Production of haploids from anther culture of banana [Musa balbisiana (BB)]

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1 Plant Cell Rep (2003) 21: DOI /s CELL BIOLOGY AND MORPHOGENESIS A. Assani F. Bakry F. Kerbellec R. Haïcour G. Wenzel B. Foroughi-Wehr Production of haploids from anther culture of banana [Musa balbisiana (BB)] Received: 17 May 2002 / Revised: 15 October 2002 / Accepted: 19 October 2002 / Published online: 17 December 2002 Springer-Verlag 2002 Abstract We report here, for the first time, the production of haploid plants of banana Musa balbisiana (BB). Callus was induced from anthers in which the majority of the microspores were at the uninucleate stage. The frequency of callus induction was 77%. Callus proliferation usually preceded embryo formation. About 8% of the anthers developed androgenic embryos. Of the 147 plantlets obtained, 41 were haploids (n=x=11). The frequency of haploid production depended on genotypes used: 18 haploid plants were produced from genotype Pisang klutuk, 12 from Pisang batu, seven from Pisang klutuk wulung and four from Tani. The frequency of regeneration was 1.1%, which was based on the total number of anthers cultured. Diploid plants (2n=2x=22) were also observed in the regenerated plants. The haploid banana plants that were developed will be important Communicated by H. Lörz A. Assani ( ) R. Haïcour Université de Paris Sud XI, Laboratoire de Morphogenèse Végétale Expérimentale, Bâtiment 360, Orsay Cedex, France akym.assani@u-bourgogne.fr Tel.: , Fax: F. Bakry F. Kerbellec CIRAD-FLHOR, Avenue du Val Montferrand, BP 5035, Montpellier Cedex, France G. Wenzel Lehrstuhl für Pflanzenbau und Pflanzenzüchtung, Technische Universität München, Freising-Weihenstephan, Germany B. Foroughi-Wehr Bundesanstalt für Züchtungsforschung an Kulturpflanzen, Institut für Resistenzgenetik, Graf-Seinsheim-Strasse 23, Grünbach, Germany A. Assani Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l Alimentation, Laboratoire de Génie des Procédés Alimentaires et Biotechnologiques 1, Esplanade Erasme, Dijon, France material for the improvement of banana through breeding programmes. Keywords Musa balbisiana Anther culture Androgenesis Haploids Plant regeneration Abbreviations BAP: Benzylaminopurine CIRAD: Centre de coopération internationale en recherche agronomique pour le développement DAPI: 4,6-Diamidino-2-phenylindole IAA: Indole-3-acetic acid MOPS: 3-(Nmorpholino)propanesulfonic acid Introduction Haploids can result through natural parthenogenesis (development of haploid plants from unfertilised eggs) or androgenesis (development of haploid sporophytes from pollen). They can also be artificially induced through the culture of ovaries (Muren 1989), ovules (Hansen et al.1994), anthers (Foroughi-Wehr et al. 1982), microspores (Köhler and Wenzel 1985) and sexual hybridisation [e.g. cross between cultivated barley and the wild species (Kasha and Kao 1970)]. However, anther or microspore cultures have been found to be the most efficient techniques for obtaining a large number of haploid plants (De Buyser and Henry 1980). The regenerated haploid plants are generally sterile, requiring chromosome doubling for use in breeding programmes. Chromosomes can be doubled either spontaneously or artificially, and haploid plantlets are usually treated with colchicine as a means of inducing chromosome doubling (Foroughi-Wehr and Friedt 1984). Whereas many years are needed to obtain inbreds by conventional breeding methods, homozygous lines can be produced in only 1 year by chromosome doubling of haploid plantlets obtained by anther culture (Foroughi-Wehr et al. 1982; Foroughi-Wehr and Wenzel 1989). The inbred line produced by anther culture has fixed genotypes so that continued selection for an inter-

2 512 esting gene combination is not required (Wan and Widholm 1993). Since a reproducible method for producing haploid plants is available in banana, it may be used to produce haploid plants which could be used in conventional breeding programmes for banana improvement. Further, the establishment of haploid cell cultures may be useful in somatic fusion procedures (unconventional breeding) to obtain directly triploid cells starting from diploid and haploid cells. Kerbellec (1996) was first to report successful haploid plant regeneration in banana Musa acuminata (AA). However, to date, no report is available on haploid plant production in M. balbisiana (BB). In the present investigation, we report, for the first time, the successful regeneration of haploid plants (x=n=11) of four genotypes of the species M. balbisiana (BB), which is known to carry resistant genes against economically important banana diseases. Materials and methods Plant materials The plants, diploid (2n=2x=22) Pisang klutuk (BB), Pisang klutuk wulung (BB), Pisang batu (BB) and Tani (BB), all derived from the diploid wild species Musa balbisiana (BB), which contributes the B genome, were provided by CIRAD-Guadeloupe. Fig 1 A Male flower bud of banana (adapted from Champion 1963). Bar 6 cm. B Male flower of banana (adapted from Champion 1963). Bar 2cm Methods Anther cultures The anthers were isolated according to procedure described by Kerbellec (1996), with some modifications. The male flower bud (Fig. 1A), which contains male flowers in all developmental stages (immature and mature flowers), was used as donor material for anther isolation. For sterilisation, male flower buds cm in length were immersed in 95% (v/v) ethanol, followed by flaming. This sterilisation procedure was repeated three times. The bract was separated from the immature male flowers (Fig. 1B) and eliminated. An immature male flower (5 6 cm), which contained five stamens with fully developed anthers was isolated and transferred into a petri dish (diameter: 9.5 cm) containing filter paper that had been moistened with sterile water. The surrounding tepals (compound and free tepals) and the ovary on which the anthers were fixed were eliminated. One anther from each flower was squashed in 2.8% NaH 2 PO 4 buffer or 1% aceto-carmine in order to determine the appropriate microspore developmental stage, i.e. uninucleate stage (Fig 2A). Banana anthers are relatively large cm so that their isolation and transfer onto culture medium can be done with the naked eye. Ten anthers (each approx. 1 cm in length) containing microspores at the uninucleate stage were placed in a petri dish (diameter: 9.5 cm) containing 30 ml medium. The solid culture medium contained MS macro- and micro-nutrients (Murashige and Skoog1962), vitamins of Morel (Morel and Wetmore1951), 500 mg l -1 casein hydrolysate, 73 mm sucrose, 4.4 µm BAP, 2.3 µm IAA and 6 g l -1 agarose (type II; Sigma, St. Louis, Mo.). The ph of the medium was adjusted to 5.7 before autoclaving. Cultures were kept at 27 C in darkness. Anthers were maintained on the same culture medium without subculture until callus formation, which occurred after 4 months on the culture medium. Androgenic embryos were formed 6 months after the anthers had been plated. Plant regeneration Androgenic embryos were transferred onto regeneration medium containing MS macro- and micro-nutrients, vitamins of Morel, 88 mm sucrose, 2.2 µm BAP, 2.3 µm IAA, 7.5 g l -1 agarose sea plaque (Sigma) (ph 5.7). The cultures were kept at 27 C in the dark. Plant regeneration occurred 8 months after the anthers had been plated. Regenerated plantlets were transferred to solidified growth regulator-free MS medium with 1.2 mm NH 4 NO 3 (ph 5.7) and cultured at 27 C under a 16/8-h (day/night) photoperiod (light intensity: 65 µmol m -2 s -1 ). Determination of ploidy level The flow cytometric technique was used to determine the ploidy level of the anther-derived plants. Leaf pieces (1 1 cm) from in vitro plants derived from anther cultures were chopped with a razor blade in 600 µl buffer solution consisting of 45 mm MgCl 2, 30 mm Tri-sodium citrate (Na 3 C 6 H 5 O 7, 2H 2 O), 20 mm MOPS, 1% triton X-100, 10 mm sodium-metabisulfite (Na 2 S 2 O 5 ) (ph 7) to obtain a nuclei suspension. The whole sample was sieved through a 40-µm mesh nylon filter and stained with 6 µg ml -1 DAPI solution. A diploid parental plant was used as an internal standard. Nuclei analysis was done using a Partec CA II flow cytometer. Results Androgenic callus About 4 months after being transferred onto culture medium, banana anthers formed the first androgenic calli. Callus was only initiated from cultured anthers between

3 Fig. 2 A Microspores isolated from banana anthers. Bar 14 µm. B Development of callus from anthers after 5 months on induction medium. Bar 2 mm. C Androgenic embryos formed on calli. Bar 4 mm. D Haploid plantlets regenerated from anthers. Bar 3cm 513

4 514 Table 1 Frequency of embryo formation in anther culture of four genotypes of banana Musa balbisiana (BB) a Number of anthers forming callus/total number of anthers cultured b Number of embryos formed/ total number of anthers cultured Genotype Number of Anthers forming calli Embryos formed: cultured anthers Number Percentage a Number Percentage b Pisang klutuk wulung Pisang klutuk 1,650 1, Pisang batu 1, Tani ,890 2, Table 2 Frequency of plant regeneration in anther culture of four genotypes of banana Musa balbisiana (BB) a Number of plantlets/total number of anthers cultured Genotype Total number Diploid plantlets: Haploid plantlets: of plantlets Number Percentage a Number Percentage a Pisang klutuk wulung Pisang klutuk Pisang batu Tani scales 17 and 28 relative to the floral apex (floral apex has a scale of 0). Squashes of anthers ( cm) from these scales showed microspores at the uninucleate stage (Fig. 2A). A subculture of the anthers before the induction of first calli led to necrosis; therefore, a subculture of anthers before callus formation should be avoided. The calli formed were either friable or compact (Fig. 2B). There was no correlation between the type of calli and the genotype; friable and compact calli were observed in all genotypes. Anther-derived calli were produced from all four of the genotypes tested. The number of anthers producing calli was generally very high (Table 1) and appeared to be genotype-dependent. Among the genotypes studied, the best results with respect to callus formation were obtained with Pisang klutuk wulung (88.5%) and Pisang klutuk (81.5%), with the average rate being 76.8%. Androgenic embryos Androgenic embryos (Fig. 2C) were formed on calli after 6 months of anther culture. The frequency of embryo formation was found to be genotype-dependent (Table 1). The highest relative embryo number per anther was obtained with Pisang klutuk wulung (9.2%) followed by Pisang batu (8.7%), Pisang klutuk (7.3%) and Tani (4.8%). The average rate of embryos formed was 7.9% based on the total number of anthers cultured. Haploid plantlet regeneration Fig. 3 Flow scheme for anther culture of banana Plant regeneration occurred after 8 months of anther culture. Of the 147 plantlets obtained, 41 were haploid plantlets (Table 2): four (1.4%, based on the total number of anthers cultured) were from Tani, seven (1.3%) from Pisang klutuk wulung, 18 (1.1%) from Pisang klutuk and 12 (0.8%) from Pisang batu. The average relative number of haploid plantlets that developed from the cultured anthers was 1.1%. Flow cytometry analysis revealed that diploid plants were also present among the regenerated plants. The relative number of diploid plants

5 formed was 2.7%, based on the total number of anthers cultured. The haploid plants (Fig. 2D) obtained have been kept as an in vitro collection, and some were transplanted to the field for further testing. A flow scheme for anther culture of banana is presented in Fig. 3. Discussion The results of the investigation reported here show, for the first time, that haploid plants can be produced efficiently in Musa balbisiana (BB). This species plays an important role in banana breeding because it contains resistant genes against banana diseases (Fouré 1993). To the best of our knowledge, there is only one report on haploid plant regeneration in banana M. acuminata (AA) (Kerbellec 1996). We showed that callus formation was obtained if the cultured anthers contained microspores at the uninucleate stage. This has also been observed in banana M. acuminata (Kerbellec 1996) and in many other monocots like barley (Foroughi-Wehr et al. 1982), wheat (Hu and Kasha 1997), maize (Wan et al. 1991) and rice (Alemanno and Guiderdoni 1994). The average frequency of calli formed was 76.8% under our culture conditions, which is higher than that obtained for rice (20%) (Sathish et al. 1995). Some of the androgenic calli were also embryogenic. The embryos produced were similar to those obtained in previous studies on embryogenesis in banana (Assani et al. 2001, 2002). The percentage of anthers developing into embryos was 7.9%, which is similar to the value obtained for wheat (Stober and Hess 1997). The frequency of haploid plant regeneration was 1.1%. The differential response among the genotypes with respect to plant regeneration suggests that genetic factors affect anther cultures. According to Powell (1988), the genotype dependency of anther culture response is the major limitation to a wider exploitation of anther culture in breeding. However, genotype difference can be overcome by crossing a highly responsive genotype to a non-responsive genotype (Hou et al. 1994). However, this is only relevant for conventional cross breeding. The presence of diploid plants was also observed among the regenerants. While this could be a consequence of the regeneration of diploid anther tissues such as anther wall or connective tissue, in monocots the regeneration of somatic anther tissue has been very rarely reported. The other possibility is spontaneous chromosome doubling in haploid cells. The possible factors leading to spontaneous doubled-haploid plants could be nuclear fusion in the early divisions of the microspores, endomitosis, endoreduplication or multipolar mitosis during the callus phase (Chen et al.1982; De Buyser and Henry1986). Spontaneous chromosome doubling in anther cultures is considered to be advantageous because artificially induced chromosome doubling through colchicine treatment of haploid plants is then not necessary. Conclusion Haploid plant production can be achieved in banana M. balbisiana (BB). Our results show that: (1) the callus phase usually preceded embryo formation, (2) the frequency of haploid plant regeneration was genotypedependent, (3) diploid plants were present among the regenerants; this could be a result of spontaneous chromosome doubling occurring during androgenesis. The haploid plants developed here may be important for the improvement of banana through breeding programmes. Acknowledgements We thank Dr. M.V. Rajam, University of Delhi South Campus, New Delhi for reading the manuscript and Mr. D. Froger for his help with the photography. This investigation was generously supported by the European Union (INCO- DC-Contract no. IC18-CT ). 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