PLANT REGENERATION BY SOMATIC EMBRYOGENESIS AND ORGANOGENESIS IN COMMERCIAL PINEAPPLE (ANANAS COMOSUS L.) SUNEERAT SRIPAORAYA † , ROBERT MARCHANT, J. BRIAN POWER, AND MICHAEL R. DAVEY* Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK (Received 19 February 2002; accepted 2 January 2003; editor C. Chetsanga) Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker- based (MT) medium with 2.0 mg l 21 (9.8 mM) indolebutyric acid, 2.0 mg l 21 (10.74 mM) naphthaleneacetic acid, and 2.0 mg l 21 (9.29 mM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mg l 21 (8.87 mM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mg l 21 (2.26 mM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg l 21 (8.87 mM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mg l 21 (12.42 mM) 4-amino-3,5,6- trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mg l 21 (4.44 mM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mg l 21 (4.14 mM) picloram or 1.0 mg l 21 (4.52 mM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mg l 21 (4.44 mM) BA. All regenerated shoots rooted on growth regulator- free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms. Key words: Ananas comosus; pineapple; plant regeneration; organogenesis; somatic embryogenesis. Introduction Pineapple is an important crop cultivated in tropical regions of the world, with Thailand as a major area of production in recent years (FAO, 1998). The fruit is utilized in the processing and canning industries. Nutritionally, freshly harvested pineapple is an inexpensive source of vitamins A, B, and C, calcium, phosphorus, and iron. Vegetative propagation of pineapple from suckers and crowns is often restricted in some cultivars by the limited availability of propagules. Crowns may remain attached to the fruits at the time of sale, while suckers are often restricted in number with their inevitable variation in size resulting in erratic flowering and fruit formation on plants derived from them. In addition, the major Thai pineapple cultivars still lack disease resistance. Weed control is also a major problem. Since cultivar improvement requires at least 5 yr by sexual hybridization and selection, tissue culture- based technologies provide a crucial adjunct not only to conventional breeding but also for the propagation and genetic improvement of pineapple. In previous studies of the tissue culture of pineapple, Mapes (1973) obtained plants and protocorm-like structures from cultured shoot tips of the cv. Smooth Cayenne, while Lakshmi Sita et al. (1974) cultured meristems excised from slips. Other workers have cultured axillary buds from the crowns of mature fruit (Zepeda and Sagawa, 1981), or both lateral and axillary buds (Mathews and Rangan, 1979; Cabral et al., 1984; De Wald, 1988). Khatun et al. (1997) micropropagated pineapple cultivars specific to Bangladesh. More recently, Rahman et al. (2001) used crown tips from mature fruits to propagate the commercial cv. Giant Kew and the Bangladeshi cv. Khulna Local. To date, there is only one report of somatic embryogenesis in pineapple (Daquinta et al., 1996), in which dicamba was used as the growth regulator in the culture medium to induce embryogenic callus from leaves of the cvs. Red Spanish and Smooth Cayenne. However, only 42% and 55% of leaf explants of these cultivars, respectively, responded by producing slow-growing embryogenic callus. This report describes a simple procedure for the establishment in vitro of pineapple germplasm, together with procedures for efficient plant regeneration by organogenesis from leaf bases and by somatic embryogenesis from leaf base-derived callus and cell suspensions. These procedures will facilitate the exploitation of somatic cell techniques for pineapple multiplication and subsequent improvement. Materials and Methods Introduction and maintenance of shoots in vitro. Suckers, each 1–2 mo. old and about 30 cm in height with approximately 20 leaves, were excised †Present address: Faculty of Agriculture Nakhonsithammarat, Rajamangala Institute of Technology, Thungyai, Nakhonsithammarat 80240, Thailand. *Author to whom correspondence should be addressed: Email mike. davey@nottingham.ac.uk In Vitro Cell. Dev. Biol.—Plant 39:450–454, September – October 2003 DOI: 10.1079/IVP2003445 q 2003 Society for In Vitro Biology 1054-5476/03 $18.00+0.00 450