Vargas Isabel Beatriz de, Droste Annette
Rev Biol Trop. 2014 Mar;62(1):299-308. doi: 10.15517/rbt.v62i1.3661.
Cyathea atrovirens occurs in a wide range of habitats in Brazil, Paraguay, Uruguay and Argentina. In the Brazilian State of Rio Grande do Sul, this commonly found species is a target of intense exploitation, because of its ornamental characteristics. The in vitro culture is an important tool for propagation which may contribute toward the reduction of extractivism. However, exogenous contamination of spores is an obstacle for the success of aseptic long-term cultures. This study evaluated the influence of different sterilization methods combined with storage conditions on the contamination of the in vitro cultures and the gametophytic development of C. atrovirens, in order to establish an efficient propagation protocol. Spores were obtained from plants collected in Novo Hamburgo, State of Rio Grande do Sul, Brazil. In the first experiment, spores stored at 7 degrees C were surface sterilized with 0.5, 0.8 and 2% of sodium hypochlorite (NaClO) for 15 minutes and sown in Meyer's culture medium. The cultures were maintained in a growth room at 26 +/- 1 degrees C for a 12-h photoperiod and photon flux density of 100 micromol/m2/s provided by cool white fluorescent light. Contamination was assessed at 60 days, and gametophytic development was scored at 30, 60, 120 and 130 days of in vitro culture, analyzing 300 individuals for each treatment. There was no significant difference in culture contamination among the different sodium hypochlorite concentrations tested, and all treatments allowed for the development of cordiform gametophytes at 130 days of culture. In the second experiment, spores stored at 7 and -20 degrees C were divided into two groups. Half of the spores were surface sterilized with 2% of NaClO for 15 minutes and the other half was not sterilized. All spores were sown in Meyer's medium supplemented with one of the following antibiotics: nystatin, Micostatin and actidione. The culture conditions and the procedures used for evaluating contamination and gametophytic development were the same described for the first experiment. No contamination was observed in spores stored at -20 degrees C and treated with NaCIO and actidione. In all treatments, cordiform gametophytes presenting antheridia were observed at 120 days. The percentages of these gametophytes increased from 120 to 130 days and no significant differences were observed among treatments. Archegonia were observed on cordiform gametophytes at 130 days. The findings provide data relevant to in vitro propagation procedures of this species, which may increase the availability of plants for ornamental purposes, therefore contributing to the reduction of the exploitation of endangered tree ferns species.
黑绿桫椤生长在巴西、巴拉圭、乌拉圭和阿根廷的多种生境中。在巴西南里奥格兰德州，这种常见物种因其观赏特性而成为过度开发的对象。离体培养是一种重要的繁殖工具，有助于减少对其的采挖。然而，孢子的外源污染是无菌长期培养成功的障碍。本研究评估了不同灭菌方法与储存条件对黑绿桫椤离体培养污染及配子体发育的影响，以建立高效的繁殖方案。孢子取自巴西南里奥格兰德州新汉堡市采集的植株。在第一个实验中，将保存在7摄氏度的孢子用0.5%、0.8%和2%的次氯酸钠（NaClO）进行表面灭菌15分钟，然后接种到迈耶培养基上。培养物在生长室中于26±1摄氏度、12小时光周期以及由冷白色荧光灯提供的100微摩尔/平方米/秒的光子通量密度下培养。在60天时评估污染情况，在离体培养30、60、120和130天时对配子体发育进行评分，每个处理分析300个个体。在所测试的不同次氯酸钠浓度之间，培养污染没有显著差异，并且所有处理在培养130天时都能使心形配子体发育。在第二个实验中，将保存在7摄氏度和 -20摄氏度的孢子分成两组。一半孢子用2%的NaClO进行表面灭菌15分钟，另一半不灭菌。所有孢子都接种到添加了以下抗生素之一的迈耶培养基中：制霉菌素、米可定和放线菌酮。培养条件以及用于评估污染和配子体发育的程序与第一个实验相同。保存在 -20摄氏度并用NaClO和放线菌酮处理的孢子未观察到污染。在所有处理中，在120天时观察到有心形且带有雄器的配子体。这些配子体的百分比从120天到130天增加，各处理之间未观察到显著差异。在130天时在心形配子体上观察到颈卵器。这些发现为该物种的离体繁殖程序提供了相关数据，这可能会增加用于观赏目的的植株数量，从而有助于减少对濒危树蕨物种的采挖。
