Cryopreservation of Arachis pintoi (leguminosae) somatic embryos.

In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

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.(AU)

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.

Cryopreservation of seeds and in vitro-cultured protocorms of Oncidium bifolium sims. (Orchidaceae) by encapsulation-dehydration.

Encapsulation-dehydration was employed for cryopreserving seeds and in vitro-cultured protocorms of Oncidium bifolium. Freshly harvested seeds, 120 days after pollination, were encapsulated in beads containing 1/2 MS medium with 3% sucrose and 3% calcium alginate and subsequently pretreated in agitated (80 rpm) liquid medium supplemented with 0.15 M sucrose (24 h) followed by 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h). The beads with seeds were dehydrated with silica gel for 5 h to 19.2% moisture content and immersed in liquid nitrogen for 1 h, thawed at 30 degrees C for 2 min, post-treated using the same series of liquid media [0.5 M sucrose (24 h), 0.25 M sucrose (48 h), 0.15 M sucrose (24 h)], and recultured on 1/2 MS medium with 0.1M sucrose and 0.7% percent agar. As much as 4.8% of the cryopreserved seeds produced complete plants. In-vitro cultured protocorms were successfully cryopreserved following the same procedure, allowing 11.3% of them to produce plants.

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.

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.

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. (AU)

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. (AU)

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.

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.

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.

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.

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. (AU)

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.

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

Micropropagation of Ilex dumosa var. dumosa R. ([quot ]yerba señorita[quot ]) 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.

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 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.

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.

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.(AU)