Plant Cell Reports

Plant Cell Reports (1985) 4:274-276

© Springer-Verlag 1985

Electrofusion, a simple and reproducible technique in somatic hybridization of Nicotiana plumbaginifolia mutants K. J. Puite, P. van Wikselaar, and H. Verhoeven Research Institute ITAL, Postbox 48, NL-6700 AA Wageningen, The Netherlands Received August 16, 1985 -Communicated by I. Potrykus

ABSTRACT Electrofusion of protoplasts from two complementary nitrate reductase deficient mutants of Nicotiana plumbaginifolia has resulted in somatic hybrid lines. Mesophyll protoplasts isolated from the cofactor mutant CNX 20 and fluorescein diacetate stained protoplasts derived from a cell suspension culture of the NA 36 line, being defective in the apoenzyme, were used in the fusion experiments. In total, 594 lines were recovered which could proliferate on a selective medium with nitrate as the sole nitrogen source. This is including 141 putative hybrid lines which were obtained after transfer of 1048 heterokaryons with a micromanipulator one day after electrofusion. The hybrid character of some of the selected lines was confirmed by nitrate reductase activity measurements. Plants were grown from hybrid calli. Abbreviations NR FDA 2,4-D BAP NAA NED PEG AC DC

- nitrate reductase fluorescein diacetate - 2,4-dichlorophenoxyacetic acid benzylaminopurine - naphthaleneacetic acid N-l-naphtyl-ethylenediamide hydrochloride - polyethylene glycol - alternating current - direct current

-

-

INTRODUCTION Protoplast fusion in an electric field has been referred to by Senda et al. 1979 and Zimmermann and Scheurich 1981. Most of the information until now is restricted to the instrumentation part (Bates et al. 1983; Watts and King 1984; Zachrisson and Bornman 1984) and results on the biology part are scarce (Tempelaar and Jones 1985). Recently, somatic hybridization of two auxotrophic tobacco lines by electrofusion has been established (Kohn and Schieder 1984). As electrofusion may offer possibilities for mass production of heterokaryons in a reproducible way we initiated fusion experiments using a parallel plate multi-electrode electrofusion chamber. In this report we describe the successful somatic hybridization of Nicotiana plumbaginifolia protoplasts from two NR deficient lines (Mart6n et al. 1982b).

Offprint requests to: K.J. Puite

MATERIALS

AND METHODS

Plant Material and Protoplast

Isolation

Protoplasts were isolated from 2 to 4 weeks old plantlets of the N. plumbaginifolia CNX 20 line (Negrutiu et al. 1983) and from a cell suspension culture of the N__t plumbaginifolia NA 36 line (Mart6n et al. 1982a). CNX 20 is a NR deficient cell line mutated in the cofactor, whereas the NA 36 line exhibits a deficiency in the apoenzyme. Both lines have an absolute requirement for reduced nitrogen. The CNX 20 plants were maintained on ½ strength MS (Murashige and Skoog 1962) medium supplemented with I0 g/l sucrose, I0 mM ammonium succinate and 0.2 mM sodium molybdate, and solidified with 0.8% agar. They were grown in the growth chamber at 24°C with 16 h light (I0 klux) and 8 h darkness. The NA 36 suspension culture was maintained on RMOP medium (Sidorov et al. 1981) supplemented with i0 mM ammonium succinate on a rotary shaker at 28°C in the dark. It was subcultured once in 7 to i0 days by adding fresh medium to the cells and twofold dilution. The enzyme mixture used for protoplast isolation consisted of 1.5% cellulase Onozuka RI0, 0.3% macerozyme RIO and 9% mannitol in modified K3 medium (Sidorov and Maliga 1982) at a pH of 5.6, diluted with 9% mannitol to 15% and 65% of the original strength for the CNX 20 and NA 36 cells, respectively. Protoplasts were isolated by overnight incubation in the enzyme solution. The NA 36 cells were incubated on a shaker (40 rpm). The protoplasts were collected on 0.5 M sucrose. FDA was added to the cell suspension protoplasts to a concentration of 8 ~g/ml. After a staining period of 30 min, free FDA was removed by washing with W5 solution (Sidorov et al. 1981) and 0.5 M mannitol.

Electrofusion A i:I mixture of mesophyll protoplasts an~ FDA stained cell suspension protoplasts (i to 2xlO /ml) was pipetted into the electrofusion chamber (Fig.l). The multi-electrode system had a 3 mm distance between the parallel plate electrodes, was open underneath and placed inside a 5 cm petri-dish. Cohesion of the medium, and the polished electrodes ensured that the medium did not leak out of the compartments during fusion.

275

, ,[

I---- +I+00 V R~

fusion~

chclmber

30 Vp-p I MHz

R3

:'z D ~7

Fig. 2. Electronic circuit used in the electrofusion experiments. RI, 2, 3, 4: 56, 68, 390 and 5600 ohms, respectively; CI: 0.i PF, I000 V; Z: Zenerdiode 2 x 27 V; D: diode IN 4002.

Fig. i. Multi-electrode electrofusion chamber. total content of the four compartments about I ml.

The is

The K 3 and MS media used for viability controls were supplemented with 250 mg/l casein hydrolysate and I0 mM ammonium succinate, respectively (non-selective media). NR assay

With an AC field of 1 MHz and 150 V/cm protoplasts were collected along the field lines in chains of 5 to 15 protoplasts. After a DC pulse (duration 50 Us, field strength 1.2 to 2kV/cm), fusion between protoplasts occurred (Fig. 2). The fusion process was followed under a fluorescence microscope (Zeiss filterset G 365, FT 395 and LP 420). Directly after fusion the electrode array was transferred to a next dish and 2.5 ml of non-selective culture medium (modified K3 medium without 2,4-D and supplemented with 250 mg/l casein hydrolysate) was added to the fused protoplast mixture. One day after fusion heterokaryons were selected manually. In other experiments selective media were used to collect putative hybrid calli. Heterokaryon

Selection

Pieces of calli (0.05 to 0.15 g) were incubated in 5 ml of a medium consisting of 0.08 M phosphate buffer (pH 7.5), 0.02 M KNO~ and 4% isopropanol at 30°C. Nitrite released in t~e medium was determined at zero time and after 0.5 h by adding i ml of 1% sulfanilamide in 3 M HCI and I ml of 0.02% NED to i ml aliquots. After I h the optical density at 546 nm was measured (Jaworski 1971).

NIEOTIANAPLUMBAI31NIFOLIA CNX20 I NA36 mesophy[~ I profoptQsfs I profoptosfs from suspension ~u~ure

and Culture

Heterokaryons were selected using a Leitz micromanipulator, as described by Patnaik et al. 1982 and Hein et al. 1983 in PEG-fusion experiments. Heterokaryons were easily identified showing both red (chloroplast) and green-yellow (FDA) fluorescence and were transferred into 0.2 ml of non-selective culture medium in the center part of a 5 cm Falcon dish no. 3037. A cell suspension culture of NA 36 cells in the outer part of the dish served as a feeder system. Culture of Fused Protoplast Mixtures The fused protoplast mixtures were diluted after one week with the same non-selective medium but with 0.3 M instead of 0.4 M glucose and with one tenth of the hormone concentrations. After I to 2 weeks this medium was replaced by a selective dilution medium (Fig. 3) based on the normal dilution medium but with nitrate as the sole nitrogen source and adding i mM K^SO. to compensate for SO. ions. Calli of a few mm z q in size appeared after 3 weeks and were transferred to selective MS medium also with nitrate as the sole nitrogen source, solidified with 0.8% agar and containing 3% sucrose, 1.0 mg/l BAP and 0.2 mg/l NAA.

T

T

llt

solid medium---~ 111

1" Ii 1"

i, I 2

3

5

2 7

8910

Fig. 3. Experimental set up of the somatic hybridization studies. (i) + (9): viability controls, (2) + (I0): NRcontrols, (3) + (8): self-fusion controls, (5) cross-feeding controls.

276 RESULTS AND DISCUSSION Electrofusion In the AC field pearl-chain formation of the protoplasts by mutual dielectrophoresis occurred due to field disturbances evoked by the presence of the protoplasts in the field (Fig. 4). After the DC pulse fusion bodies were observed which became sphere-shaped in a few minutes. The frequency of heterokaryon formation was reproducible, being 3.8 + 0.4 (standard deviation) % in seven independent e~periments. Somatic hybrids In total, 1048 heterokaryons were selected using the micromanipulator in nine independent fusion experiments at a rate of about 100/h. Counting all heterokaryons immediately after transfer showed that 80% survived the mechanical treatment. After 3 to 4 weeks the 179 calli present were transferred to selective solidified MS medium. Most of the calli (141) were able to grow on this medium. The NR activity was measured of 26 of these calli, of which 22 were NR positive. Seven calli showed an activity of at least 60% of the wild type N. plumbaginifolia calli, growing on selective medium, which was 0.95 ~M NO 2 /h.g. Somatic hybrid lines were also recovered using the selective5dilution and selective solid media. From 2.7 x I0 protoplasts submitted to fusion in eight experiments, 453 nitrate utilizing lines were obtained (Table i). NR activity was determined in 30 lines and was found in 24 of 28 calli which were obtained after two transfers on selective media. Only one callus had an activity of more than 60% of the wild type. Plantlets could be obtained from some of the hybrid calli. Control experiments with 2 x 105 protoplasts, in mono- and cocultures as well as in homofusion experiments, resulted in three calli from cocultures growing on selective medium, while numerous calli were obtained after culture on non-selective media. Additional experiments showed that potato protoplasts could also easily be fused in the multi-electrode fusion chamber. In conclusion, the results reported here show that the technique of electrofusion has to be considered as profitable in somatic hybridization studies.

Fig. 4. Arrangement field.

of the protoplasts

in an AC

ACKNOdLEDGEMENT We thank dr. I. Negrutiu for providing the CNX 20 line and dr. L. Mart6n for the NA 36 line. The feeder system used was suggested by dr. T. Hein. The critical review of the manuscript by drs. L. Gilissen, B. de Groot, S. Roest and A. de Laat is appreciated.

REFERENCES Bates GW, Gaynor JJ, Shekhawat NS (1983) Plant Physiol 7 2 : 1 1 1 0 - 1 1 1 3 Hein T, Przewo~ny T, Schieder 0 (1983) Theor Genet 6 4 : 1 1 9 - 1 2 2

Appl

Jaworski EG (1971) Biochem Biophys Res Commun 43: 1274-1279 Kohn H, Schieder 0 (1984) Int Symp Plant Tissue and Cell Culture, p 89, Olomouc Csechoslovakia, abstract. Mart6n L, Dung TM, Mendel RR, Maliga P (1982a) Mol Gen Genet 1 8 2 : 3 0 1 - 3 0 4 Mart6n L, Sidorov V, Biasini G, Maliga P (1982b) Mol Gen Genet 1 8 7 : 1 - 3 Murashige T, Skoog F, (1962) Physiol Plant 15: 473-497 Negrutiu I, Dirks R, Jaeobs M, (1983) Theor Appl Genet 6 6 : 3 4 1 - 3 4 7

Table I: Nitrate utilizing

lines from fused protoplast Patnaik G, Cocking EC, Hamill J, Pental D (1982) 24: 105-110

mixtures~ Experiment Estimated number of heternkaryons

1,2,3 4 5 6 7 8 4 to 8

not determined 175 418 453 583 1728 3357

Number of nitrate utilizing ealli

51 5 56 59 37 245 402

Frequency of calli refutation

3 13 13 6 14 12

Senda M, Takeda J, Abe S, Nakamura T (1979) Plant Cell Physiol 2 0 : 1 4 4 1 - 1 4 4 3 Sidorov VA, Maliga P (1982) Mol Gen Genet 186: 328-332 Sidorov VA, Menczel L, Nagy F, Maliga P (1981) Planta 152:341-345 Tempelaar MJ, Jones MKG (1985) Plant Cell Reports 4: 92-95 Watts JW, King JM (1984) Bioscience Reports 4: 335-342 Zimmermann U, Seheurieh P (1981) Planta 1 5 1 : 2 6 - 3 2 Zachrisson A, Bornman CH (1984) Physiol Plant 61: 314-320