Plant Cell, Tissue and Organ Culture 28: 231-233, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands. Micropropagation note Micropropagation of Tetraclinis articulata (Vahl) Masters (Cupressaceae) M.A. Morte ~, M. Honrubia I & A. Piqueras 2 ~Plant Biology Department (Botany), University of Murcia, Murcia, Spain; 2CEBAS, C. S. I. C., Murcia, Spain Received 25 February 1991; accepted in revised form 3 October 1991 Key words: cypress, in vitro propagation, root production Greater attention has been focused recently on the use of Cartagena cypress (Tetraclinis ar- ticulata (Vahl) Masters), a member of Cupres- saceae, for reafforestation programmes in the coastal zone of southern Spain. This is an en- demic species from the Mediterranean forest of Murcia (Spain) and North Africa, and is increas- ingly important as a forestry species in South- East Spain. The induction of shoot proliferation from apices or axillary buds to produce conifer multi- ple shoots that are rooted is now recognized as a viable technique for plant propagation. To our knowledge there are no reports on the in vitro propagation of this conifer. Our research was conducted to develop practical methods for mi- cropropagation of Cartagena cypress. Shoot tips, approximately 0.5 cm long, were excised from one-year-old seedlings from El Valle tree nursery (Murcia, Spain). These initial explants were washed under running tap water for 30 min. Surface disinfestation procedures in- volved 15 min agitation in 30% sodium hypo- chlorite and 0.05% Tween 20 (1:1 , v/v) solu- tion, followed by collective washing in 10% hy- drogen peroxide (30%) for 5 to 8 s, and then in 80% ethanol for 10 s, three rinses of 5 min each in sterile distilled water, light rinsing in 80% ethanol followed by sterile distilled water, and then transfer to basal medium. Basal medium consisted of Schenk & Hilde- brandt (1972) salts supplemented with myo-ino- sitol (1000 mgl-1), thiamine-HCl (0.5 mgl-l), nicotinic acid (5.0 mg 1-I), pyridoxine-HC1 (0.5 mg 1-1) and 3% sucrose. This medium was selected because it appears to be the most suit- able for culture of conifers such as Juniperus sp. (G6mez et al. 1987). The pH was adjusted to 5.8 before autoclaving. The medium was gelled with 7 g 1-1 Panreac agar and autoclaved for 20 min at 121°C, and 103 kPa. For shoot proliferation tri- als, 6-benzyladenine (BA) at 0.44, 2.2, 4.4, 6.7 or 8.9 ixM and kinetin at 0.46, 2.3, 4.6, 6.9 or 9.3 IxM, were added to the basal medium with or without naphthaleneacetic acid (NAA) at 0.054, 0.27, 1.33 or 8.05 p,M. Twenty-five cultures per treatment were grown at 25 +2°C and 40 ixmol m -2 s-1 Growlux fluorescent lighting on a 16-h photoperiod. Experiments were repeated three times at least. Shoot clumps were transferred to basal medium (minus plant growth regulators) for ap- proximately 4 weeks to enhance shoot elongation prior to utilization in root induction medium. Following shoot multiplication and elongation, shoots 15-25 mm long were transferred to root induction medium, consisting of SH basal medium containing NAA and/or IBA at various concentrations. After 4 weeks in auxin treat- ment, shoots were transferred to auxin-free SH basal medium with different concentrations of sucrose (1.5, 2, 3%). After 6 weeks, plantlets with well-developed roots were potted in a peat- perlite mixture (1:1 v/v). The plantlets were adapted to growth chamber conditions (26-+ 2°C) with a 16-h photoperiod. They were gradu- ally exposed to reduced relative humidity by progressively removing a plastic cover during a period of 2 to 3 weeks. Once the acclimatization was accomplished, the plants were transferred to the greenhouse. After six weeks in SH medium without growth