Plant Cell Reports

Plant Cell Reports (1990) 9:411-414

9 Springer-Verlag 1990

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3) Marie-H~l~ne Chaput 1, Darasinh Sihaehakr t, Georges Duereux l, Dominique Marie 2 and Nasrine Barghi 1 1 Morphogtn~se Vtgttale Exptrimentale, C.N.R.S. U R A 115, Brit. 360, Universit6 Paris Sud, F-91405 Orsay Cedex, France z Cytomttrie, Institut des Sciences Vtgttales, C.N.R.S., F-91190 Gif sur Yvette, France Received June 10, 1990/Revised version received August 10, 1990 - Communicated by A. M. Boudet

ABSTRACT In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BFI5 (HI), were eleetrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with "BFI5 + Aminca" were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions "BFI5 + Aminca" and "BFI5 + Cardinal". Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase

(ICD).

dihaploid clones Aminca and Cardinal. Herein, we describe the production and characterization of somatic hybrid plants of BFI5 with Aminca and Cardinal using protoplast electrofusion. MATERIALS AND METHODS Plant materials Three clones from dihaploid potato (Solanum tuberosum L.) (2n=2x=24), BFI5 (HI), Aminca (H6) and Cardinal (H3), obtained by the Institut National de Recherche Agronomique (I.N.R.A.), and kindly provided by Dr. P. Rousselle, I.N.R.A. Landerneau (France), were used. Plants were maintained in vitro by subculturing leafy node cuttings on a MS basal medium (Murashige and Skoog 1962) supplemented with vitamins (Morel and Wetmore 1951), 2% (w/v) sucrose and solidified with 0.7% (w/v) agar. Environmental conditions were 12h/day illumination at 55 pE/m /s, 20 ~ C and 60% humidity. Protoplast isolation

INTRODUCTION In potato the use of dihaploids accelerates the selection process considerably and it is gaining importance in breeding programs ( Ross 1986 ) . Because of their low agronomic value, dihaploids have to be brought back to the tetraploid level. This can be performed by somatic hybridization which has the advantage of doubling ploidy level, of combining qualitatively and quantitatively inherited characters and of maximizing heterozygosity in the resulting hybrids (Wenzel et al. 1982). Particularly, intraspecific fusions in potato may give rise to the final hybrid cultivar displaying desirable agronomic traits inherited from both parents without intensive backcrosses (Deimling et al. 1988). In this case, the first attempt by Austin et al. (1985) has led to limited success in producing intraspecific somatic hybrids, probably due to the lack of a selection system. Debnath and Wenzel (1987) then used the hybrid vigour of the regenerated calli for the early selection of hybrids. Recently, Masson et al. (1989) developed an effective selection system by using transformed plants as protoplast source. Moreover, Waara et al. (1989) used several isoenzyme systems for the effective identification of hybrids. While the French cultivar BFI5 is widely grown, representing one of the main crop potatoes recommended to French growers, its susceptibility to virus Y and particularly to X, to nematodes (ROi) and also the regrowth of tubers could endanger its culture. Therefore, in a joint project with the I.N.R.A. at Landerneau on the improvement of the cultivar BFI5, protop!ast fusions are being used for the elimination of these traits using two

Offprint requests to: D. Sihachakr

Leaves taken from 4 to 5 week-old cuttings were scarified and placed abaxial face down on an enzyme solution composed of cell and protoplast washing (CPW) salts (Frearson et al. i[973), 1% (w/v) cellulase R-10 (Yakult, Tokyo, Japan), 0.05% (w/v) pectolyase Y-23 (Sheishin, Tokyo, Japan), 9.1% (w/v) mannitol and 0.05% (w/v) 2(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.5. Incubation was carried out overnight in darkness at 27 ~ C. Released protoplasts were then passed through a metallic sieve (i00 ~m mesh) and centrifuged at 55 x g for 5 min. The supernatant was removed and the protoplasts were suspended in a CPW solution containing 21% (w/v) sucrose, then centrifuged at ~20 x g for i0 min. Floating protoplasts were collected and washed twice in a CPW solution plus 0.25M mannitol and 0.125M NaCI. Prior to fusion, protoplasts were washed once in a 0.5M mannitol solution plus 0.2mM CaCl^, then they we{e suspended in thls solution at densitzes of 4-5 x i0- protoplasts/ml. Electrical apparatus and fusion procedure The electrical apparatus and fusion procedure as described in Sihachakr et al. (1988) were used. Briefly, the movable multi-electrodes were placed into a 15 x 50 mm petri dish containing 600 to 800 pl aliquot of a mixture of protoplasts of two clones. An AC field at 125 V/cm and IMHz was applied for aligning the protoplasts ; subsequently, 2 square pulses developing 1.2 kV/cm for 40 ~s each were applied to achieve protoplast fusion. Protoplast culture and plant regeneration Immediately after fusion, the electrodes were removed

412 and 6 ml culture medium were gradually added into the fused protoplast mixture. The culture medium was V-KM medium (Binding et al. 1978) supplemented with 250 mg/l polyethylene glycol 6,000 (PEG), 0.2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l zeatin and i mg/l ~-naphthaleneacetic acid (NAA), 3.3% (w/v) glucose and 3.2% (w/v) mannitol as osmotic agent and 0.05% (w/v) MES. Protoplasts were initially cultured in the dark for 7 days. Afterwards, they wer~ illuminated with a photoperiod of 12h/day at 62 pE/m /s. On day 15, cultures were diluted 10 times with the same medium, except for growth regulators replaced with 2 mg/l 6-benzylaminopurine (BAP) and 0.i mg/l 2,4-D. Twenty days later and in order to stimulate the callus growth, suspensions of microcolonies were plated onto a solid MS medium supplemented with 0.~5 mg/l NAA and 0.5 mg/1 zeatin. Afterwards, sufficiently developed calli were transferred onto the regeneration medium consisting of MS medium plus 2 mg/1 zeatin and 0.i mg/l indole-3-acetic acid (IAA). Shoots were excised from callus and rooted on hormone-free MS medium and the rooted plants were then transferrgd to the greenhouse (16h/day illumination at 180 DE/m-/s, 20-30 ~ C and 70% humidity). Identification of somatic hybrids Identification of somatic hybrid plants was based on the analysis of plant morphology, the ploidy level, and particularly the electrophoretic patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD). Isoenzyme analysis Samples of leaves taken from in vitro plants were c r u s h e d at 4 ~ C in a 0.2M tris-HCl buffer at pH 8.5 containing 20% (w/v) sucrose, 0.03% (v/v) mercaptoethanol, 0.4% (w/v) thioglycolate, 0.4% (w/v) PEG and 4% (w/v) polyvinyl pyrrolidone (PVP). After two centrifugations at 12,000 rpm for 10 min., supernatants were kept at -80 ~ C. Malate dehydrogenase (MDH) (E.C.I.I.I.37) and isocitrate dehydrogenase (ICD) (E.C.I.I.I.42) were separated by electrophoresis on a 13% starch gel. Stainings were performed as described in Shields et al. (1983). Determination of ploidy level and cytological anilysis About Icm 2 leaf material from in vitro plants was chopped with a razor blade in 400 ~i of an ice-cold buffer containing CPW salts, 0.5M mannitol, 0.25% (w/v) PEG, 0.5% (v/v) Triton X-100, 0.25% (v/v) mercaptoethanol at pH 6.5-7.0. Crude samples were filtered with a 40 ~m mesh nylon and stained with a DNA specific dye, bisbenzimide Hoechst 33342 (2 ~g/ml). Nuclei were analyzed in an Epics V flow cytometer (Spectra-Physics argon laser at i00 mW) using wavelengths of (351 + 364) nm for excitation, 408 to 530 for the emission. Each histogram was generated by the analysis of at least I0,000 nuclei. The dihaploid parental plants were used as external references to calibrate the scale of fluorescence. Cytological analysis was done on root tips taken from greenhouse-grown plants as described in Sihachakr et al. (1988). RESULTS

AND DISCUSSION

Electrofusion Protoplast fusion occurred when we applied two DC pulses of 1.2 kV/cm for 40 ~s each. Under these conditions, the fusion rate, defined as the percentage of fused protoplasts compared with the number of aligned protoplasts, was estimated at 51%. Most of the fusion products were multifusions with more than 2 fused protoplasts since only 15% overall fusions were binary fusions. Large fused protoplasts either burst or plasmolyzed after a few hours in ~ e culture medium. The addition of small amounts of Ca ions and keeping the mixture of p[otoplasts on ice significantly improved the

frequency of binary fusions up to 17% and maintained a constant rate of fusion in routine experiments. This was probably d~$ to the stability of membranes in the presence of Ca ions and a limited release of salts into the medium, as reported by several authors (Tempelaar et al. 1987; Fish et al. 1988; Sihachakr et al. 1988). Protoplast culture and plant regeneration Since protoplasts from Aminca and Cardinal did not divide when cultured in V-KM medium, these protoplasts and those from BF15 were mixed in a ratio 5:4 respectively. After eleetrofusion the first protoplasts divided on day 4, and the division frequency rose to 6% on day 7. Sustained calli grew rapidly when they were plated on the growth medium. The plating efficiency, defined as the number of calli on growth medium compared with the number of protoplasts initially cultured in V-KM medium, was estimated at less than 0.05%. Calli turned green within 3 weeks on regeneration medium. Although the period of shoot regeneration ranged from 3 to 6 months after protoplast isolation, plants were regenerated from both fusion experiments with an average of 7 and 2% (defined as the number of regenerating calli compared to the total number of calli on the regeneration medium) for the fusions "BFI5 + Aminca" and "BFI5 + Cardinal" respectively. Generally, regenerating calli bore i-i0 shoots/callus. Excised shoots were multiplied into 3 copies by subculturing leafy node cuttings on MS medium. Flow cytometry and cytological analysis Since at the stage of in vitro culture, we were not able to distinguish hybrid from parental plants, the preliminary selection of hybrids was based on the ploidy level of regenerated plants. This was determined by using flow cytometry. DNA histogram were measured from in vitro plants, and the ploidy level was determined by comparing the position of the peaks between regenerated and parental plants (Fig. i). The analysis of plants recovered from the fusion experiment with "BF15 + Amiuca" showed that 10% of them were diploids (Tab. I). They were all identified as individuals from BE15 protoplasts by examining their morphology in the greenhouse and particularly their electrophoretic patterns for MDH which were found to discriminate somatic hybrids from both parents (Fig. 3A). 14% of regenerated plants were triploids. Most of them (63%) were at the expected tetraploid level, and very few were mixoploids or had a higher level of ploidy (Tab. i). Tetraploid plants could arise from homo or heterofusions, but unfortunately also from chromosome doubling during the culture. It has been shown that cultured protoplasts from dihaploid potato frequently give rise to tetraploid plants following chromosome doubling (Gill et al. 1986). Chromosome counts made on root tips of a random sample of plants confirmed their ploidy level determined by flow cytometry (Fig. 2C and D). Cytometry has been reported to be efficient for the determination of ploidy level (Petit et al. 1986; De Laat et al. 1987) with a strong correlation between nuclear DNA content and chromosome number (Fahleson et al. 1988). Only plants with a ploidy level higher than that of diploids were retained and transferred into a greenhouse for further analysis. Finally, we have retained 106 and 30 plants regenerated from the fusions "BFI5 + Aminca" and "BFI5 + Cardinal'[ respectively. These plants were assessed for their hybrid vigour and morphological characters in the greenhouse. Identification of somatic hybrids through morphological analysis The selected plants were grown to maturity in a greenhouse. Using stem and leaf morphology and vigour in their growth as selection criteria (Fig. 2A and B), we were able to select a total of 24 putative hybrid plants,

413 Table i. Distribution of plants regenerated from electrofusion experiments with mesophyll protoplasts from BFI5 (2x) and Aminca (2x) according to their ploidy level.

.= =~

zc

zc Ploidy level

c 5O

100

150

200

50

100

150

200

m

Number of plants

Diploid (2x) Triploid (3x) Tetraploid (4x) Pentaploid (5x) Hexapleid (6x) Octoploid (8x) Mixoploid Total

, , . ;':%

50

Fig.

1.

., . , , 150 200 50 100 Fluorescence i n t e n s i t y ( a r b i t r a r y

12 16 74 4 6 I 5 118

% 10.2 13.6 62.7 3.4 5.1 0.8 4.2

Number of hybrids 0 0 15 0 4 I 0 20

. . . . . . . . . . .

100

Histograms

of

fluorescence

intensities

150 units)

200

associa-

with 10,000 nuclei isolated from leaves of in vitro plants of dihaploid BFI5 (top left) and A~ninca (top right). After bisbenzimide staining, fluorescence is proportional to nuclear DNA and the position of the dominant GO and G1 peak reflects the ploidy level. Two somatic hybrids may accordindly be assessed as tetraploid (bottom left) and hexaploid (bottom right). ted

namely 20 and 4 hybrids (accounting for 12% and 13% of all regenerants) from the fusions "BFI5 + Aminca" and "BFI5 + Cardinal" respectively (Tab. i). The fact that hybrids were recovered with a high frequency may be due to the hybrid vigour of hybrid calli which was expressed in the ability to regenerate plants. Similar results were obtained from the protoplast fusions in potato (Debnath and Wenzel 1987) and in eggplant (Sihachakr et al. (1988, 1989).Among the 20 hybrid plants recovered after fusion between BFI5 and Aminca protoplasts, 15 were tetraploids, 4 hexaploids and i octoploid. Anlong the 4 somatic hybrid plants of BFI5 with Cardinal, 3 were tetraploids and I hexaploid. These results were in agreement with those of Debnath and Wenzel (1987) and Fish et al. ( 1988 ) with regard to the regeneration of mostly tetraploid somatic hybrids after intra- and interspecific fusions of dihaploid potatoes respectively.

On the contrary,Waara et al. (1989) obtained only hexaploid and mixoploid hybrids from the fusion experiments with clones from dihaploid potato. Only hexaploid and octoploid hybrids were also recovered from the fusion between a dihaploid potato and S. phureja (2x) (Puite et al. 1986). The putative hybrid plants recovered in this study exhibited several traits intermediate to those of the parents, including habit, the number of leaflets, leaf colour and form. Tetraploid somatic hybrids grew vigorously and had larger leaves, thicker and taller stems when compared with those of the parents (Fig. 2A and B). The larger size of the hybrids was partially due to an increase in the ploidy level, but essentially to the hybrid vigour since plants from BFI5, Aminca or Cardinal which were either the original heterozygote tetraploids or returned to the tetraploid level by homofusion of dihaploid protoplasts were less vigorous than the somatic hybrids. Increased vigour was also reported by Austin et al. (1985) and Debnath and Wenzel (1987) for somatic hybrid plants of dihapleid potato. Isoenzyme analysis Finally, the hybrid nature of the selected plants was confirmed by examining the electrophoretic patterns for malate dehydrogenase (MDH) (Fig. 3A) and isocitrate dehydrogenase (ICD) (Fig. 3B) which were found to distinguish hybrids of BFI5 with Aminca and those of BFI5 with Cardinal from both parents respectively.

Fig. 2. Leaves (A) and plants (B) of BFIS, Aminca (Am), Cardinal (Car) and their respective somatic hybrids (BFI5 + Am) and (BFI5 + Car). A root tip cell of dihaploid BFI5 2n=2x=24 (C) and a tetraploid somatic hybrid 2n=4x=48 (D).

414

Fig. 3. Isoenzyme patterns for malate dehybrodgenase (MDH) of somatic hybrid plants of BF15 with Aminca (Am), lanes i-4 (A) and isocitrate dehydrogenase (ICD) of those of BFI5 with Cardinal (Car), lanes i-4 (B)

The somatic hybrid pattern for MDH contained the bands which were identical to those found in the mixed extracts of the parents (Fig. 3A). A little difference was found in the isoenzyme pattern of ICD between the mixed extracts of BFlS-Cardinal and their somatic hybrids. The latters showed a four-banded phenotype, while the mixed extracts contained three specific bands (two from Cardinal and a large elongated band from BFI5) (Fig. 3B). Probably due to different electrophoretic mobility, the large elongated band in BFI5 may give rise to two separate bands in the somatic hybrids. Isoenzymes with new mobilities were also observed in hybrid cells, probably as a result of the formation of hybrid enzymes (Maliga et al. 1977). Based upon the analysis of isoenzymes and morphological markers, particularly the exhibition of intermediate traits and strong hybrid vigour in their growth, the 24 selected plants were considered to be somatic hybrids. Both the stability of the ploidy level determined from leaves and root tips by flow cytometry and cytological analysis respectively, and the fact that only BFI5 protoplasts were capable of dividing in the culture medium used (those from Aminca and Cardinal did not divide) would exclude the possibility of the regeneration of chimeric plants. Conclusion Many somatic hybrid plants were recovered after electrofusion between mesophyll protoplasts from dihaploid potatoes. No selection system has been necessary to recover somatic hybrids, probably due to the high rate of protoplast fusions induced by electrofusion. Similar results were reported by Fish et al. (1988) and Sihachakr et al. (1988, 1989) for protoplast electrofusion in potato and eggplant respectively. Moreover, morphological markers of parents made hybrid identification simple as reported by others (Schieder 1978; Austin et al. 1985). Most of the hybrids were tetraploids and exhibited strong hybrid vigour in their growth. The evaluation for the inheritance of desirable agronomic traits is in progress, and in the case of positive tests such plants may give rise to the final tetraploid hybrid cultivars. Acknowledgements We would like to thank Dr. P. Rousselle for providing plant materials, Dr. L. Rossignol and Dr. S. B r o w n for helpful discussion, Mrs A. Servaes for technical assistance, M. J.L. David for the greenhouse culture and M. D. Froger for the photography.

REFERENCES Austin S, Baer M, Ehlenfeldt M, Kazmierczak PJ, Helgeson JP (1985) Theor Appl Genet 71:172-175 Binding H, Nehls R, Schieder O, Sopory SK, Wenzel G (1978) Physiol Plant 43:52-54 De Laat AMM, GShde W, Vogelzang MJD (1987) Plant Breeding 99:303-307 Debnath SC, Wenzel G (1987) Potato Res 30:371-380 Deimling S, Zitzisperger J, Wenzel G (1988) Plant Breeding 101:181-189 Fahleson J, Dixelius J, Sundberg E, Glimelius K (1988) Plant Cell Rep 7:74-77 Fish N, Karp A, Jones MGK (1988) Theor Appl Genet 76:260-266 Frearson EM, Power JB, Cocking EC (1973) Dev Biol 33:130-137 Gill BS, Kam-Morgan LNW, Shepard JF (1986) The Journal of Heredity 777:13-16 Maliga P, Lazar G, Nagy AH, Menczel L (1977) Mol Gen Genet 157:291-296 Masson J, Lancelin D, Dellini C, Lecerf M, Guerche P, Pelletier G (1989) Theor Appl Genet 78:153-159 Morel G, Wetmore RH (1951) Am J Bot 38:141-143 Murashige T, Skoog F (1962) Physiol Plant 15:473-479 Petit P, Conia J, Bergounioux C (1986) Biofutur 5:128-137 Puite KJ, Roest S, Pijnacker LP (1986) Plant Cell Rep 5:262-265 Ross H (1986) In: Potato breeding - Problems and perspectives. Verlag Pub Berlin 132p Schieder O (1978) Mol Gen Genet 162:113-119 Shields CR, Orton TJ, Stuber CW (1983) In: Tanskley SD, Orton TJ (eds) Isoenzymes in Plant Genetics and Breeding Part A. Elsvier Sci Pub BY, Amsterdam pp443-516 Sihachakr D, Haicour R, Serraf I, Barrientos E, Herbreteau C, Ducreux G, Rossignol L, Souvannavong V (1988) Plant Sci 57:215-223 Sihachakr D, Haicour R, Chaput MH, Barrientos E, Ducreux G, Rossignol L (1989) Theor Appl Genet 77:1-6 Tempelaar MJ, Duyst A, De Vlas SY, Krol G, Symmonds C, Jones MGK (1987) Plant Sci 48:99-105 Waara S, Tegelstr6m H, Wallin A, Eriksson T (1989) Theor Appl Genet 77:49-56 Wenzel G, Bapart VA, Uhrig H (1982) In: Giles KL, Sen SK (eds) Plant Cell Culture in Crop Improvement. Plenum Pub Corp pp337-349