Influence of donor material and genotype on protoplast regeneration

Plant Science 162 (2002) 355– 362 www.elsevier.com/locate/plantsci

Influence of donor material and genotype on protoplast regeneration in banana and plantain cultivars (Musa spp.) Akym Assani a,d,*, Robert Haı¨cour a, Gerhard Wenzel c, Ba¨rbel Foroughi-Wehr d, Fre´de´ric Bakry b, Franc¸ois-Xavier Coˆte b, Georges Ducreux a, Annick Ambroise a, Agne`s Grapin b a

Laboratoire de Morphogene`se Ve´ge´tale Expe´rimentale, Uni6ersite´ de Paris Sud XI, Baˆtiment 360, F-91405 Orsay Cedex, France b CIRAD-FLHOR, A6enue du Val Montferrand, BP: 5035, F-34032 Montpellier Cedex, France c Technische Uni6ersita¨t Mu¨nchen, Lehrstuhl fu¨r Pflanzenbau und Pflanzenzu¨chtung, D-85350 Freising-Weihenstephan, Germany d Bundesanstalt fu¨r Zu¨chtungsforschung an Kulturpflanzen, Institut fu¨r Resistenzgenetik, Graf-Seinsheim-Str. 23, D-85461 Gru¨nbach, Germany Received 14 August 2001; received in revised form 23 October 2001; accepted 23 October 2001

Abstract Among the various strategies for genetic improvement in banana, somatic hybridisation appears to be a promising complement to classical breeding since protoplasts are amenable to plant regeneration. Therefore, the present investigation was undertaken to improve protoplast regeneration in banana. Protoplasts were isolated from young leaves, calli and embryogenic cell suspensions. The highest protoplast yield was obtained with cell suspensions. Mesophyll protoplasts and callus-derived protoplasts did not divide. However, protoplasts isolated from cell suspension developed into plants. Feeder cells and protoplast culture at high density were required for plant regeneration from protoplasts. Plant regeneration through somatic embryogenesis was achieved in Grande Naine and Gros Michel (Musa spp. Sub-Group ca6endish AAA), Currare Enano and Dominico (Sub-Group plantain AAB), SF265, IRFA903 and Col49 (Sub-Group AA). The frequency of embryo formation and plant regeneration was genotype-dependent. Large number of somatic embryos and regenerated plants were produced in SF265 (AA) followed by Dominico (AAB) and Grande Naine (AAA). © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Musa spp.; Protoplasts; Feeder cells; Cell suspension; Somatic embryogenesis; Plant regeneration

1. Introduction Banana and plantains (Musa spp.) are a staple food in tropical countries that ranks fourth among fruits produced in the world and second in fruit trade. Banana and plantain are threatened by numerous pathogens and pests, therefore, the creation of new banana varieties resistant to the diseases and pests with good fruit quality (better control of ripening, taste, shape, Abbre6iations: BAP, benzylaminopurine; CIRAD, centre de coope´ration internationale en recherche agronomique pour le de´veloppement; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; MES, 2-(N-morpholino) ethanesulfonic acid; NAA, a-naphthaleneacetic acid; PCV, packed cell volume. * Corresponding author. Tel.: + 33-169-1577-19; fax: +33-1698554-90. E-mail address: [email protected] (A. Assani).

colour, pulp rigidity, etc.) is important for its improvement. Genetic improvement by classical breeding methods often encounters the problem of sterility, which characterise the majority of banana and plantain genotypes. Somatic hybridisation through the fusion of protoplasts would be a potential alternate to circumvent the barriers of sterility. Somatic hybrids have been routinely produced in some important crops like potato [1–3], eggplants [4] and rape [5]. More recently, this technique has successfully been used to introduce resistant traits, including disease resistance from wild relatives to cultivated varieties [6,7]. Very few publications exist on protoplast technology in banana. Callus formation from protoplasts of cell suspensions in wild banana Long Ta6oy (AA) has been initiated [8]. Plant regeneration from embryogenic cell suspension-derived protoplasts of cooking banana

0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 6 2 - 3

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A. Assani et al. / Plant Science 162 (2002) 355–362

(Bluggoe ABB), which has only local importance has been reported for the first time [9,10]. Embryogenic cell suspensions can be established from pseudostem and rhizome tissues [11], meristematic shoot tips [12], immature zygotic embryos [13], immature male flowers [14–16] and recently immature female flowers [17]. The establishment of a reproducible regeneration protocol of protoplasts applicable to various economically important varieties is required for somatic cell fusion. We report here for the first time protoplast regeneration via somatic embryogenesis for seven banana genotypes, Grande Naine and Gros Michel (Musa spp. Sub-Group ca6endish AAA); Currare Enano and Dominico (Sub-Group plantain AAB); SF265 ; Col49 ; and IRFA903 (Sub-Group AA). Cavendishs and plantains represent the dessert and the cooking bananas, respectively, which are the most commonly produced in the world. The behaviour of protoplasts isolated from different plant tissues, calli, young leaves and cell suspensions and the genotype effects on the somatic embryogenesis of cell suspension-derived protoplasts have been investigated.

2. Materials and methods

2.1. Plant materials The triploid plants Grande Naine (AAA), Gros Michel (AAA), Currare Enano (AAB), Dominico (AAB) and diploid SF265 (AA), IRFA903 (AA), Col49 (AA), Colatino Ouro (AA), Pisang Klutuk (BB), Pisang Klutuk Wulung (BB), Pisang Batu (BB) and Tani (BB) were derived from the diploid wild species Musa acuminata (AA) which contributes the A genome and M. balbisiana (BB) which contributes the B genome. This plant material was provided by centre de coope´ ration internationale en recherche agronomique pour le de´ veloppement (CIRAD).

2.2. Initiation and maintenance of cell suspension cultures Immature male flowers were excised and cultured in M1 medium as previously described [18]. M1 medium consisted of MS [19] micro-and macronutrients and vitamins, complemented with 4.1 mM biotin, 18 mM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.7 mM a-naphthaleneacetic acid (NAA), 87 mM sucrose and 7 g l − 1 agarose Type II (Sigma) (pH 5.7). After 5– 6 months of culture, friable white and compact yellow calli were formed. Only friable calli with embryos were selected and cultured in liquid M2 medium [14], which consisted of basic MS medium with 4.1 mM biotin, 4.5 mM 2,4-D, 680 mM glutamine, 100 mg l − 1 malt extract (Sigma)

and 130 mM sucrose (pH 5.3). Cultures were kept in 250 ml Erlenmeyer flasks on a gyratory shaker at 100 rpm, in 16 h photoperiod, at 65 mmol m − 2 s − 1, 27 °C. From the callus stage, embryogenic and stable cell suspension cultures were established within 3–4 months. The cell suspensions of Grande Naine (AAA), Gros Michel (AAA), Currare Enano (AAB), Dominico (AAB), SF265 (AA), IRFA903 (AA) and Col49 (AA) were subcultured every week at 1.5% (v/v) in 50 ml M2 medium.

2.3. Callus culture and in 6itro plants The diploid in vitro plants Tani (BB) and Colatino Ouro (AA) were initiated through shoot tip cultures [12]. They were subcultured in growth regulator-free solidified MS medium with 1.2 mM NH4NO3 [20]. Haploid calli of Pisang Klutuk (B), Pisang Klutuk Wulung (B) and Pisang Batu (B) were initiated from anthers of male flowers [21]. These calli were cultured on MS medium containing 500 mg casein hydrolysate, 11.4 mM indole-3-acetic acid (IAA), 8.9 mM benzylaminopurine (BAP), Morel vitamins [22] (pH 5.7). Regenerated plants from haploid calli of Pisang Klutuk (B) were transferred onto MS medium containing 1.2 mM NH4NO3 and these plants served as the source of protoplasts. The calli or in vitro plants were transferred onto fresh medium every 4 weeks. The plants were maintained in 16 h photoperiod (65 mmol m − 2 s − 1) at 27 °C. Embryogenic calli were maintained in the same conditions but in the dark.

2.4. Isolation of protoplasts from cell suspensions Embryogenic suspension cultures of Gros Michel (AAA), Grande Naine (AAA), Currare Enano (AAB), Dominico (AAB), SF265 (AA), IRFA903 (AA), and Col49 (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 mm stainless mesh. About 2 ml enzyme solution containing 1.5% cellulase RS (Yakult Honsha Co., Tokyo, Japan), 0.15% pectolyase Y 23 (Seishin Pharmaceutical Co., Tokyo, Japan), 204 mM KCl, 67 mM CaCl2 (pH 5.6), was added to 1 ml cell suspensions. The mixture, enzyme and cell suspension were incubated overnight (12–14 h) at 27 °C without shaking.

2.5. Isolation of protoplasts from callus and lea6e Embryogenic calli or young leaves from in vitro plants (4–5 weeks old) were cut into pieces (1 mm thickness) and transferred into 150 ml Erlenmeyer flask provided with a side nozzle connected to a Millipore filter (22 mm pore size, millex GS filters, Millipore corporation). One gram of calli or leaves were mixed


with 10 ml enzyme solution. The enzyme solution used for callus digestion was composed of 1.5% cellulase RS (Yakult Honsha Co.), 0.15% pectolyase Y 23 (Seishin Pharmaceutical Co.), 1% macerozyme (Sigma), 204 mM KCl, 67 mM CaCl2 (pH 5.6). The enzyme solution used for leave digestion consisted of 1% cellulase RS, 0.15% pectolyase Y 23, 1% macerozyme, 204 mM KCl, 67 mM CaCl2, 0.5 M mannitol (pH 5.6). The mixture was incubated on a gyratory shaker (30 rpm) for 12– 14 h at 27 °C in the dark.

2.6. Purification of protoplasts The digestion mixture was filtered through 100/25 mm metallic mesh combination to remove the debris and large cell colonies. Protoplasts were washed through centrifugation at 66 g for 5 min. The pellet was washed again two times (centrifugation at 66 g for 5 min). The washing solution consisted of 204 mM KCl, 67 mM CaCl2. Protoplast viability was determined by fluorescein diacetate (FDA) [23]. Protoplasts yield was estimated using a Nageotte hematocymeter.

2.7. Culture of protoplasts To induce cell divisions in cultured protoplasts, nurse cultures have been used. They were prepared a day before the protoplast isolation. Cell suspensions of Grande Naine (AAA) and IRFA903 (AA) were used for nurse cell culture, which was prepared as follow, (1) cell suspensions were sieved through a 250 mm metallic mesh in order to select only small cell aggregates. (2) The PCM medium, which consisted of MS salts, 9 mM 2,4-D, Morel vitamins, 2.8 mM glucose, 278 mM maltose, 116 mM saccharose, 2.5 mM myo inositol (pH 5.7) was sterilised by filtration. (3) Sieved cell suspensions were mixed with 100 ml double concentrated PCM liquid medium, to obtain a final cell concentration of 10%; (4) 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 PCM medium containing nurse cells; (5) 10 –12 ml of the mixture were poured into small Petri

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dishes (5.5 cm diameter). The medium was covered with sterilised nitro-cellulose filter (AA type, Millipore corporation); (6) 0.5× 106 of freshly isolated protoplasts were suspended in 0.5 ml liquid culture medium (1.0× 106 protoplasts per ml) and transferred onto nitro-cellulose filter. The protoplast culture medium was composed of N6 salts [24], vitamins, organic acids and sugar alcohol [25], Morel vitamins, 117 mM sucrose, 0.4 M glucose, 0.5 mM 2-(N-morpholino) ethanesulfonic acid (MES), 1.9 mM KH2PO4, 2.3 mM zeatine, 0.9 mM 2,4-D and 4.4 mM NAA (pH 5.7) and sterilised by filtration. The cultures were maintained at 27 °C in the dark. Cell wall regeneration was observed with calcofluor white (fluorescent brightener) under UV microscope [26].

2.8. Somatic embryogenesis Protoplast-derived microcalli were individually picked up from feeder layer and gently transferred onto regeneration medium A0.4B0.5 containing MS salts, Morel vitamins, 88 mM saccharose, 2.3 mM IAA, 2.2 mM BAP, and 7.5 g l − 1 agarose sea plaque (pH 5.7). 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 (pH 5.7). The plants were cultured under 16 h photoperiod (65 mmol m − 2 s − 1) at 27 °C. After reaching the size of 10 cm, the plants were transplanted in soil for field experimentation.

3. Results

3.1. Isolation of protoplasts The highest yield of protoplasts was obtained when the donor material was cell suspension (Fig. 1A). Among the cell suspensions studied, Dominico (AAB; 31.4×106 protoplasts per ml packed cell volume (PCV)), SF265 (AA; 28.4×106 protoplasts per ml PCV) and Grande Naine (AAA; 27.5× 106 protoplasts per ml PCV) gave the best results (Table 1). The viability range of freshly isolated suspension proto-

Table 1 Characterisation of cell suspension-derived-protoplasts Genotype

Grande Naine

Gros Michel

Currare Enano

Dominico

IRFA903

SF265

Col49

Genome Number (106 ml−1)a Viability (%)b Size (mm)

AAA 27.5 9 6.4 85.3 93.8 15–25

AAA 9.09 5.1 89.4 94.6 10–20

AAB 7.7 9 4.1 76.8 95.1 15–20

AAB 31.4 912.6 91.0 92.0 15–20

AA 20.0 9 14.7 82.4 9 1.7 10–25

AA 28.4 919.5 71.0 93.4 10–25

AA 10.09 3.5 84.69 1.3 10–22

The data was based on three independent experiments with two to three replicates. a The number of protoplasts isolated per ml cell suspension. b Protoplast viability was checked 1 h after isolation.

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Fig. 1. (A) Freshly isolated cell suspension-derived protoplasts of SF265 (AA). Bar, 30 mm. (B) Freshly isolated protoplasts from leaves of Tani (BB). Bar, 25 mm. (C) Microcalli from cell suspensions-derived protoplasts of SF265 (AA). Bar, 120 mm. (D) Microcalli from cell suspensionderived protoplasts on feeder layer IRFA903 (AA). Bar, 1.7 cm. (E) Somatic embryos from protoplasts of Grande Naine (AAA). Bar, 10 mm. (F) Somatic embryos of IRFA903 (AA). Bar, 5 mm. (G) Regenerated plants from protoplast cultures of Grande Naine (AAA). Bar, 5 mm. (H) Protoplast-derived plants of Grande Naine (AAA) in greenhouse.


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Table 2 Characterisation of protoplasts isolated from calli (c) and leaves (l) Genotype

Pisang Klutuk (c)

Pisang Klutuk Wulung (c)

Pisang Batu (c)

Pisang Klutuk (l)

Tani (l)

Colatino Ouro (l)

Genome Number (106 g−1)a Viability (%)b Size (mm)

B 1.79 0.2 309 1.7 15–20

B 2.4 90.6 30 92.2 15–25

B 2.8 90.4 35 95.7 10–25

B 1.1 9 0.7 31 96.0 10–20

BB 1.2 90.9 40 93.5 12–20

AA 2.1 91.3 27 9 3.5 10–22

The data was based on three independent experiments with two to three replicates. a The number of protoplasts isolated per g calli (c) or leaves (l). b Protoplast viability was observed 1 h after protoplast isolation.

plasts was 71–91%. The yield of protoplasts from leaves or calli (Fig. 1B) did not exceed 2.8× 106 protoplasts per g leaves or calli (Pisang Batu (B)) (Table 2). The viability of newly isolated protoplasts from leaves and calli was low (27– 40%) as compared with those isolated from cell suspensions. Mesophyll protoplasts (85% with a diameter of 20 mm, 10% 10– 15 mm, 5% 22 mm) were more uniform in size than suspension or callus protoplasts (69% with a diameter of 20– 25 mm, 17% 10 mm, 10% 15 mm and 4% 12 mm).

3.2. Culture of protoplasts Protoplasts from leaves or calli did not divide, however, they were alive for 3 weeks while on the feeder layer. Only cell suspension-derived protoplasts were able to divide. The first cell divisions occurred on feeder layers 6–8 days after initiation of protoplast cultures. To optimise cell division rate and the formation of microcalli from cell suspension derived-protoplasts, nurse cells of either Grande Naine (AAA) and IRFA903 (AA) were tested. In all cases, the best results were obtained with Grande Naine (AAA) as the feeder layer (Table 3). Depending on the genotype used, the number of cell divisions per Petri dish was 45– 125× 103 (9– 25% of the number of protoplasts plated) on Grande Naine (AAA) nurse cells, and only 10– 45 × 103 (2 –9% protoplasts plated) with IRFA903 (AA). Microcallus formation (Fig. 1C and D) was observed 14 –21 days after initiation of protoplast culture. The number of microcalli formed per Petri dish was 9– 43 × 103 (1.8–8.6% protoplasts plated) on nurse cells Grande Naine (AAA) and 1.6 – 4.7 × 103 (0.3 – 0.9% protoplasts plated) on IRFA903 (AA) (Table 3). The highest number of cell divisions was observed with protoplasts of genotype SF265 (AA) (125×103 per Petri dish) and Grande Naine (AAA) (80 × 103 per Petri dish) on Grande Naine (AAA) as nurse cells.

3.3. Somatic embryogenesis Microcalli were individually picked up on feeder cells and transferred onto regeneration A0.4B0.5 soli-

dified medium. The first somatic embryos (Fig. 1E and F) were observed 8–10 weeks after initiation of protoplast cultures. The total number of embryos formed depended on the genotype used. SF265 (AA) produced the highest number of embryos (4800 embryos) followed by Grande Naine (AAA; 3900 embryos) and Dominico (AAB; 3600 embryos) (Table 4).

3.4. Regeneration of plants Somatic embryos were transferred onto the same regeneration solidified A0.4B0.5 medium. Plants were observed 11–12 weeks after the initiation of protoplast culture. Out of 13 360 plants obtained (Fig. 1G and H), 3290 belongs to genotype SF265 (AA), 2250 to Grande Naine (AAA), 1830 to Gros Michel (AAA), 1740 to Col49 (AA), 1550 to Dominico (AAB), 1450 to Currare Enano (AAB) and 1250 to IRFA903 (AA; Table 4).

4. Discussion In the present study, a successful plant regeneration protocol from protoplasts has been established and extended to seven other banana and plantain genotypes, Grande Naine and Gros Michel (Sub-Group ca6endish AAA), Dominico and Currare Enano (SubGroup plantain AAB), SF265, IRFA903 and Col49 (Sub-Group AA). Previously, protoplast regeneration was described only for Bluggoe (Musa spp. Sub-Group ABB) [9,10]. Protoplast donor material and genotype have been investigated regarding their influence on the efficiency of isolation and development of protoplasts. The isolation of protoplasts in banana depends significantly on both donor material and genotype used. It was essential to design an enzymatic mixture for each donor material (calli, leaves and cell suspensions) in order to optimise the yield of protoplasts. This suggested that cell wall components are different depending of donor material. The higher yield of cell suspension-derived protoplasts and their higher viability rate as compared


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with those of callus and mesophyll protoplasts showed that the appropriate donor material for efficient protoplast isolation in banana are cell suspension cultures. In other monocotyledons such as rice [27], maize [28], wheat [29] and barley [30], cell suspension cultures have been mainly used as the source for protoplast isolation. Unfortunately, the induction of embryogenic suspension cultures in banana requires more than 1 year [12], and so far the number of the genotypes which can be used to initiate suspension cultures is limited [14,13,15– 17]. Among the genotypes tested, Dominico (AAB), SF265 (AA) and Grande Naine (AAA) suspensions produced the highest yield of protoplasts. The best results obtained with these genotypes may be correlated with their rapidly growing cell suspensions and their fine structures. Protoplast yield of Duboisia could be increased by using cell suspensions with small cell aggregates and high division rate [31]. The poor yield of mesophyll or callus-derived protoplasts and their lower viability rate could be linked to the sensitivity of leaves and callus tissues to enzymatic stress, resulting in more damage during enzymatic maceration. For some interesting genotypes (for example Pisang Klutuk (BB), Pisang Batu (BB)) in which embryogenic and stable cell suspensions are not available or difficult to establish, calli and leaves may be actually a suitable alternative to cell suspensions for protoplast isolation for somatic fusion experiments. In our investigation, in contrast to leave and callus-derived protoplasts, cell divisions were only obtained with cell suspension-derived protoplasts. Except in the case of rice [32], successful culture from mesophyll protoplasts has not been reported in monocotyledons. Callus has not produced regenerable

protoplasts indicating that active cell divisions do not occur from callus derived-protoplasts. For induction and sustained cell divisions from cell suspension derived-protoplasts, feeder cells were required. The best response was obtained by using Grande Naine (AAA) as nurse culture; this could be due to the higher mitotic activity of this cell line as compared with IRFA903 (AA). The use of feeder cells to induce cell division from protoplasts was previously reported in barley [30], maize [33] and banana [8–10], which are known to be recalcitrant to protoplast culture. Somatic embryos were obtained in all genotypes in which cell divisions were observed. However, the genotypes SF265 (AA), Grande Naine (AAA) and Dominico (AAB) produced the highest rate of somatic embryos and plantlets.

5. Conclusion A reproducible and efficient protoplast regeneration protocol has been developed which allowed us to regenerate seven new banana genotypes. There is a close correlation between the quality of cell suspensions, cell division rate, number of microcalli formed, number of embryos and number of regenerating plants. The regenerated plants will be evaluated in the field with respect to fruit quality, yield, etc. Since protoplast regeneration is a prerequisite for somatic hybridisation, which could be an important alternate tool to breeding to produce new cultivars, the present efficient regeneration from protoplasts would be important for banana improvement through protoplast fusion.

Table 3 Effect of feeder layer source (Grande Naine and IRFA903 ) on cell division and microcolony formation from cell suspension-derived protoplasts Genotype

Genome

Feeder layer source

Number of cell divisions (103) per Petri dish

Number of Microcalli (103) per Petri dish

Grande Naine

AAA

Gros Michel

AAA

Currare Enano

AAB

Dominico

AAB

SF265

AA

Col49

AA

IRFA903

AA

Grande Naine IRFA903 Grande Naine IRFA903 Grande Naine IRFA903 Grande Naine IRFA903 Grande Naine IRFA903 Grande Naine IRFA903 Grande Naine IRFA903

8095.2 209 3.0 7093.4 1295.0 629 2.0 209 4.0 549 5.7 309 6.0 1259 5.0 459 7.0 459 8.9 109 3.6 609 3.5 359 9.5

18 9 2.1 1.6 9 0.4 10 9 1.5 1.89 0.3 15 9 2.7 2.1 9 0.3 12 92.5 1.8 9 0.3 43 9 6.2 2.3 90.6 9 9 2.0 1.4 9 0.4 15 9 2.5 4.79 0.3

The data was based on two independent experiments with three replicates. Each replicate consisted of a Petri dish containing 0.5×106 protoplasts.


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Table 4 Total number of plants regenerated from cell suspension-derived protoplasts Genotype

Genome

Number of embryos

Number of plants

Rate of plant regeneration (%)

Grande Naine Gros Michel Currare Enano Dominico SF265 Col49 IRFA903

AAA AAA AAB AAB AA AA AA

3900 2400 2700 3600 4800 3400 2500 23 300

2250 1830 1450 1550 3290 1740 1250 13 360

57.7 76.3 53.7 43.1 68.5 51.2 50.0 57.2

The number of protoplasts plated was 1.5×106 (three Petri dishes) per genotype

Acknowledgements This work was supported by European Union (INCO-DC-Contract No. IC18-CT97-02-04). We thank Dr M.V. Rajam for their comments on the manuscripts.

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