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, 0.7 5 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.

Influence of genotype and explant source on indirect organogenesis by in vitro culture of leaves of Melia azedarach L

In vitro regeneration of shoots from leaf explants of the Paradise tree (Melia azedarach L.) was studied. Three different portions (proximal portion, distal portion and rachis of the leaflets) of three developmental stages (folded, young still expanding and completely expanded) of leaves of 10-15 year old plants of seven genotypes were cultured on Murashige and Skoog (1962) medium (MS) supplemented with 1 mg x l(-1) benzylaminopurine (BAP) + 0.1 mg x l(-1) kinetin (KIN) + 3 mg x l(-1) adenine sulphate (ADS). The rachis of the leaflets of the completely expanded leaves was found to be the most responsive tissue, in most of the genotypes employed. Shoot regeneration occurred in leaf explants of all the genotypes tested. The best genotype for shoot regeneration was clone 4. Rooting was induced on MS medium supplemented with 2.5 mg x l(-1) 3-indolebutyric acid, IBA, (4 days) followed by subculture on MS lacking growth regulators (26 days). Complete plants were transferred to soil.

Genetic stability in rice micropropagation

An efficient clonal propagation procedure for six rice varieties cultivated in Argentina was developed by using shoot tip cultures, and the genetic stability of the micropropagated plants was verified by isozyme analysis. One week old seedlings obtained on MS medium were sectioned and subcultured on MS medium (0.75% agar) supplemented with different combination and concentrations of cytokinins (BAP and KIN) and auxins (2,4-D and NAA). After four weeks of culture, multiple shoots were obtained. The best response was observed on MS supplemented with BAP 5 mg l(-1). Shoot clumps were multiplied in MS liquid medium containing BAP 5 mg l(-1). Profuse rooting was obtained after transfer to MS medium lacking growth regulators and with sucrose 8% (w/v). Complete plants were successfully transferred to soil and grown to maturity. ADH and EST patterns of micropropagated rice plants showed polymorphisms compared with plants of the original varieties. However, the zymograms of the seed derived progeny of the micropropagated plants were similar to that of the original varieties. These results indicate the maintenance of the genetic stability in the sexual progeny of micropropagated plants.

Plant regeneration from Ilex spp. (Aquifoliaceae) in vitro

In vitro plant regeneration from nodal segments (containing one axillary bud) of seven species of the genus Ilex (I. argentina, I. brevicuspis, I. dumosa, I. microdonta, I. pseudoboxus, I. taubertiana and I. theezans) were readily achieved through three steps: 1) shoot regeneration by in vitro culture of nodal segments in MS medium at 1/4 strength, plus 3 sucrose and 0.65 agar (1/4MS) and 0.5 microM BA (45 days of culture); 2) Induction of rooting from regenerated shoots with 1/4MS (solidified with 2.5 g.L-1 "Phytagel") with 7.3 microM IBA (7 days) and, 3) subculture of shoot on a fresh medium (1/4MS lacking plant growth regulators) during 21 days. Shoot regeneration of other three species (I. aquifolium, I. brasiliensis and I. integerrima) were also obtained by in vitro culture of nodal segments. Shoot regeneration of I. aquifolium, I. brasiliensis, I. integerrima, I. microdonta, I. pseudoboxus, and I. taubertiana were also obtained by culture shoot tips on 1/4MS and 0.5 microM BA. Shoot regeneration from meristems of I. argentina, I. brevicuspis, I. dumosa, and I. theezans were readily achieved by in vitro culture on the same medium.