Isolation and culture of protoplasts from anthers of apple (Malus pumila Mill.) rootstock ‘M9’ By M. F. ABREU-TARAZI, L. R. LEITE, A. C. M. DANTAS and M. P. GUERRA* Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Caixa Postal 476 CEP 88.040-900, Florianópolis, SC, Brazil (e-mail: mpguerra@cca.ufsc.br) (Accepted 4 May 2009) SUMMARY This report describes a reliable method for callus induction, isolation, and the culture of anther-derived protoplasts from the apple rootstock, ‘M9’, and provides information on the development of a protoplast-to-plant regeneration system. Callus induction occurred in anthers when cultivated on Nitsch and Nitsch (NN) or on AT3 media. Suspension cells were established from friable, anther-derived callus formed on NN basal medium. Protoplasts were isolated from callus and anther-derived suspension cells.The highest viability value for freshly isolated protoplasts (62.67 ± 1.53%) was achieved using a 4 h digestion period with suspension cells on Kao and Michayluk (KM) basal medium diluted in an enzyme solution primarily composed of [1% (w/v)] cellulase ‘Onozuka’ RS in combination with [1% (w/v)] macerase and [2% (w/v)] pectolyse Y-23. Protoplasts were cultured using three methods: agarose bead, hanging drop, or liquid culture. The first protoplast division was observed after 8 d of cultivation. The liquid culture method led to sustained division of callus and suspension-derived protoplasts. Micro-colonies were formed within 21 d, and microcalli were observed after 28 d of cultivation. I n vitro production of haploid plants associated with protoplast techniques is an important tool for breeders. Protoplast fusion provides opportunities for bringing together the genomes of taxonomically divergent species that cannot be combined sexually due to incompatibility barriers (Liu et al., 2007). Through the fusion of haploid and diploid protoplasts, new forms of intra- and inter-specific hybrids, with lower ploidy levels, have been obtained. The generation of gameto-somatic hybrid plants also has considerable potential in terms of gene introgression (Davey et al., 2005). However, due to the general difficulty of obtaining haploid plants from anther-derived protoplasts (Davey et al., 2005), and despite the success of gene-transfer protocols for many crops (Pati et al., 2004), this technology has received a setback and limited attention. At the same time, recent studies related to the production of somatic hybrids have revealed the potential role of this technique in enriching plant germplasm, as well as for the production of novel varieties (Ananthakrishnan et al., 2006; Trojak-Goluch and Berbe´ c, 2007; Yermishin et al., 2008). The application of somatic hybridisation may widen the gene pool available for apple breeding and improvement, and may be particularly important for one of the most important dwarfing apple rootstocks used worldwide, the cultivar ‘M9’ (Malus pumila Mill.; Höhnle and Weber, 2007). ‘M9’ rootstock presents a long reproductive cycle, a high degree of heterozygosity, and frequent self-incompatibility (Zhang and Lespinasse, 1992), which place limitations on the use of conventional breeding methods. However, the establishment of a protocol for haploid protoplast isolation and culture is a prerequisite for an efficient somatic hybridisation programme. Haploid production through apple anther culture has shown low levels of induction of embryogenesis, and is considered highly genotype-dependent (Höfer, 1997). Efforts to regenerate apple plants by means of this technique have been performed since the 1970s, but successful friable anther-derived calli and plant regeneration were described mostly in Malus pumila Mill. and M.  domestica Borkh. (Patat-Ochatt et al., 1993; Germanà, 2006). Despite many studies, only Niizeki et al. (1983) have reported an efficient system that allowed protoplast isolation from haploid callus derived from anther cultures of apple. The aim of this study was to establish a reliable protocol for the isolation and culture of anther-derived protoplasts from the apple rootstock, ‘M9’. MATERIALS AND METHODS Plant material Experiments were carried out using the apple rootstock ‘M9’ (Malus pumila Mill.) from the Experimental Station of Santa Catarina Agricultural Research and Rural Extension Enterprise (EPAGRI), Sáo Joaquim, Santa Cata, Brazil. Branches with flower buds were collected after dormancy break, before the petals turned pink, and transported to the laboratory where they were stored in the dark at 4ºC for 10 d. Buds were surface-sterilised with 96% (v/v) ethanol for 20 min, followed by 15% (w/v) sodium hypochlorite solution [4% (w/v) available chlorine] for 15 min, then rinsed in sterile distilled water. Anthers were excised *Author for correspondence. Journal of Horticultural Science & Biotechnology (2009) 84 (5) 513–518