Histodifferentiation and ultrastructure of nodular cultures from seeds of Vriesea friburgensis Mez var. paludosa (L.B. Smith) L.B. Smith and leaf explants of Vriesea reitzii Leme & A. Costa (Bromeliaceae).

Micropropagation via induction, multiplication and development of nodular cultures (NCs) is an efficient regeneration system for Bromeliaceae, a family of endangered monocot plants with ornamental value. Therefore, the present work aimed to induce NCs from seeds and leaf explants of Vriesea in order to characterize the morphological and histochemical aspects of induction and formation of these cultures. Seeds of Vriesea friburgensis var. paludosa were sterilized and inoculated into liquid culture media supplemented with different concentrations and combinations of growth regulators. Leaf explants of Vriesea reitzii were inoculated into medium supplemented with 4 µM α-naphthalene acetic acid (NAA) and 2 µM 6-benzylaminopurine (BAP). The addition of NAA (4 µM) in the culture medium used for seeds led to an induction rate of 72% in NCs. First, the embryo began to germinate, and afterwards, nodular structures started to form. While NCs formed from seeds is associated with root and shoot meristems, the formation of NCs from leaf explants involves the intercalary meristem. Meristematic cells generate an appropriate response in the induction medium, producing NCs by the proliferation of small cells with meristematic characteristics and large vacuolated cells. These results provide a better understanding of morphogenetic responses in bromeliads and, hence, the opportunity to develop optimized micropropagation protocols. Abbreviations: BAP, 6-benzylaminopurine; 2-iP, N6 (2-isopentyl) adenine; CBB, Coomassie Brilliant Blue; CLSM, confocal laser scanning microscopy; MSB, MS basal medium; NAA, α-Naphthalene acetic acid; NCs, nodular cultures; PAS, Periodic Acid-Schiff; SEM, scanning electron microscopy; TDZ, N-phenyl-N'-1,2,3-thidiazol-5-ylurea; TB-O, Toluidine Blue O; TEM, Transmission electron microscopy.

Ultra-low temperature conservation of Brazilian pine embryogenic cultures.

This study aimed to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, enabling the ex situ conservation of the species. Embryogenic cultures were established from immature seeds and treated with variations of the cryoprotectant solutions SuDG, SoD and PVS2 prior to immersion in liquid nitrogen. Cell viability was evaluated after 30, 60 and 90 days of re-growth. The highest re-growth without morphological alterations and with normal biochemical composition was obtained with the PVS2 solution with 40 min immersion in ethanol (-20 °C). This procedure opens new horizons for the ex situ conservation of the species genetic.