Micropropagation of a giant ornamental bromeliad Puya berteroniana through adventitious shoots and assessment of their genetic stability through ISSR primers and flow cytometry

An efficient micropropagation protocol has been developed for Puya berteroniana, a giant Chilean bromeliad with attractive turquoise flowers and great horticultural potential. Plant material for the experiment was multiplied via repeated sub-cultures of adventitious shoots, originally derived from a single in vitro germinated seedling, on a half-strength Murashige and Skoog (MS) medium (Murashige and Skoog in Physiol Plant 15:473-497, 1962) containing 0.44 A mu M 6-benzyladenine (BA). For the in vitro propagation experiment, the shoots were cultured on a half-strength MS medium supplemented with BA (0.04-2.22 A mu M) or zeatin (0.05-2.28 A mu M) alone or in combination with alpha-naphthaleneacetic acid (NAA) (0.54 A mu M). The maximum shoots per explant (5.5), was obtained on a medium containing 0.44 A mu M BA. Rooting of the shoots was tested on a medium supplemented with NAA (0.54-2.69 A mu M) or indole-3-acetic acid (0.57-2.85 A mu M). The best rooting was achieved on a medium containing 2.69 or 1.61 A mu M NAA. The rooted plantlets were transferred ex vitro, with 98.3 % survival rate. Inter simple sequence repeat (ISSR), flow cytometry, and karyological analysis were used to evaluate true-to-type of the in vitro regenerants. Ten randomly chosen plants and control plant were used. Twenty ISSR primers produced 95 clear, distinct, and reproducible bands per analysed sample. All amplified products were monomorfic, and no polymorphism was detected. Similarly, flow cytometric analysis confirmed that the ploidy level in all plantlets was stable. Karyological analysis revealed number of somatic chromosomes 2n = 50. As no somaclonal variation was detected in culture, this micropropagation protocol can be used for the mass production of P. berteroniana plants.