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 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 degrees 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.

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.

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.(AU)

Cryopreservation of ilex immature zygotic embryos.

Tropical Ilex species have recalcitrant seeds. This chapter describes protocols for long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. microdonta, I. integerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos are aseptically removed from the seeds and precultured (7 days) in the dark at 27±2°C on solidified quarter-strength Murashige and Skoog medium with 3% sucrose and 0.1 mg/L zeatin. The embryos are 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). The beads are dehydrated for 5 h with silica gel to 25% water content (fresh weight basis) and then placed in sterile 5-mL cryovials. Then the beads are either plunged rapidly in liquid nitrogen where they are kept for 1 h (rapid cooling), or cooled at 1°C/min to -30°C and then immersed in liquid nitrogen for 1 h (slow cooling). After cryopreservation, the beads are rewarmed by immersion of the cryovials for 1 min in a water bath at 30°C. Finally, the beads are transferred onto culture medium (1/4MS, 3% sucrose, and 0.1 mg/L zeatin, solidified with 0.8% agar) and incubated in a growth room at 27±2°C under a 14-h light (116 µmol/m2/s) and 10-h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on the species and the treatment) were obtained with the cryopreserved embryos.

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.

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.

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.(AU)

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.

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.

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.(AU)

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.

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.

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.

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.(AU)