Plant Cell, Tissue and Organ Culture (2005) 83: 145–151 Springer 2005 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 1Department of Plant Agriculture, University of Guelph, Bovey Building, N1G 2W1, Guelph, Ontario, Canada; 2Faculte´ 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¨tMu¨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])

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

Key words: electrofusion, Musa spp., polyethylene glycol, 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 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 interploid crosses with other diploid lines or for direct release of triploid somatic hybrids by haplo/ The main factor hampering progress in banana diploid protoplast fusion (Assani et al., 2003). Since breeding using conventional genetic improvement a protoplast regeneration system in banana has methods is the sterility of most edible varieties due already been established (Megia et al., 1993; Panis et to triploidy. Integration of somatic fusion technol- al., 1993; Matsumoto and Oka, 1998; Assani et al., ogy, which is the way of combining two different 2001; Assani et al., 2002); the use of plant asexually, would be a potential tool techniques in banana breeding had become a to overcome sterility and increase genetic variabil- realizable objective. ity. A goal of banana improvement is the release of In monocots, most of the reported work on pro- disease-resistant triploid hybrids (Bakry et al., toplast technology has been focused on embryoge- 2001), protoplast fusion is considered as useful nic cell suspension-derived (Vasil et al., complementary tool for the production of somatic 1990: ; Hahne et al., 1990: oat, Foroughi- tetraploid parents that could be used in Wehr et al., 1982: barley; Shillito et al., 1989: maize; 146

Datta et al., 1992: rice; Megia et al., 1993: banana). Initiation and maintenance of cell suspension Cell suspension cultures of banana can be establis- cultures hed from pseudo-stem and rhizome tissues (Novak et al., 1989), meristematic shoot tips (Dhed’a et al., The initiation and maintenance of banana 1991), immature zygotic embryos (Marroquin embryogenic cell suspension cultures has been et al., 1993); immature male flowers (Ma, 1991; described by Ma (1991) and Coˆ te et al. (1996). Coˆ te et al., 1996; Grapin et al., 1996) and immature female flowers (Grapin et al., 2000). For genotypes Isolation of protoplasts in which cell suspension cultures are not available, embryogenic callus or leaf can be used to produce Embryogenic suspension cultures of cv. Gros protoplasts (Assani et al., 2002; Assani et al., 2003). Michel (AAA) and cv. SF265 (AA), 3–4 days after Numerous somatic hybrid plants have been the last subculture, were used as donor material for obtained in dicots solanaceae like (Debnath the isolation of protoplasts. The cell suspensions and Wenzel, 1987), eggplant (Sihachakr et al., were sieved through 200 lm stainless mesh in order 1994) and in citrus (Crosser et al., 2000). Con- to select only small cell aggregates. 2 ml versely, within the monocots like poaceae (cereals) solution containing 1.5% (w/v) cellulase RS (Yak- and musaceae (banana), somatic hybridisation has ult Honsha Co., Tokyo, Japan), 0.15% (w/v) proven to be very difficult. However, Tabaiezadeh pectolyase Y 23 (Kyowa Chemical Products Co., et al., (1986) obtained somatic hybrid embryos of Osaka, Japan), 204 mM KCl, 67 mM CaCl2 (pH sugarcane (Saccharum officinarum L.) and pearl 5.6), was added to 1 ml sieved cell suspensions. The millet (Pennisetum americanum L.), but plantlet enzyme–cell suspension mixture was incubated formation of somatic hybrid embryos was not overnight (12–14 h) at 27 C without shaking. mentioned. More recently, regeneration of somatic hybrid plants of rice (Oryza sativa L.) and barley Purification of protoplasts (Hordeum vulgare L.) has been reported (Kisaka et al., 1998). In banana, somatic hybrids have also The digestion mixture was filtered through 100/ been reported (Matsumoto et al., 2002). However, 25 lm metallic mesh combination to remove the before somatic fusion can be routinely used in debris and large cell colonies. Protoplasts were banana breeding, the optimising of fusion param- then washed through centrifugation at 66 g for eters is required. In the present study, we com- 5 min. The washing solution consisted of 204 mM pared a standard chemical fusion procedure KCl, 67 mM CaCl2. The pellet was washed again (polyethylene glycol: PEG) and electric technique two times (centrifugation at 66 g for 5 min). in protoplast fusion in banana (Musa spp.). Protoplast viability was determined by fluorescein diacetate (FDA) according to Widholm (1972). Protoplasts yield was estimated using a Nageotte Materials and methods hematocymeter.

Plant material Fusion of protoplasts The plants, Musa spp. triploid cv. Gros Michel (AAA) and diploid cvs. SF265 (AA), all derived Electric technique from the diploid wild species Musa acuminata which contributes to the A (AA). Cultivar Gros Both fusion partners were mixed in equal propor- Michel (AAA) is a natural ‘‘dessert’’ worldwide tion (1:1) in a fusion solution containing 0.5 M banana variety whereas cv. SF265 is a natural mannitol and 0.5 mM CaCl2 in order to obtain a Ôcooking’ variety originated from Papua New final concentration of 5 · 105 protoplasts per ml. Guinea. Gros Michel is genetically and morpholog- For each fusion experiment, a total number of ically very distant from cv. SF265 clone. Cultivar 20 · 105 protoplasts were used. The protoplasts Gros Michel (AAA) was used for fusion experi- were distributed in equal numbers in eight Petri ments and SF265 (AA) for nurse cell culture and dishes (i.e. 2.5 · 105 protoplasts per Petri dish). fusion experiments. The movable multi-electrodes were placed in Petri 147 dishes (5.5 cm diameter) containing 500 ll fusion – the PCM medium, which consisted of MS salts solution with protoplasts of both fusion partners. (Murashige and Skoog, 1962), 9 lM 2.4-D, An alternating current (AC) field of 1 MHz and Morel vitamins (Morel and Wetmore, 1951), 230 V cm)1 output voltage was applied for 30 s to 2.8 mM glucose, 278 mM maltose, 116 mM align the protoplasts, and 10–20 direct current sucrose, 2.5 mM myo inositol (pH 5.7) was (DC) pulses (pulse width: 30–40 ls) of 1.5–2.3 kV sterilized by filtration. cm)1 was used to induce membrane fusion. After – sieved cell suspensions were mixed with 100 ml the application of pulses, the AC field was reduced double-concentrated PCM liquid medium, to to 20 V cm)1 to maintain the alignment of the obtain a final cell concentration of 10%. protoplast chains. Five minutes after the fusion – 1.2 g agarose sea plaque (Sigma) was dissolved procedure, the fusion solution was replaced by a in 100 ml double distilled water and then auto- liquid protoplast culture medium composed of N6 claved (pH 5.7). When the temperature of aga- salts (Chu et al., 1975), vitamins, organic acids and rose solution decreased to 30–35 C, it was sugar alcohol (Kao and Michayluk, 1975), Morel gently mixed with 100 ml double-concentrated vitamins (Morel and Wetmore, 1951), 117 mM PCM medium containing nurse cells. sucrose, 0.4 M glucose, 0.5 mM MES, 1.9 mM – 10–12 ml of the mixture was poured into small KH2PO4, 2.3 lM zeatin, 0.9 lM 2.4-D and Petri dishes (5.5 cm diameter). The medium 4.4 lM NAA (pH 5.7) and sterilized by filtration. was covered with sterilized nitro cellulose filter (AA type, Millipore Corporation) to prevent Chemical procedure (polyethylene glycol: PEG) mixing with fusion protoplasts. – After fusion (chemical procedure or electric Protoplasts of the two fusion partners, at a density technique), 0.5 · 106 protoplasts were mixed of 5 · 105 protoplasts per ml were mixed in equal with 0.5 ml liquid culture medium as described proportion in a fusion solution containing 0.5 M above (i.e. 1.0 · 106 protoplasts per ml) and mannitol and 0.5 mM CaCl2. Drops of 300 ll transferred onto the nitrocellulose filter. suspension containing 1.5 · 105 protoplasts were The cultures were maintained at 27 C in the dark. transferred into each Petri dish (5.5 cm diameter). Cell wall regeneration was observed with calcoflu- For each fusion experience, 12 · 105 protoplasts or white (fluorescent brightener) under UV micro- were used (i.e. eight Petri dishes). After the scope as described by Galbraith (1981). protoplasts had settled, 4–6 drops of 50 ll each of PEG solution (50% PEG, 0.5 M mannitol and Somatic embryogenesis 0.5 mM CaCl2) were slowly added. The protop- lasts adhered to the Petri dish 20–30 min after Protoplast-derived microcalluses were individually polyethylene glycol treatment. The PEG-solution picked from the feeder layer and gently transferred was gently diluted with 3 ml fusion solution. Ten onto A B regeneration medium containing MS minutes after dilution, the diluted PEG-solution 0.4 0.5 salts, Morel vitamins, 88 mM sucrose, 2.3 lM IAA, were gently removed and replaced with liquid ) 2.2 lMBAand7.5gl 1 agarose sea plaque (pH 5.7) culture medium as described above. The fusion (Assani et al., 2001). The cultures were maintained at process was observed microscopically to estimate 27 C in the dark. Regenerated plants were trans- the number of binary fusions. ferred onto solidified growth regulator-free MS medium with 1.2 mM NH4NO3. The plants were Culture of protoplasts ) ) cultured under 16-h photoperiod (65 lmol m 2 s 1) at 27 C. The plants were transplanted in soil for We used nurse cultures to induce cell divisions in field-testing, when larger than 10 cm. cultured protoplasts. They were prepared a day before the protoplast isolation. Cell suspensions of SF 265 (AA) were used for cell nurse culture, Identification of somatic hybrids which was prepared as follows: – cell suspensions were sieved through a 250 lm Morphological markers of in vitro plants like leaf metallic mesh in order to select only small cell size, leaf colour, leaf thickness and pseudo stem aggregates. thickness are used as first step for pre-screening of 148 putative hybrids. In the second step, flow cytometry ploid, pentaploid, hexaploid, heptaploid). The (Assani et al., 2003) has been used to determine plant issued from protoplast fusion has a larger, the level and to select the somatic hybrids. intensive-green leaf and thicker pseudo stem. As second step, the pre-selected plants through mor- Statistics phological marker were analysed using flow cytometry. This demonstrated that 4 plants were The results were obtained in three independent pentaploid, 1 hexaploid, 19 tetraploid, 2 triploid, experiments, each with three or four replicates. 74 diploid and 1 heptaploid (Table 2).

Results Discussion

As shown in Figure 1a, the application of an Somatic hybridization could be an excellent tool alternating current field led to the formation of for the breeding of this important cultivated plant. protoplast chains, which are required for electric Unfortunately, there are very few reports of technique fusion. Regarding fusion efficiency, the protoplast fusion technology in monocots. Cell average rate of binary fusion (Figure 1b) was 10% fusion of banana protoplasts has been achieved in (25 · 103 protoplasts per Petri dish) with electric this report. In the present study, we compared two technique and 17% (42 · 103 protoplasts per Petri of the most widely used fusion methods, i.e. dish) with chemical procedure (Table 1). This treating protoplasts with the fusogen PEG or suggested that PEG procedure led to better fusion electrofusion. Protoplasts treated with PEG gave rate. To reach the highest rate of binary fusion higher frequency of binary fusions than those using electric fusion process, it was necessary to fused electrically. On the other hand, the electro- apply up to 20 direct current pulses (30–40 ls) of fusion process is easier to control. 1.5–2.3 kV cm)1 and 230 kV cm)1 alternative Concerning rate, the best results current field. When more than 20 current pulses were obtained with electrofusion. The lower rate were applied, many multifusions were obtained. of cell divisions of protoplasts treated with PEG A higher than 50% PEG concentration, or the suggests that PEG leads to perturbations of application of PEG longer than 30 min led to mitotic activities. Due to its toxicity, PEG is serious protoplast damage. known to affect protoplast viability (Mercer and A comparative study regarding mitotic cell Schlegel, 1979). PEG treatment also affects the activities showed that cell division rate was higher development of fusion products in organized (35%) after electric fusion technique than after structures, resulting in higher numbers of calluses PEG treatment (24%) (Table 1). Moreover, the formed and lower numbers of somatic embryos. number of microcalluses formed was 18 · 103 with Conversely, electric field pulse technique seems to electrical technique and 13 · 103 with chemical stimulate the somatic embryogenesis, since the (PEG) procedure (Table 1). Protoplasts treated large number of embryos and plantlets formed was with PEG needed 21 days to form microcalluses higher with this technique, compared to PEG and those fused electrically needed only 14 days fusion. It has been shown in our comparative (Table 1). investigations that concerning to fusion frequen- PEG treatment seemed to affect the somatic cies, fusion with fusogen PEG has a superior embryogenesis and the duration of the formation effect. In contrast, electrofusion was found to be of embryos (Figure 1c). As indicated in Table 1, better with respect to mitotic activities, somatic 2400 embryos and 1600 plantlets were formed after embryogenesis and plantlet regeneration rate. electrical fusion, versus 1500 embryos and 924 plantlets after the chemical procedure. The dura- tion required for formation of plantlets was Acknowledgements 120 days for protoplast treated with PEG, and 90 days for protoplasts electrically fused (Table 1). This work was generously supported by the Morphological marker allowed the pre-screening European Union (INCO-DC-Contract N IC18- of 101 plants among the fusion products (tetra- CT97-02-04). 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

Electrical technique Chemical procedure

Rate (%) of binary fusion 10 ± 1.7 17 ± 3 Rate of first cell division 35 ± 4.5 24 ± 3.6 Number of microcalluses 18 000 ± 700 13 000 ± 100 Duration (days) microcallus formation 21 ± 3 14 ± 2 Number of embryos 2400 ± 265 1500 ± 100 Number of plantlets 1600 ± 100 924 ± 46 Duration (days) of plantlets formation 90 ± 5 120 ± 10

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

Plant ploidy level Plant number Percentage

Diploid 74 73 Triploid 2 2 Tetraploid 19 19 Pentaploid 5 5 Hexaploid 1 1 Heptaploid 1 1 R 101

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