Adventitious Bud Formation and Plantlet Regeneration of Achyrocline satureoides-A Multipurpose Medicinal Plant.

Adventitious bud regeneration was achieved from hypocotyls, cotyledons and leaf explants of Achyrocline satureioides. Organogenesis was induced from every explant cultured on Murashige and Skoog semisolid medium (plus sucrose 30g·L-1) containing different combinations of 6-benzyladenine (BA) and -naphtalenacetic acid (NAA) under 116mol·m2·s-1 photosynthetic photon flux density (PPFD), photoperiod 14 h and at 272ºC. The regeneration was similar for every tested explant and varied between 64 and 83%. The number of buds formed per regenerative explants was similar in every treatment (5-8 shoots/explant). In order to stimulate In vitro rooting, regenerative leaves were sub cultured from the best induction medium in MS lacking plant growth regulators for the same periods. Every plantlet raised In vitro was phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 60-day-old in vitro regenerated plants was established.

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.

Micropropagation of Ilex dumosa (Aquifoliaceae) from nodal segments in a tissue culture system

Micropropagation of Ilex dumosa var. dumosa R. ("yerba señorita") from nodal segments containing one axillary bud was investigated. Shoot regeneration from explants of six-year-old plants was readily achieved in 1/4 strength Murashige and Skoog medium (1/4 MS) plus 30 gr x L(-1) sucrose and supplemented with 4.4 microM BA. Further multiplication and elongation of the regenerated shoots were obtained by subculture in a fresh medium of similar composition with 1.5 gr x L(-1) sucrose. Rooting induction from shoots were achieved in two steps: 1) 7 days in 1/4 MS (30 gr x L(-1) sucrose, 0.25% Phytagel) with 7.3 microM IBA and 2) 21 days in the same medium without IBA and 20 microM of cadaverine added. Regenerated plants were successfully transferred to soil. This micropropagation schedule can be implemented in breeding programs of Ilex dumosa.

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.