Plant Cell Tissueand Organ Culture 40: 49-54, 1995. (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands. Increased frequency of dividing microspores and improved maintenance of multicellular microspores of Coffea arabica in medium with coconut milk B. Neuenschwander 1 & T. W. Baumann 2 1Centro Agron6mico Tropical de Investigaci6n y Enseaanza, Turrialba, Costa Riea; 21nstitute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland Received6 April 1994;accepted in revisedform 17 August 1994 Key words: androgenesis, coconut milk, Coffea arabica, microspore culture, microspore division, multicellular microspores Abstract The number of dividing microspores of Coffea arabica L. cv. Catuai and Catimor could be drastically increased in microspore media containing 16% (w/v) coconut milk, allowing cell divisions to continue in the microspore and multicellular microspores to survive until day 60. After a cold treatment, the microspores were mechanically isolated prior to cultivation in Murashige and Skoog medium supplemented with sucrose and maltose and (mg 1-l) 2,4-D: 2, BAP: I or a combination of kinetin: .5, 2,4-9:.5 and NAA: .5 as stationary suspension at a density of 1,200/ml. The crucial stage during microsporogenesis suitable for in vitro androgenesis proved to be mid uninucleate till early binucleate in flowerbuds with the size of 13-15 mm two to three days before anthesis. The initial steps of androgenesis were determined. Abbreviations: BAP - 6-benzylaminopurine, 2,4-D - 2,4-dichlorophenoxy-acetic acid, NAA - 1-naphthaleneacetic acid Introduction In vitro androgenesis of isolated microspores and sub- sequent plant regeneration has been reported for vari- ous mono- and dicots including Hordeum vulgare (Wei et al. 1986; Ziauddin et al. 1990), Triticum aestivum (Datta & Wenzel 1987; Tuvesson & 0hlund 1993), Zea mays (Pescitelli et al. 1989; Coumans et al. 1989), Brassica napus (Lichter 1982), Oryza sativa (Cho & Zapata 1988) and Nicotiana tabacum (Nitsch 1974). The microspores are either isolated mechanically prior to culture initiation or allowed to dehisce from anthers floating in liquid medium. In a limited number of trop- ical fruit trees, haploidization by in vitro androgenesis using anther cultures resulted in haploid plants from Carica papaya, Litchi chinensis, Euphorbia longan and Annona squamosa (Zhang 1992). Irregularities during meiosis occur naturally in dif- ferent coffee species (Medina 1977; B oaventura 1990). In C. canephora, cold treatment during meiosis causes the formation of aneuploid and polyploid microspores of which a portion develops into pollen containing two or more vegetative nuclei (Lanaud 1983). For various coffee species including C. arabica, the most cold- sensitive developmental stages during microsporoge- nesis proved to be immediately after tetrad formation forming pollen with two or three vegetative nuclei, and at first mitosis resulting in the same type of abnormal microspores or in microspores containing one repro- ductive nucleus and up to nine vegetative nuclei sig- nifying the beginning of an androgenetic development (Lanaud & Parvais 1980). Haploid plants from C. arabica (2n = 4x = 44) were obtained spontaneously by polyembryony (Dublin & Parvais 1976). An efficient system for haploidization would allow the production of homogeneous hybrids of wild diploid coffee species (2n = 2x = 22) which are needed to introduce desirable genes to C. arabi- ca. Whereas the gametophytic development has been investigated extensively, only few reports on haploid