Micropropagation of paradise tree (Melia azedarach) by in vitro culture of axillary buds.

Paradise tree (Melia azedarach L.) is a multipurpose ornamental and timber tree, and its extracts are used to make insecticides and fungicides. Conventional propagation is done by seeds; however, sexual reproduction results in wide genetic variability. Therefore, clonal propagation is desirable to reduce genetic variation. This chapter describes a protocol for in vitro propagation of paradise tree by axillary buds. There are major steps for this protocol. Firstly, shoot induction by in vitro culture of axillary buds, excised from potted plants obtained by rooting of cuttings of 10-15-year-old adult trees. The initiation medium was composed of Murashige and Skoog medium (MS) supplemented with 0.5 mg/L BAP (benzylaminopurine), 0.1 mg/L IBA (indolebutyric acid), and 0.1 mg/L GA(3) (gibberellic acid). Secondly, multiplication of the regenerated shoots on MS medium amended with 0.5 mg/L BAP and 0.1 mg/L GA(3). Thirdly, rooting of the regenerated shoots on MS medium containing 0.1 mg/L IBA. Fully well-developed plants were transferred to pots containing sand, peat moss, and perlite (1:1:1), and maintained initially in the greenhouse or plastic tunnels.

Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi.

A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53 percent and 56 percent of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16 percent of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.

A cryopreservation protocol for immature zygotic embryos of species of Ilex (Aquifoliaceae).

Tropical Ilex species have recalcitrant seeds. This work describes experiments demonstrating the feasibility of long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and precultured (7 days) in the dark, at 27 +/- 2 degrees C on solidified (0.8% agar) 1/4MS medium, [consisting of quarter-strength salts and vitamins of Murashige and Skoog (1962) medium] with 3% sucrose and 0.1 mg/l Zeatin. The embryos were 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). Beads were dehydrated for 5 h with silicagel to 25% water content (fresh weight basis) and then placed in sterile 5 ml cryovials. Then the beads were either plunged rapidly in liquid nitrogen were they were kept for 1 h (rapid cooling) or cooled at 1 degrees C min(-1) to -30 degrees C. Then the beads were immersed in liquid nitrogen for 1 h (slow cooling). The beads were rewarmed by immersion of the cryovials for 1 min in a water bath thermostated at 30 degrees C. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l zeatin, solidified with 0.8% agar) and incubated in a growth room at 27 +/- 2 degrees C under a 14 h light (116 micromol. m(-2) x s(-1))/ 10 h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on de the species and the treatment) were obtained with the cryopreserved embryos.