ABSTRACT
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.
Subject(s)
Cell Survival , Cryopreservation/methods , Ilex/embryology , Ilex/physiology , Seeds , Seeds/physiology , Germination , Tissue Culture TechniquesABSTRACT
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.
Subject(s)
Plant Shoots/growth & development , Plant Leaves/growth & development , Melia azedarach/growth & development , Organogenesis/genetics , Regeneration/genetics , Plant Shoots/drug effects , Plant Shoots/genetics , Plant Leaves/drug effects , Plant Leaves/genetics , Genotype , Melia azedarach/drug effects , Melia azedarach/genetics , Organogenesis/drug effects , Regeneration/drug effects , Plant Growth Regulators/pharmacology , Growth Substances/pharmacologyABSTRACT
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.