Springer 2005

Plant Cell, Tissue and Organ Culture (2005) 83: 145–151 DOI 10.1007/s11240-005-4633-9

Protoplast fusion in banana (Musa spp.): comparison of chemical (PEG: polyethylene glycol) and electrical procedure Akym Assani1,*, Djamila Chabane2, Robert Haı¨ cour3, Fre´de´ric Bakry4, Gerhard Wenzel5 & Ba¨rbel Foroughi-Wehr6 1

Department of Plant Agriculture, University of Guelph, Bovey Building, N1G 2W1, Guelph, Ontario, Canada; Faculte´ des Sciences Biologiques, Universite´ des Sciences et de Technologie, B.P: 32 El Alia, 16111, Alger, Alge´rie; 3Ecologie, Syste´matique et Evolution, Universite´ de Paris Sud XI, UMR 8079, Baˆtiment 362, F-91405, Orsay Cedex, France; 4CIRAD-FLHOR, TA 50/PS4, Boulevard de la Lironde, F-34398, Montpellier Cedex 5, France; 5Lehrstuhl fu¨r Pflanzenbau und Pflanzenzu¨chtung, Technische Universita¨t Mu¨nchen, D-85350, Freising-Weihenstephan, Germany; 6Institut fu¨r Resistenzgenetik, Bundesanstalt fu¨r Zu¨chtungsforschung an Kulturpflanzen, Graf-Seinsheim-Str. 23, D-85461, Gru¨nbach, Germany (*requests for offprints; Phone: +1-519-824-4120 ext. 52727; Fax: +1-519-767-0755; E-mail: [email protected]) 2

Received 28 December 2004; accepted in revised form 24 March 2005

Key words: electrofusion, Musa spp., polyethylene glycol, protoplast fusion

Abstract Optimization of protoplast fusion parameters is a prerequisite for the establishment of somatic fusion technology for banana breeding. In the present investigations, we compared the most frequently used fusion methods: the electrofusion technique and chemical procedure (polyethylene glycol). With regard to frequency of binary fusion, protoplast fusion with the fusogen polyethylene glycol was best. Conversely, electric fusion was found to be better with respect to mitotic activities, somatic embryogenesis and plantlet regeneration rate. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; BA – benzylaminopurine; CIRAD – Center for International Cooperation in Agricultural Research for Development; IAA – indole-3-acetic acid; MES – 2-(N-morpholino) ethanesulfonic acid; NAA – a-naphthaleneacetic acid; PCV – packed cell volume; PEG – polyethylene glycol

Introduction The main factor hampering progress in banana breeding using conventional genetic improvement methods is the sterility of most edible varieties due to triploidy. Integration of somatic fusion technology, which is the way of combining two different plant genomes asexually, would be a potential tool to overcome sterility and increase genetic variability. A goal of banana improvement is the release of disease-resistant triploid hybrids (Bakry et al., 2001), protoplast fusion is considered as useful complementary tool for the production of somatic hybrid tetraploid parents that could be used in

interploid crosses with other diploid lines or for direct release of triploid somatic hybrids by haplo/ diploid protoplast fusion (Assani et al., 2003). Since a protoplast regeneration system in banana has already been established (Megia et al., 1993; Panis et al., 1993; Matsumoto and Oka, 1998; Assani et al., 2001; Assani et al., 2002); the use of cell fusion techniques in banana breeding had become a realizable objective. In monocots, most of the reported work on protoplast technology has been focused on embryogenic cell suspension-derived protoplasts (Vasil et al., 1990: wheat; Hahne et al., 1990: oat, ForoughiWehr et al., 1982: barley; Shillito et al., 1989: maize;

146 Datta et al., 1992: rice; Megia et al., 1993: banana). Cell suspension cultures of banana can be established from pseudo-stem and rhizome tissues (Novak et al., 1989), meristematic shoot tips (Dhed’a et al., 1991), immature zygotic embryos (Marroquin et al., 1993); immature male flowers (Ma, 1991; Coˆte et al., 1996; Grapin et al., 1996) and immature female flowers (Grapin et al., 2000). For genotypes in which cell suspension cultures are not available, embryogenic callus or leaf can be used to produce protoplasts (Assani et al., 2002; Assani et al., 2003). Numerous somatic hybrid plants have been obtained in dicots solanaceae like potato (Debnath and Wenzel, 1987), eggplant (Sihachakr et al., 1994) and in citrus (Crosser et al., 2000). Conversely, within the monocots like poaceae (cereals) and musaceae (banana), somatic hybridisation has proven to be very difficult. However, Tabaiezadeh et al., (1986) obtained somatic hybrid embryos of sugarcane (Saccharum officinarum L.) and pearl millet (Pennisetum americanum L.), but plantlet formation of somatic hybrid embryos was not mentioned. More recently, regeneration of somatic hybrid plants of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) has been reported (Kisaka et al., 1998). In banana, somatic hybrids have also been reported (Matsumoto et al., 2002). However, before somatic fusion can be routinely used in banana breeding, the optimising of fusion parameters is required. In the present study, we compared a standard chemical fusion procedure (polyethylene glycol: PEG) and electric technique in protoplast fusion in banana (Musa spp.).

Materials and methods

Initiation and maintenance of cell suspension cultures The initiation and maintenance of banana embryogenic cell suspension cultures has been described by Ma (1991) and Coˆte et al. (1996). Isolation of protoplasts Embryogenic suspension cultures of cv. Gros Michel (AAA) and cv. SF265 (AA), 3–4 days after the last subculture, were used as donor material for the isolation of protoplasts. The cell suspensions were sieved through 200 lm stainless mesh in order to select only small cell aggregates. 2 ml enzyme solution containing 1.5% (w/v) cellulase RS (Yakult Honsha Co., Tokyo, Japan), 0.15% (w/v) pectolyase Y 23 (Kyowa Chemical Products Co., Osaka, Japan), 204 mM KCl, 67 mM CaCl2 (pH 5.6), was added to 1 ml sieved cell suspensions. The enzyme–cell suspension mixture was incubated overnight (12–14 h) at 27 C without shaking. Purification of protoplasts The digestion mixture was filtered through 100/ 25 lm metallic mesh combination to remove the debris and large cell colonies. Protoplasts were then washed through centrifugation at 66 g for 5 min. The washing solution consisted of 204 mM KCl, 67 mM CaCl2. The pellet was washed again two times (centrifugation at 66 g for 5 min). Protoplast viability was determined by fluorescein diacetate (FDA) according to Widholm (1972). Protoplasts yield was estimated using a Nageotte hematocymeter.

Plant material Fusion of protoplasts The plants, Musa spp. triploid cv. Gros Michel (AAA) and diploid cvs. SF265 (AA), all derived from the diploid wild species Musa acuminata which contributes to the A genome (AA). Cultivar Gros Michel (AAA) is a natural ‘‘dessert’’ worldwide banana variety whereas cv. SF265 is a natural Ôcooking’ variety originated from Papua New Guinea. Gros Michel is genetically and morphologically very distant from cv. SF265 clone. Cultivar Gros Michel (AAA) was used for fusion experiments and SF265 (AA) for nurse cell culture and fusion experiments.

Electric technique Both fusion partners were mixed in equal proportion (1:1) in a fusion solution containing 0.5 M mannitol and 0.5 mM CaCl2 in order to obtain a final concentration of 5 · 105 protoplasts per ml. For each fusion experiment, a total number of 20 · 105 protoplasts were used. The protoplasts were distributed in equal numbers in eight Petri dishes (i.e. 2.5 · 105 protoplasts per Petri dish). The movable multi-electrodes were placed in Petri

147 dishes (5.5 cm diameter) containing 500 ll fusion solution with protoplasts of both fusion partners. An alternating current (AC) field of 1 MHz and 230 V cm)1 output voltage was applied for 30 s to align the protoplasts, and 10–20 direct current (DC) pulses (pulse width: 30–40 ls) of 1.5–2.3 kV cm)1 was used to induce membrane fusion. After the application of pulses, the AC field was reduced to 20 V cm)1 to maintain the alignment of the protoplast chains. Five minutes after the fusion procedure, the fusion solution was replaced by a liquid protoplast culture medium composed of N6 salts (Chu et al., 1975), vitamins, organic acids and sugar alcohol (Kao and Michayluk, 1975), Morel vitamins (Morel and Wetmore, 1951), 117 mM sucrose, 0.4 M glucose, 0.5 mM MES, 1.9 mM KH2PO4, 2.3 lM zeatin, 0.9 lM 2.4-D and 4.4 lM NAA (pH 5.7) and sterilized by filtration. Chemical procedure (polyethylene glycol: PEG) Protoplasts of the two fusion partners, at a density of 5 · 105 protoplasts per ml were mixed in equal proportion in a fusion solution containing 0.5 M mannitol and 0.5 mM CaCl2. Drops of 300 ll suspension containing 1.5 · 105 protoplasts were transferred into each Petri dish (5.5 cm diameter). For each fusion experience, 12 · 105 protoplasts were used (i.e. eight Petri dishes). After the protoplasts had settled, 4–6 drops of 50 ll each of PEG solution (50% PEG, 0.5 M mannitol and 0.5 mM CaCl2) were slowly added. The protoplasts adhered to the Petri dish 20–30 min after polyethylene glycol treatment. The PEG-solution was gently diluted with 3 ml fusion solution. Ten minutes after dilution, the diluted PEG-solution were gently removed and replaced with liquid culture medium as described above. The fusion process was observed microscopically to estimate the number of binary fusions. Culture of protoplasts We used nurse cultures to induce cell divisions in cultured protoplasts. They were prepared a day before the protoplast isolation. Cell suspensions of SF 265 (AA) were used for cell nurse culture, which was prepared as follows: – cell suspensions were sieved through a 250 lm metallic mesh in order to select only small cell aggregates.

– the PCM medium, which consisted of MS salts (Murashige and Skoog, 1962), 9 lM 2.4-D , Morel vitamins (Morel and Wetmore, 1951), 2.8 mM glucose, 278 mM maltose, 116 mM sucrose, 2.5 mM myo inositol (pH 5.7) was sterilized by filtration. – sieved cell suspensions were mixed with 100 ml double-concentrated PCM liquid medium, to obtain a final cell concentration of 10%. – 1.2 g agarose sea plaque (Sigma) was dissolved in 100 ml double distilled water and then autoclaved (pH 5.7). When the temperature of agarose solution decreased to 30–35 C, it was gently mixed with 100 ml double-concentrated PCM medium containing nurse cells. – 10–12 ml of the mixture was poured into small Petri dishes (5.5 cm diameter). The medium was covered with sterilized nitro cellulose filter (AA type, Millipore Corporation) to prevent mixing with fusion protoplasts. – After fusion (chemical procedure or electric technique), 0.5 · 106 protoplasts were mixed with 0.5 ml liquid culture medium as described above (i.e. 1.0 · 106 protoplasts per ml) and transferred onto the nitrocellulose filter. The cultures were maintained at 27 C in the dark. Cell wall regeneration was observed with calcofluor white (fluorescent brightener) under UV microscope as described by Galbraith (1981). Somatic embryogenesis Protoplast-derived microcalluses were individually picked from the feeder layer and gently transferred onto A0.4B0.5 regeneration medium containing MS salts, Morel vitamins, 88 mM sucrose, 2.3 lM IAA, 2.2 lM BA and 7.5 g l)1 agarose sea plaque (pH 5.7) (Assani et al., 2001). The cultures were maintained at 27 C in the dark. Regenerated plants were transferred onto solidified growth regulator-free MS medium with 1.2 mM NH4NO3. The plants were cultured under 16-h photoperiod (65 lmol m)2 s)1) at 27 C. The plants were transplanted in soil for field-testing, when larger than 10 cm. Identification of somatic hybrids Morphological markers of in vitro plants like leaf size, leaf colour, leaf thickness and pseudo stem thickness are used as first step for pre-screening of

148 putative hybrids. In the second step, flow cytometry (Assani et al., 2003) has been used to determine the ploidy level and to select the somatic hybrids.

The results were obtained in three independent experiments, each with three or four replicates.

ploid, pentaploid, hexaploid, heptaploid). The plant issued from protoplast fusion has a larger, intensive-green leaf and thicker pseudo stem. As second step, the pre-selected plants through morphological marker were analysed using flow cytometry. This demonstrated that 4 plants were pentaploid, 1 hexaploid, 19 tetraploid, 2 triploid, 74 diploid and 1 heptaploid (Table 2).

Results

Discussion

As shown in Figure 1a, the application of an alternating current field led to the formation of protoplast chains, which are required for electric technique fusion. Regarding fusion efficiency, the average rate of binary fusion (Figure 1b) was 10% (25 · 103 protoplasts per Petri dish) with electric technique and 17% (42 · 103 protoplasts per Petri dish) with chemical procedure (Table 1). This suggested that PEG procedure led to better fusion rate. To reach the highest rate of binary fusion using electric fusion process, it was necessary to apply up to 20 direct current pulses (30–40 ls) of 1.5–2.3 kV cm)1 and 230 kV cm)1 alternative current field. When more than 20 current pulses were applied, many multifusions were obtained. A higher than 50% PEG concentration, or the application of PEG longer than 30 min led to serious protoplast damage. A comparative study regarding mitotic cell activities showed that cell division rate was higher (35%) after electric fusion technique than after PEG treatment (24%) (Table 1). Moreover, the number of microcalluses formed was 18 · 103 with electrical technique and 13 · 103 with chemical (PEG) procedure (Table 1). Protoplasts treated with PEG needed 21 days to form microcalluses and those fused electrically needed only 14 days (Table 1). PEG treatment seemed to affect the somatic embryogenesis and the duration of the formation of embryos (Figure 1c). As indicated in Table 1, 2400 embryos and 1600 plantlets were formed after electrical fusion, versus 1500 embryos and 924 plantlets after the chemical procedure. The duration required for formation of plantlets was 120 days for protoplast treated with PEG, and 90 days for protoplasts electrically fused (Table 1). Morphological marker allowed the pre-screening of 101 plants among the fusion products (tetra-

Somatic hybridization could be an excellent tool for the breeding of this important cultivated plant. Unfortunately, there are very few reports of protoplast fusion technology in monocots. Cell fusion of banana protoplasts has been achieved in this report. In the present study, we compared two of the most widely used fusion methods, i.e. treating protoplasts with the fusogen PEG or electrofusion. Protoplasts treated with PEG gave higher frequency of binary fusions than those fused electrically. On the other hand, the electrofusion process is easier to control. Concerning cell division rate, the best results were obtained with electrofusion. The lower rate of cell divisions of protoplasts treated with PEG suggests that PEG leads to perturbations of mitotic activities. Due to its toxicity, PEG is known to affect protoplast viability (Mercer and Schlegel, 1979). PEG treatment also affects the development of fusion products in organized structures, resulting in higher numbers of calluses formed and lower numbers of somatic embryos. Conversely, electric field pulse technique seems to stimulate the somatic embryogenesis, since the large number of embryos and plantlets formed was higher with this technique, compared to PEG fusion. It has been shown in our comparative investigations that concerning to fusion frequencies, fusion with fusogen PEG has a superior effect. In contrast, electrofusion was found to be better with respect to mitotic activities, somatic embryogenesis and plantlet regeneration rate.

Statistics

Acknowledgements This work was generously supported by the European Union (INCO-DC-Contract N IC18CT97-02-04).

149

Figure 1. (a) Formation of ‘‘;protoplast chains’’ after application of alternative current. Bar = 120 lm. (b) Binary fusions induced by polyethylene glycol. Bar = 30 lm. (c) Somatic embryos formed after fusion with polyethylene glycol. Bar = 1.8 cm. (d) Somatic embryos formed after electric fusion. Bar = 10 mm.

150 Table 1. Comparison of electrical technique and chemical procedure in banana protoplast fusion

Rate (%) of binary fusion Rate of first cell division Number of microcalluses Duration (days) microcallus formation Number of embryos Number of plantlets Duration (days) of plantlets formation

Electrical technique

Chemical procedure

10 35 18 000 21 2400 1600 90

17 24 13 000 14 1500 924 120

± ± ± ± ± ± ±

1.7 4.5 700 3 265 100 5

± ± ± ± ± ± ±

3 3.6 100 2 100 46 10

Table 2. Ploidy variability of fusion products of combination Gros Michel (+) SF265 after flow cytometry analysis Plant ploidy level

Plant number

Percentage

Diploid Triploid Tetraploid Pentaploid Hexaploid Heptaploid R

74 2 19 5 1 1 101

73 2 19 5 1 1

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