RESEARCH NOTE Micropropagation of a Casuarina hybrid (Casuarina equisetifolia L. 3 Casuarina glauca Sieber ex Spreng) following facilitated seed germination Xiuli Shen William S. Castle Frederick G. Gmitter Jr Received: 29 October 2008 / Accepted: 29 December 2008 Ó Springer Science+Business Media B.V. 2009 Abstract A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. 9 Casuarina glauca Sieber ex Spreng (C. e. 9 C. g.), was developed. When seeds without seed coats were cultured on 4 germi- nation media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 lM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium sup- plemented with 17.8 lM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 lM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 lM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse. Keywords Casuarina hybrid (C. e. 9 C. g.) Á Micropropagation Á Seed germination Abbreviations BA 6-Benzylaminopurine C. e. 9 C. g. Casuarina equisetifolia L. 9 Casuarina glauca Sieber ex Spreng IBA Indole-3-butyric acid MS Murashige and Skoog (1962) PGRs Plant growth regulators Casuarina species of the family Casuarinaceae are tropical and subtropical trees native to Australia, southeastern Asia and islands of the western Pacific Ocean. Among other uses, Casuarina trees are widely used as windbreaks for rehabilitating and stabilizing dunes, as ornamental trees, and for timber and firewood production (Beadle 1981; El-Lakany 1983a, b; Kondas 1983; Turnbull 1990; Midg- ley et al. 1983, National Academy of Science 1984; Castle 2008a, b). Seed propagation of Casuarina, as with many plants reproduced by seed, requires rapid, uniform germination to achieve efficient nursery production. Seeds of Casuarina have germination percentages that range from 10 to 50% (El-Lakany and Shepherd 1983; Turnbull and Martensz 1982; El-Lakany et al. 1989; Goh et al. 1995, Boland et al. 1996). The reasons for poor seed performance have been attributed to the existence of a large number of shrunken, insect damaged, or empty seeds in seed lots, and to the barrier imposed by the seed coat (Sivakumar et al. 2007). In our earlier study of the causes of poor germination and techniques to improve the germination of a Casuarina hybrid and C. cunninghamiana Miq., we also experienced poor germination (unpublished data). Seeds classified as filled had the highest germination rate. Filled seeds gen- erally sank when seeds were separated by a petroleum ether technique. We hypothesized that the germination perfor- mance of Casuarina seeds could be further improved by culturing only filled seed with their seed coat removed. In vitro seed germination offers an alternative means to propagate recalcitrant seeds and it has the potential to X. Shen Á W. S. Castle (&) Á F. G. Gmitter Jr Citrus Research and Education Center (CREC), Institute of Food and Agricultural Sciences (IFAS), University of Florida (UF), 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA e-mail: bcastle@ufl.edu 123 Plant Cell Tiss Organ Cult DOI 10.1007/s11240-009-9502-5