are inside sealed vessels. All nonsterile glassware and solutions (unless filter-sterilization is specified) must be autoclaved at 121 °C and 20 psi for 20 min. Scalpels must be disin­fected by heat with a Bunsen burner or an electric incinerator. Researchers should wear surgical gloves during all operations.

2. Protoplasts are very fragile. Be very gentle when pipeting protoplasts and resuspending a protoplast pellet. Be sure to use only wide-bore pipets and allow the protoplast suspension to drain gently from the pipet with force.

3. Perform electrofusion immediately after protoplast preparation, since pro­toplasts start regenerating cell walls as soon as they are out of the digesting solution.

4. Use disposable, sterile electroporation cuvets for electrofusion. 5. An efficient system for selecting somatic hybrids is critical for the suc­

cessful application of protoplast fiision. Early selection at the cellular level or callus level is more efficient than late selection at the plant level. Select­able markers, such as antibiotic or herbicide resistance, can be introduced into fusion partners using gene-transfer techniques. Somatic hybrid selec­tion based on this system has been demonstrated successfully in potato (7). Also, potato mutants resistant to ammo acid analogs have been used as the fusion partners to facilitate somatic hybrid selection (8). To avoid the time-

Electrofusion in Potato 187

consuming task of producing genetically selectable fusion partners, cer­tain endogenous traits can be used conveniently for somatic hybrid selec­tion or screening. For example, hybrid growth vigor in p-calli may be used in vitro for somatic hybrid selection, and some morphological characteris­tics can be used for hybrid screening. In our laboratory, hybrid growth vigor in p-calli was used to select somatic hybrids between a Colorado-potato-beetle-resistant clone of S. chacoense {In = 2x = 24) and a S tuberosum dihaploid (2« = 2x = 24). As a tetraploid hybrid, the p-calli grew faster than the p-calli from either fusion partner. Selection was fur­ther defined during shoot regeneration, since p-calli from S. chacoense did not regenerate shoots under the defined culture conditions, whereas p-calli from S. tuberosum regenerted readily. The production of visible anthocyanin pigments m the shoots and leaves of the S chacoense clone were used as an additional marker for hybrid identification. Those that regenerated shoots with anthocyanins were identified as somatic hybrids, and those that were completely green were regenerates of S tuberosum. This system was very effective, since the putative hybrids were confirmed by peroxidase isozyme electrophoresis, morphological differences, alka­loid profiles, and insect bioassays. Similar characterizations could be used in other potato species and genotypes, and should be considered when choosing fusion partners.

When no selectable or screenable marker is available, a manual system may be considered. In such a system, protoplasts prepared from herbicide-bleached leaves (or suspension cultures and dark-grown calli, as suggested by the present authors) without chlorophyll are stained with fluorescein diacetate (FDA) and then fiised with normal chlorophyll-containing proto­plasts. After fusion treatments, those protoplasts showing both red and green fluorescence are identified as heterokaryons and hand-picked under a fluorescent microscope equipped with a micromanipulator (9). This sys­tem requires low protoplast density culture techniques, which may not be feasible with other potato species.

6. Selected somatic hybrids need to be analyzed to confirm their hybrid nature. Isozyme markers (10,11) or DNA markers (12) may be used for this purpose. In addition, distinct morphological differences between the two fiision partners are also informative for hybrid identification. Cyto-logical studies may be conducted to investigate the ploidy level and chro­mosomal numbers of confirmed somatic hybrids.

References 1. Negrutiu, I., de Brouwer, D., Watts, J. W., Sidorov, V. I., Dirks, R., and Jacobs, M.

(1986) Fusion of plant protoplast: a study using auxotrophic mutants of Nicotiana plumbagmifoba, Viviani. Theor Appl Genet 72,279-286.

188 Cheng and Saunders

2. Bates, G. W., Saunders, J. A., and Sowers, A. E, (1987)Electrofusion". pnnciples and applications, in Cell Fusion (Sowers, A E , ed.). Plenum, New York, pp. 367-395

3 Saunders, J. A. and Bates, G. W. (1987) Chemically induced fusion of plant proto­plasts, in Cell Fusion (Sowers, A E , ed ), Plenum, New York, pp. 497-520

4. Fish, N , Karp, A , and Jones, M G K (1988) Production of somatic hybrids by electrofusion in Solanum Theor Appl Genet. 76, 260—266

5 San, H. L., Vedel, F , Sihachakr, D., and Remy, R (1990) Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by proto­plast, electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill zaA Lycopersicon peruvianum MiW Mol. Gen Genet 221, 17—26.

6 Saunders, J. A , Smith, C. R., and Kaper, J. M. (1989) Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts. Biotech-niguesl(10), 1124-1131

7 Masson, J , Lancelin, D , Bellini, C , Lecerf, M., Guerche, P., and Pelletier, G. (1989) Selection of somatic hybrids between diploid clones of potato (Solanum fMZ?ero5Mm L) transformed by direct gene transfer. Theor Appl. Genet 78,153—159.

8 de Vries, S. E , Jacobsen, E., Jones, M. G K , Loonen, A E H M., Tempelaar, M J , and Wijbrandi, J (1987) Somatic hybridization of amino acid analogue-resis­tant cell lines of potato {Solanum tuberosum L.) by electrofusion. Theor Appl Genet 73,451^58

9. Puite, K. J., Roest, S., and Pijnacker, L. P. (1986) Somatic hybrid potato plants after electrofusion of diploid Solanum tuberosum and Solanum phurej a Plant Cell Rep 5,262-265

10. Desborough, S. L. (1983) Potato {Solanum tuberosum L) , in Isozymes in Plant Genetics and Breeding, Part B (Tanksley, S. D. and Orton, T. J , eds ), Elsevier, Amsterdam, pp. 167—188.

11. Shields, C. R., Orton, T J., and Stuber, C W. (1983) An outline of general resource needs and procedures for the electrophoretic separation of active enzymes from plant tissue, m Isozymes m Plant Genetics and Breeding, Part A (Tanksley, S. D. and Orton, T J , eds ), Elsevier, Amsterdam, pp 443—468

12 Landry, B. S. and Michelmore, R. W (1987) Methods and applications of restnc-tion fragment length polymorphism analysis to plants, m Tailoring Genes for Crop Improvement An Agricultural Perspective (Bruening, G., Harada, J , and Hollaender, A., eds.). Plenum, New York, pp. 25—44