Plant Science 162 (2002) 355–362

Influence of donor material and genotype on protoplastregeneration in banana and plantain cultivars (Musa spp.)

Akym Assani a,d,*, Robert Haıcour a, Gerhard Wenzel c, Barbel Foroughi-Wehr d,Frederic Bakry b, Francois-Xavier Cote b, Georges Ducreux a, Annick Ambroise a,

Agnes Grapin b

a Laboratoire de Morphogenese Vegetale Experimentale, Uni�ersite de Paris Sud XI, Batiment 360, F-91405 Orsay Cedex, Franceb CIRAD-FLHOR, A�enue du Val Montferrand, BP: 5035, F-34032 Montpellier Cedex, France

c Technische Uni�ersitat Munchen, Lehrstuhl fur Pflanzenbau und Pflanzenzuchtung, D-85350 Freising-Weihenstephan, Germanyd Bundesanstalt fur Zuchtungsforschung an Kulturpflanzen, Institut fur Resistenzgenetik, Graf-Seinsheim-Str. 23, D-85461 Grunbach, 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 complementto classical breeding since protoplasts are amenable to plant regeneration. Therefore, the present investigation was undertaken toimprove 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 notdivide. However, protoplasts isolated from cell suspension developed into plants. Feeder cells and protoplast culture at highdensity were required for plant regeneration from protoplasts. Plant regeneration through somatic embryogenesis was achieved inGrande Naine and Gros Michel (Musa spp. Sub-Group ca�endish 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-depen-dent. Large number of somatic embryos and regenerated plants were produced in SF265 (AA) followed by Dominico (AAB) andGrande Naine (AAA). © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Musa spp.; Protoplasts; Feeder cells; Cell suspension; Somatic embryogenesis; Plant regeneration

www.elsevier.com/locate/plantsci

1. Introduction

Banana and plantains (Musa spp.) are a staple foodin tropical countries that ranks fourth among fruitsproduced in the world and second in fruit trade. Ba-nana and plantain are threatened by numerous patho-gens and pests, therefore, the creation of new bananavarieties resistant to the diseases and pests with goodfruit quality (better control of ripening, taste, shape,

colour, pulp rigidity, etc.) is important for its improve-ment. Genetic improvement by classical breeding meth-ods often encounters the problem of sterility, whichcharacterise the majority of banana and plantain geno-types. Somatic hybridisation through the fusion of pro-toplasts would be a potential alternate to circumventthe barriers of sterility. Somatic hybrids have beenroutinely produced in some important crops like potato[1–3], eggplants [4] and rape [5]. More recently, thistechnique has successfully been used to introduce resis-tant traits, including disease resistance from wild rela-tives to cultivated varieties [6,7].

Very few publications exist on protoplast technologyin banana. Callus formation from protoplasts of cellsuspensions in wild banana Long Ta�oy (AA) has beeninitiated [8]. Plant regeneration from embryogenic cellsuspension-derived protoplasts of cooking banana

Abbre�iations: BAP, benzylaminopurine; CIRAD, centre de coop-eration internationale en recherche agronomique pour le developpe-ment; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-aceticacid; MES, 2-(N-morpholino) ethanesulfonic acid; NAA, �-naph-thaleneacetic acid; PCV, packed cell volume.

* Corresponding author. Tel.: +33-169-1577-19; fax: +33-169-8554-90.

E-mail address: akym.assani@mve.u-psud.fr (A. Assani).

0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 6 8 -9452 (01 )00562 -3

A. Assani et al. / Plant Science 162 (2002) 355–362356

(Bluggoe ABB), which has only local importance hasbeen reported for the first time [9,10].

Embryogenic cell suspensions can be establishedfrom pseudostem and rhizome tissues [11], meristematicshoot tips [12], immature zygotic embryos [13], imma-ture male flowers [14–16] and recently immature femaleflowers [17]. The establishment of a reproducible regen-eration protocol of protoplasts applicable to variouseconomically important varieties is required for somaticcell fusion.

We report here for the first time protoplast regenera-tion via somatic embryogenesis for seven banana geno-types, Grande Naine and Gros Michel (Musa spp.Sub-Group ca�endish AAA); Currare Enano and Do-minico (Sub-Group plantain AAB); SF265; Col49; andIRFA903 (Sub-Group AA). Cavendishs and plantainsrepresent the dessert and the cooking bananas, respec-tively, which are the most commonly produced in theworld. The behaviour of protoplasts isolated from dif-ferent plant tissues, calli, young leaves and cell suspen-sions and the genotype effects on the somaticembryogenesis of cell suspension-derived protoplastshave been investigated.

2. Materials and methods

2.1. Plant materials

The triploid plants Grande Naine (AAA), GrosMichel (AAA), Currare Enano (AAB), Dominico (AAB)and diploid SF265 (AA), IRFA903 (AA), Col49 (AA),Colatino Ouro (AA), Pisang Klutuk (BB), Pisang KlutukWulung (BB), Pisang Batu (BB) and Tani (BB) werederived from the diploid wild species Musa acuminata(AA) which contributes the A genome and M. bal-bisiana (BB) which contributes the B genome. Thisplant material was provided by centre de cooperationinternationale en recherche agronomique pour le devel-oppement (CIRAD).

2.2. Initiation and maintenance of cell suspensioncultures

Immature male flowers were excised and cultured inM1 medium as previously described [18]. M1 mediumconsisted of MS [19] micro-and macronutrients andvitamins, complemented with 4.1 �M biotin, 18 �M2,4-dichlorophenoxyacetic acid (2,4-D), 5.7 �M �-naph-thaleneacetic acid (NAA), 87 mM sucrose and 7 g l−1

agarose Type II (Sigma) (pH 5.7). After 5–6 months ofculture, friable white and compact yellow calli wereformed. Only friable calli with embryos were selectedand cultured in liquid M2 medium [14], which consistedof basic MS medium with 4.1 �M biotin, 4.5 �M 2,4-D,680 �M glutamine, 100 mg l−1 malt extract (Sigma)

and 130 mM sucrose (pH 5.3). Cultures were kept in250 ml Erlenmeyer flasks on a gyratory shaker at 100rpm, in 16 h photoperiod, at 65 �mol m−2 s−1, 27 °C.From the callus stage, embryogenic and stable cellsuspension cultures were established within 3–4months. 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 �itro plants

The diploid in vitro plants Tani (BB) and ColatinoOuro (AA) were initiated through shoot tip cultures[12]. They were subcultured in growth regulator-freesolidified MS medium with 1.2 mM NH4NO3 [20].Haploid calli of Pisang Klutuk (B), Pisang KlutukWulung (B) and Pisang Batu (B) were initiated fromanthers of male flowers [21]. These calli were culturedon MS medium containing 500 mg casein hydrolysate,11.4 �M indole-3-acetic acid (IAA), 8.9 �M benzy-laminopurine (BAP), Morel vitamins [22] (pH 5.7).Regenerated plants from haploid calli of Pisang Klutuk(B) were transferred onto MS medium containing 1.2mM NH4NO3 and these plants served as the source ofprotoplasts. The calli or in vitro plants were transferredonto fresh medium every 4 weeks. The plants weremaintained in 16 h photoperiod (65 �mol m−2 s−1) at27 °C. Embryogenic calli were maintained in the sameconditions 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), andCol49 (AA), 3–4 days after the last subculture, wereused as donor material for the isolation of protoplasts.The cell suspensions were sieved through 200 �m stain-less 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, en-zyme and cell suspension were incubated overnight(12–14 h) at 27 °C without shaking.

2.5. Isolation of protoplasts from callus and lea�e

Embryogenic calli or young leaves from in vitroplants (4–5 weeks old) were cut into pieces (1 mmthickness) and transferred into 150 ml Erlenmeyer flaskprovided with a side nozzle connected to a Milliporefilter (22 �m pore size, millex GS filters, Milliporecorporation). One gram of calli or leaves were mixed

A. Assani et al. / Plant Science 162 (2002) 355–362 357

with 10 ml enzyme solution. The enzyme solution usedfor callus digestion was composed of 1.5% cellulase RS(Yakult Honsha Co.), 0.15% pectolyase Y 23 (SeishinPharmaceutical Co.), 1% macerozyme (Sigma), 204 mMKCl, 67 mM CaCl2 (pH 5.6). The enzyme solution usedfor leave digestion consisted of 1% cellulase RS, 0.15%pectolyase Y 23, 1% macerozyme, 204 mM KCl, 67mM CaCl2, 0.5 M mannitol (pH 5.6). The mixture wasincubated on a gyratory shaker (30 rpm) for 12–14 h at27 °C in the dark.

2.6. Purification of protoplasts

The digestion mixture was filtered through 100/25 �mmetallic mesh combination to remove the debris andlarge cell colonies. Protoplasts were washed throughcentrifugation at 66 g for 5 min. The pellet was washedagain two times (centrifugation at 66 g for 5 min). Thewashing solution consisted of 204 mM KCl, 67 mMCaCl2. Protoplast viability was determined by fluores-cein diacetate (FDA) [23]. Protoplasts yield was esti-mated using a Nageotte hematocymeter.

2.7. Culture of protoplasts

To induce cell divisions in cultured protoplasts, nursecultures have been used. They were prepared a daybefore the protoplast isolation. Cell suspensions ofGrande Naine (AAA) and IRFA903 (AA) were used fornurse cell culture, which was prepared as follow, (1) cellsuspensions were sieved through a 250 �m metallicmesh in order to select only small cell aggregates. (2)The PCM medium, which consisted of MS salts, 9 �M2,4-D, Morel vitamins, 2.8 mM glucose, 278 mM mal-tose, 116 mM saccharose, 2.5 mM myo inositol (pH5.7) was sterilised by filtration. (3) Sieved cell suspen-sions were mixed with 100 ml double concentratedPCM liquid medium, to obtain a final cell concentra-tion of 10%; (4) 1.2 g agarose sea plaque (Sigma) wasdissolved in 100 ml double distilled water and thenautoclaved (pH 5.7). When the temperature of agarosesolution decreased to 30–35 °C, it was gently mixedwith 100 ml PCM medium containing nurse cells; (5)10–12 ml of the mixture were poured into small Petri

dishes (5.5 cm diameter). The medium was covered withsterilised nitro-cellulose filter (AA type, Millipore cor-poration); (6) 0.5×106 of freshly isolated protoplastswere suspended in 0.5 ml liquid culture medium (1.0×106 protoplasts per ml) and transferred onto nitro-cellu-lose filter. The protoplast culture medium wascomposed of N6 salts [24], vitamins, organic acids andsugar alcohol [25], Morel vitamins, 117 mM sucrose,0.4 M glucose, 0.5 mM 2-(N-morpholino) ethanesul-fonic acid (MES), 1.9 mM KH2PO4, 2.3 �M zeatine, 0.9�M 2,4-D and 4.4 �M NAA (pH 5.7) and sterilised byfiltration. The cultures were maintained at 27 °C in thedark. Cell wall regeneration was observed with cal-cofluor white (fluorescent brightener) under UV micro-scope [26].

2.8. Somatic embryogenesis

Protoplast-derived microcalli were individuallypicked up from feeder layer and gently transferred ontoregeneration medium A0.4B0.5 containing MS salts,Morel vitamins, 88 mM saccharose, 2.3 �M IAA, 2.2�M 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 solidifiedgrowth regulator-free MS medium with 1.2 mMNH4NO3 (pH 5.7). The plants were cultured under 16 hphotoperiod (65 �mol m−2 s−1) at 27 °C. After reach-ing the size of 10 cm, the plants were transplanted insoil for field experimentation.

3. Results

3.1. Isolation of protoplasts

The highest yield of protoplasts was obtained whenthe 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 mlPCV) and Grande Naine (AAA; 27.5×106 protoplastsper ml PCV) gave the best results (Table 1). Theviability range of freshly isolated suspension proto-

Table 1Characterisation of cell suspension-derived-protoplasts

Genotype Grande Naine Gros Michel Currare Enano Col49Dominico IRFA903 SF265

AAAAAABAABAAA AAAAAGenome7.7�4.19.0�5.127.5�6.4 31.4�12.6Number (106 ml−1)a 10.0�3.528.4�19.520.0�14.7

Viability (%)b 84.6�1.371.0�3.482.4�1.791.0�2.076.8�5.189.4�4.685.3�3.8Size (�m) 15–2010–20 10–2210–2510–2515–25 15–20

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.

A. Assani et al. / Plant Science 162 (2002) 355–362358

Fig. 1. (A) Freshly isolated cell suspension-derived protoplasts of SF265 (AA). Bar, 30 �m. (B) Freshly isolated protoplasts from leaves of Tani(BB). Bar, 25 �m. (C) Microcalli from cell suspensions-derived protoplasts of SF265 (AA). Bar, 120 �m. (D) Microcalli from cell suspension-derived 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.

A. Assani et al. / Plant Science 162 (2002) 355–362 359

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

Pisang Klutuk Wulung (c)Genotype Pisang Batu (c)Pisang Klutuk (c) Pisang Klutuk (l) Tani (l) Colatino Ouro (l)

B BGenome BB BB AA2.4�0.6 2.8�0.4 1.1�0.7 1.2�0.9Number (106 g−1)a 2.1�1.31.7�0.230�2.2 35�5.7 31�6.030�1.7 40�3.5Viability (%)b 27�3.5

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

3.2. Culture of protoplasts

Protoplasts from leaves or calli did not divide, how-ever, they were alive for 3 weeks while on the feederlayer. Only cell suspension-derived protoplasts wereable to divide. The first cell divisions occurred onfeeder layers 6–8 days after initiation of protoplastcultures. To optimise cell division rate and the forma-tion of microcalli from cell suspension derived-proto-plasts, nurse cells of either Grande Naine (AAA) andIRFA903 (AA) were tested. In all cases, the best resultswere obtained with Grande Naine (AAA) as the feederlayer (Table 3). Depending on the genotype used, thenumber of cell divisions per Petri dish was 45–125×103 (9–25% of the number of protoplasts plated) onGrande 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. Thenumber of microcalli formed per Petri dish was 9–43×103 (1.8–8.6% protoplasts plated) on nurse cellsGrande Naine (AAA) and 1.6–4.7×103 (0.3–0.9% pro-toplasts plated) on IRFA903 (AA) (Table 3). Thehighest number of cell divisions was observed withprotoplasts of genotype SF265 (AA) (125×103 perPetri dish) and Grande Naine (AAA) (80×103 perPetri dish) on Grande Naine (AAA) as nurse cells.

3.3. Somatic embryogenesis

Microcalli were individually picked up on feedercells and transferred onto regeneration A0.4B0.5 soli-

dified medium. The first somatic embryos (Fig. 1E andF) were observed 8–10 weeks after initiation of proto-plast cultures. The total number of embryos formeddepended on the genotype used. SF265 (AA) producedthe highest number of embryos (4800 embryos) fol-lowed by Grande Naine (AAA; 3900 embryos) andDominico (AAB; 3600 embryos) (Table 4).

3.4. Regeneration of plants

Somatic embryos were transferred onto the sameregeneration solidified A0.4B0.5 medium. Plants wereobserved 11–12 weeks after the initiation of protoplastculture. Out of 13 360 plants obtained (Fig. 1G andH), 3290 belongs to genotype SF265 (AA), 2250 toGrande Naine (AAA), 1830 to Gros Michel (AAA),1740 to Col49 (AA), 1550 to Dominico (AAB), 1450 toCurrare Enano (AAB) and 1250 to IRFA903 (AA;Table 4).

4. Discussion

In the present study, a successful plant regenerationprotocol from protoplasts has been established andextended to seven other banana and plantain geno-types, Grande Naine and Gros Michel (Sub-Groupca�endish AAA), Dominico and Currare Enano (Sub-Group plantain AAB), SF265, IRFA903 and Col49(Sub-Group AA). Previously, protoplast regenerationwas described only for Bluggoe (Musa spp. Sub-GroupABB) [9,10].

Protoplast donor material and genotype have beeninvestigated regarding their influence on the efficiencyof isolation and development of protoplasts. The isola-tion of protoplasts in banana depends significantly onboth donor material and genotype used. It was essen-tial to design an enzymatic mixture for each donormaterial (calli, leaves and cell suspensions) in order tooptimise the yield of protoplasts. This suggested thatcell wall components are different depending of donormaterial. The higher yield of cell suspension-derivedprotoplasts and their higher viability rate as compared

A. Assani et al. / Plant Science 162 (2002) 355–362360

with those of callus and mesophyll protoplastsshowed that the appropriate donor material for effi-cient protoplast isolation in banana are cell suspen-sion cultures. In other monocotyledons such as rice[27], maize [28], wheat [29] and barley [30], cell sus-pension cultures have been mainly used as the sourcefor protoplast isolation. Unfortunately, the inductionof embryogenic suspension cultures in banana re-quires more than 1 year [12], and so far the numberof the genotypes which can be used to initiate suspen-sion cultures is limited [14,13,15–17]. Among thegenotypes tested, Dominico (AAB), SF265 (AA) andGrande Naine (AAA) suspensions produced thehighest yield of protoplasts. The best results obtainedwith these genotypes may be correlated with theirrapidly growing cell suspensions and their fine struc-tures. Protoplast yield of Duboisia could be increasedby using cell suspensions with small cell aggregatesand high division rate [31].

The poor yield of mesophyll or callus-derived pro-toplasts and their lower viability rate could be linkedto the sensitivity of leaves and callus tissues to enzy-matic stress, resulting in more damage during enzy-matic maceration. For some interesting genotypes (forexample Pisang Klutuk (BB), Pisang Batu (BB)) inwhich embryogenic and stable cell suspensions are notavailable or difficult to establish, calli and leaves maybe actually a suitable alternative to cell suspensionsfor protoplast isolation for somatic fusion experi-ments. In our investigation, in contrast to leave andcallus-derived protoplasts, cell divisions were only ob-tained with cell suspension-derived protoplasts. Exceptin the case of rice [32], successful culture from meso-phyll protoplasts has not been reported in mono-cotyledons. Callus has not produced regenerable

protoplasts indicating that active cell divisions do notoccur from callus derived-protoplasts. For inductionand sustained cell divisions from cell suspensionderived-protoplasts, feeder cells were required. Thebest response was obtained by using Grande Naine(AAA) as nurse culture; this could be due to thehigher mitotic activity of this cell line as comparedwith IRFA903 (AA). The use of feeder cells to inducecell division from protoplasts was previously reportedin barley [30], maize [33] and banana [8–10], whichare known to be recalcitrant to protoplast culture.Somatic embryos were obtained in all genotypes inwhich cell divisions were observed. However, thegenotypes SF265 (AA), Grande Naine (AAA) and Do-minico (AAB) produced the highest rate of somaticembryos and plantlets.

5. Conclusion

A reproducible and efficient protoplast regenerationprotocol has been developed which allowed us to re-generate seven new banana genotypes. There is aclose correlation between the quality of cell suspen-sions, cell division rate, number of microcalli formed,number of embryos and number of regeneratingplants. The regenerated plants will be evaluated in thefield with respect to fruit quality, yield, etc. Sinceprotoplast regeneration is a prerequisite for somatichybridisation, which could be an important alternatetool to breeding to produce new cultivars, the presentefficient regeneration from protoplasts would be im-portant for banana improvement through protoplastfusion.

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

Number of Microcalli (103) per Petri dishGenotype Number of cell divisions (103) per Petri dishGenome Feeder layersource

Grande Naine Grande Naine 80�5.2AAA 18�2.1IRFA903 20�3.0 1.6�0.4

AAA 10�1.5Gros Michel 70�3.4Grande Naine1.8�0.312�5.0IRFA90315�2.762�2.0Grande NaineAABCurrare Enano

IRFA903 20�4.0 2.1�0.3AAB Grande NaineDominico 54�5.7 12�2.5

1.8�0.330�6.0IRFA903SF265 43�6.2AA Grande Naine 125�5.0

2.3�0.6IRFA903 45�7.09�2.045�8.9Grande NaineCol49 AA

1.4�0.4IRFA903 10�3.6Grande NaineAA 15�2.5IRFA903 60�3.5

4.7�0.335�9.5IRFA903

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

A. Assani et al. / Plant Science 162 (2002) 355–362 361

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

Number of embryos Number of plantsGenotype Rate of plant regeneration (%)Genome

3900Grande Naine 2250AAA 57.72400 1830AAA 76.3Gros Michel2700 1450Currare Enano 53.7AAB3600 1550AAB 43.1Dominico

SF265 AA 4800 3290 68.53400 1740AA 51.2Col492500 1250IRFA903 50.0AA

23 300 13 360 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 thankDr M.V. Rajam for their comments on themanuscripts.

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