A SIMPLIFIED PROTOCOL FOR MICROPROPAGATION OF GUAYULE (PARTHENIUM ARGENTATUM GRAY) † JAVIER CASTILLO ´ N ‡ AND KATRINA CORNISH * USDA, Agricultural Research Service, Western Regional Research Center, Albany, California 94710 (Received 10 November 1999; accepted 12 November 1999; editor G. C. Phillips) UMMARY A simple, efficient protocol for in vitro micropropagation of guayule is reported. Shoot cultures were maintained on MS (Murashige and Skoog, 1962) medium supplemented with 1.0 mg l -1 (4.4 mM) 6-benzylaminopurine and 0.025 mg l -1 (0.13 mM) a -naphthaleneacetic acid. Excised shoots were treated for 14–18 h with 100 mg l -1 (492.1 mM) indole-3- butyric acid in 0:5  MS salts to induce rooting. The shoots were subsequently inserted into cellulose plugs which were packed in sterile, ventilated plastic culture vessels and moistened with 0:5  MS medium without growth regulators. Use of cellulose plugs, liquid medium and ventilated culture vessels facilitated acclimation. Rooted shoots were transplanted into potting medium and acclimated to greenhouse conditions by covering with a cloche for 2 d, followed by daily watering for the first week. Key words: shoot cultures; acclimation; Sorbarod; cellulose plugs. Introduction Guayule (Parthenium argentatum Gray) is a woody desert shrub which is under development as a domestic source of hypoallergenic natural rubber (Siler and Cornish, 1994; Nakayama et al., 1996; Siler et al., 1996; Cornish, 1996, 1998). As part of this development process, existing and newly developed lines (developed either through plant breeding or genetic engineering) must be evaluated for field performance and yield. Guayule is a prolific seed producer. However, its apomictic nature and problems associated with direct seeding present obstacles for breeding, producing and evaluating large numbers of genetically identical plants (Thompson and Ray, 1989; Fangmeier et al., 1984). Micropropagation through tissue c ulture offers an alternative method for production of clonal material from improved or genetically engineered plants. There have been several reports of in vitro organogenesis and propagation of guayule that involved formation of callus on explants as an initial step (Dhar et al., 1989; Finnie et al., 1989; Zavala et al., 1982). Although these procedures did lead to regeneration of shoots and, in some cases, whole plants, induction of callus tissue often requires use of high growth regulator concentrations and can increase the occurrence of somaclonal variation (Phillips et al., 1994). Procedures for micropropagation of guayule, through in vitro multiplication of shoots followed by rooting and subsequent acclimation of plantlets, have been reported by only a few researchers (Pan et al., 1996; Trautmann and Visser, 1990; Smith, 1983). Although these methods have commonalities, they vary in terms of reported success rates as well as in terms of the time and labor that would be involved in applying these protocols to large scale micropropagation of plants. Here we report modifica- tions to these methods that improve shoot proliferation and simplify protocols for both rooting and acclimation of plantlets to greenhouse conditions. Mat erial s and Met hods Establishment and maintenance of shoot cultures. Shoot cultures of guayule lines N6-5 and hybrid line AZ101 (P. argentatum  P. tomentosum var. stramonium; Thompson and Ray, 1989) were initiated from aseptically germinated seedlings by the method of Pan et al. (1996). After 2–3 wk, shoots with three or more nodes were excised from the rest of the seedling (above the primary leaves) and transferred to shoot multiplication medium consisting of MS basal medium (Murashige and Skoog, 1962) supplemented with 3% sucrose, 1.0 mg l -1 (4.4 mM) 6-benzylaminopurine (BAP), 0.025 mg l -1 (0.13 mM) a -naphthaleneacetic acid (NAA) and 0.8% TC agar (Carolina Biological Supply Co., Burlington, NC) in Magenta GA-7 boxes (Sigma Chemical Co., St Louis, MO). Individual shoots produced multiple shoots (5–10) both from axillary buds and from callus-like tissue that grew from the cut end of the stem (Fig. 1). Shoot cultures were maintained on this medium and were subcultured every 4–6 wk by excising individual shoots and transferring these to fresh medium. Cultures were maintained in a growth chamber at 24°C under cool-white fluorescent light (80 mmol m -2 s -1 ; 16-h photoperiod). Root induction on excised shoots. Individual shoots, 10–30 mm in length and having at least two nodes, were excised from 4–6-wk-old cultures and treated with indole-3-butyric acid (IBA) to induce root formation. The IBA treatment consisted of incubating excised shoots overnight (14–18 h) at room temperature in liquid medium consisting of 0:5  MS salts (no vitamins or sucrose) and 100 mg l -1 (492.1 mM) IBA (Fig. 2a). This incubation was carried out in an open container in a transfer hood to maintain sterility while 215 In Vitro Cell. Dev. Biol.— Plant 36:215–219, May–June 2000  2000 Society for In Vitro Biology 1054-5476/00 $10.00+0.00 * Author to whom correspondence is to be addressed: Email kcornish@ pw.usda.gov ² Any mention of trade names or commercial products in this report is for informational purposes only and does not imply endorsement by the U.S. Department of Agriculture or the Agricultural Research Service. ³ Present address: USDA-ARS, USNA-FNPRU, Rm 238, Bldg. 010A, BARC-West 10300 Baltimore Ave., Beltsville, Maryland 20705