RESUMEN
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.
Asunto(s)
Ilex/embriología , Semillas , Criopreservación/métodos , Crioprotectores , Glicerol , SacarosaRESUMEN
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.
Asunto(s)
Criopreservación/métodos , Ilex/embriología , Semillas/fisiología , Supervivencia Celular , Germinación , Ilex/fisiología , Semillas/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
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.
Asunto(s)
Supervivencia Celular , Criopreservación/métodos , Ilex/embriología , Ilex/fisiología , Semillas , Semillas/fisiología , Germinación , Técnicas de Cultivo de TejidosRESUMEN
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)
Asunto(s)
Supervivencia Celular , Criopreservación/métodos , Ilex/embriología , Ilex/fisiología , Semillas , Semillas/fisiología , Germinación , Técnicas de Cultivo de TejidosRESUMEN
Nonrandom patterns of gene dispersal have been identified as possible causes of genetic structuring within populations. Attempts to model these patterns have generally focused solely on the effects of isolation by distance, but the processes involved are more complex than such modeling suggests. Here, we extend considerations of gene dispersal processes beyond simple isolation by distance effects by directly evaluating the effects of kin-structured gene dispersal mediated by the group dispersal of related seeds within fruits (i.e., kin-structured seed dispersal) by birds on genetic structure in Ilex leucoclada, a clonal dioecious shrub. To examine the genetic structure patterns, we established two 30x30 m plots (one with immature soils in old-growth forest and one in secondary forest, designated IM and SC, respectively) with different I. leucoclada stem densities. In these two plots 145 and 510 stems were found, representing 78 and 85 genets, respectively, identified by analyzing their genotypes at eight microsatellite loci. The clonal structure was stronger in the SC plot than in the IM plot. Correlograms of coancestry for genets in both plots exhibited significant, positive, high values in the shortest distance class, indicating the presence of strong genetic structure. However, Sp statistics revealed that the pattern of the genetic structure differed between the plots. In addition, to estimate the family structure within fruits, we sampled forty fruits, in total, from 15 randomly selected plants in the area around the IM and SC plots, and found that 80% of the fruits were multiseeded and 42-100% of the multiseeded fruits contained at least one pair of full sibs. Simulations based on these estimates demonstrated that the group dispersal of related seeds produced through correlated mating both within and across fruits, but not unstructured half-sib dispersal, could generate the observed magnitude and trends of genetic structure found in the IM plot. Furthermore, in addition to kin-structured seed dispersal, isolation by distance processes is also likely to promote genetic substructuring in the SC plot. After discussing possible ecological factors that may have contributed to the observed genetic structure, we contrast our results with those predicted by general isolation by distance models, and propose that kin-structured seed dispersal should promote some evolutionary phenomena, and thus should be incorporated, where appropriate, in models of gene dispersal in natural plant populations.