Claudia Verónica Luna

DIRECT SHOOT REGENERATION FROM LEAF AND INTERNODE EXPLANTS OF ALOYSIA POLYSTACHYA (GRIS.) MOLD. (VERBENACEAE)

SummaryAdventitious bud regeneration from leaf and internode explants of Aloysia polystachya was achieved. Shoots from nodal segments grown in vitro were cut into pieces and used as sources of explants. Organogenesis was induced from both explants cultured on quarter-strength Murashige and Skoog (MS) semisolid medium (plus sucrose 5 g l−1) containing different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 116 μmol m−2 s−1 photosynthetic photon flux density (PPFD), 14-h photoperiod, and at a temperature of 27±2°C. The type of explant markedly influenced organogenesis and growth of the regenerated shoots. The regeneration frequencies were higher with leaf explants, while the number of shoots formed per responsive explant was greater with internode explants. However, the growth of regenerated shoots from internodes was seriously affected by vitrification. The number of shoots produced per responsive leaf explant increased from one to seven as the percentage of leaf explants producing shoots increased from 20 to more than 80%. NAA at 0.05 μM in combination with BA at 0.5μM induced the highest regeneration rate (87±8.8%) after 20 d of culture, yielding 5.9±0.8 shoots per responsive leaf explant. Histological examination confirmed the occurrence of direct organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium without plant growth regulators for 30 d. Finally, the elongated shoots were rooted by pre-treatment in an aqueous solution of NAA at 500 μM for 2 h and transferred to 1/4 MS. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 2-yr-old in vitro-regenerated plants was established.

Cryopreservation of Ilex Immature Zygotic Embryos

Tropical Ilex species have recalcitrant seeds. This chapter describes protocols for long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. microdonta, I. integerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos are aseptically removed from the seeds and precultured (7 days) in the dark at 27±2°C on solidified quarter-strength Murashige and Skoog medium with 3% sucrose and 0.1 mg/L zeatin. The embryos are then encapsulated in 3% calcium alginate beads and pretreated at 24-h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.75, and 1 M). The beads are dehydrated for 5 h with silica gel to 25% water content (fresh weight basis) and then placed in sterile 5-mL cryovials. Then the beads are either plunged rapidly in liquid nitrogen where they are kept for 1 h (rapid cooling), or cooled at 1°C/min to -30°C and then immersed in liquid nitrogen for 1 h (slow cooling). After cryopreservation, the beads are rewarmed by immersion of the cryovials for 1 min in a water bath at 30°C. Finally, the beads are transferred onto culture medium (1/4MS, 3% sucrose, and 0.1 mg/L zeatin, solidified with 0.8% agar) and incubated in a growth room at 27±2°C under a 14-h light (116 μmol/m2/s) and 10-h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on the species and the treatment) were obtained with the cryopreserved embryos.

Anatomical and histological features of Ilex paraguariensis leaves under different in vitro shoot culture systems

Ilex paraguariensis: (Aquifoliaceae) is an evergreen tree traditionally used to prepare a caffeine-rich infusion that has several medicinal properties. The in vitro propagation of this species has been studied as an alternative to conventional methods such as cuttings and seedlings. The in vitro culture environment consists of closed flasks with high relative humidity, reduced gas exchange and low luminosity conditions, which may induce physiological, anatomical and histological disruption in the cultivated plants. The occurrence of anatomical and histological alterations to the leaves of I. paraguariensis was examined in plants cultivated under greenhouse and different in vitro conditions. The variations in the microclimate inside the culture vessels generated by the different treatments tested affected the anatomy and histology of I. paraguariensis leaves by causing alterations in their architecture, thickness, vascularization and stomatal differentiation. A temporary immersion system was the best treatment for conserving the anatomical and histological features of the leaves. This treatment led to the lowest stomatal index and an extensive system of intercellular spaces that were similar to the characteristics developed under external environment conditions. Consequently, more than 80% of rooted plantlet transferred to pots were successfully rusticated. Plants from temporary immersion had greater photosynthetic rates due to a higher stomatal conductance.

Adventitious bud formation and plantlet regeneration of Achyrocline satureoides - a multipurpose medicinal plant.

Fil: Kotik, D.. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Nordeste. Instituto de Botanica del Nordeste (i); Argentina