Cryopreservation of Cyrtopodium hatschbachii Pabst (Orchidaceae) immature seeds by encapsulation-dehydration

The aim of the present study was to investigate the efficiency of the encapsulation-dehydration technique for cryopreservation of Cyrtopodium hastchbachii Pabst seeds. Immature seeds of this species were cryopreserved by an encapsulation-dehydration technique. Seeds of five immature pods, 120 days after pollination, were encapsulated in 3% calcium alginate matrix and pretreated in liquid medium supplemented with 0.08 M sucrose (24 h), 0.15 M sucrose (24 h), 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h) in shaker at 60 rpm. Alginate beads were dehydrated 5 h in silicagel and immersed in liquid nitrogen for 12 h. Cryopreserved beads were thawed at 30°C for 1 min, rehydrated using the same liquid mediums (0.75 M sucrose (24 h), 0.5 M sucrose (24 h), 0.25 M sucrose (48 h) and 0.15 M sucrose (24 h)) and cultivated in half strength Murashige & Skoog medium (1962) with the addition of 2 g/L activated charcoal. Sixty four percent of seeds survived and developed into acclimatized plants after being cryopreserved. In this work, the encapsulation-dehydration technique was employed for first time in Cyrtopodium hatschbachii.

Effect of morphological heterogeneity of somatic embryos of Melia azedarach on conversion into plants

Embryogenic cultures were initiated from immature Melia azedarach (Meliaceae) zigotic embryos. Explants were induced on Murashige and Skoog (1962) medium with 4.54 microM thidiazuron or 0.45 microM dichlorophenoxyacetic acid. After 6 weeks of culture on induction medium, somatic embryos were categorized in four morphological classes based on the presence of single or fused embryos and if they remained united or not to the original explant; that were evaluated histologically. The somatic embryos of every category were transferred, in groups or individually, on a 1/4 MS medium. Bipolar embryos, the more typically normal ones, had well defined shoot and root apical meristems and produced single plants; subcultured individually their conversion was 28%, and subcultured in groups the conversion declined to 6.8%. Fused embryos subcultured in groups had only a 2.1% conversion and produced plants with fused stems. None conversion rate in the others classes was associated to poorly developed shoot and root meristematic areas or with their absence. The converted plants were acclimatized and transferred, in a mist, to soil, with an independent of the class 95% survival rate.

Organogenesis and plant regeneration of Arachis villosa Benth. (Leguminosae) through leaf culture

With the aim of developing an efficient plant regeneration protocol, leaflet explants of three accessions of Arachis villosa Benth. (S2866, S2867 and L97) were cultured on basic Murashige and Skoog medium supplemented with different combinations of plant growth regulators: alpha-naphthalenacetic acid, indole-3-butyric acid, 6-benzylaminopurine, kinetin and thidiazuron. The accession L97 was the only one able to differentiate buds through indirect organogenesis. The most suitable combination for bud regeneration was the basic medium added with 13.62 microM thidiazuron and 4.44 microM 6-benzylaminopurine. These results show the important role of the genotype in morphogenetic responses and the organogenetic effect of thidiazuron in Arachis villosa accession L97. A thidiazuron lacking media (only 0.54 microM alpha-naphthalenacetic acid, 13.95 microM kinetin and 13.32 microM 6-benzylaminopurine were added) promoted the elongation of the regenerated buds. Adventitious rooting was achieved 90 days after the isolated shoots were transferred to a rooting medium containing 0.54 microM alpha-naphthalenacetic acid.

Embryogenic cell suspensions from different explants and cultivars of Eragrostis curvula (Schrad. ) Nees

The aim of this work was the establishment of embryogenic calli and cell suspensions from different explants and cultivars of weeping lovegrass, Eragrostis curvula (Schrad.) Nees, to be used as targets for biolistic transformation. Calli were initiated from immature inflorescences, seeds, embryos, leaf bases and root tips. Modified MS medium (Murashige and Skoog, 1962) was used for calli induction and proliferation. Cell suspensions were established and maintained in AAF medium (Wang et al., 1993). Morphogenic calli, embryogenic cell suspensions of moderate growth rate--consisting mainly of compact proembryogenic cell clusters- and green plants were obtained from all the explants and cultivars assayed, except root tips. Both, explant and genotype were very important factors to be considered in order to obtain a morphogenic response and to establish cell suspensions from this grass. The statistical analysis detected interaction between both factors, explants and genotypes. Immature inflourescences were the best source of explant and Kromdraai was the cultivar that showed the best morphogenic response (expressed as the percentage of calli/explant and the percentage of calli with green spots--every green spot developed into green plants-) with inflourescences, embryos and leaf bases. For Morpa and Don Pablo embryos as explants were less responsive than seeds and leaf bases. There were no differences in leaf bases for all the three cultivars analysed.

Plant regeneration of Ilex paraguariensis (Aquifoliaceae) by in vitro culture of nodal segments

In vitro regeneration of complete plants from nodal single bud segments of "yerba mate" (Ilex paraguariensis St. Hil.) was studied under defined nutritional and environmental conditions. Nodal segments harvested from actively growing shoots of conventionally raised plants were cultured on nutrient medium with the mineral salts and vitamins of Murashige and Skoog medium at 1/4 strength, supplemented with various concentrations of sucrose and 6-benzyladenine (BAP). Shoot regeneration from explants of both young (2 years old) and adult (20 years old) mother plants were readily achieved in the medium supplemented with 0.04-0.09 M sucrose with or without BAP. As many as 60-65 of the nodal segments cultured formed shoots. Rooting of regenerated shoots was observed in 50 of the explants harvested from young plants, whereas 25 of the explants rooted when the nodal explants were harvested from adult plants. The best rooting induction was achieved on 1/4 strength MS medium with vermiculite as the substrate and supplemented with 1-1.5 IBA (indolebutyric acid) and 1-2 PPZ (3-methyl-1-phenyl-2 pyrazolin-5-one). Plantlets were successfully transferred to soil.