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Dose response curves fitted to growth data from the constant exposure experiment at day 25 for a) Bryum rubens, b) Campylopus introflexus, and c) Polytrichum formosum, and when mean diameter of the control replicates was four times its mean initial size for a) B. rubens (25 days), c) C. introflexus (20 days), e) P. formosum (39 days). EC 50 (g a.i. l 1 ), raw and mean corrected R 2 values are shown, Wald 95% confidence intervals are in parentheses, (n 50). Non-linear regression model: Y L((U L)/(1exp(m(log 10 (asulam concentration) log 10 (EC 50 ))))), where Y mean diameter growth, L lower asymptote, U upper asymptote, and m slope.
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Protonemata of three mosses (Bryum rubens Mitt., Campylopus introflexus (Hedw.) Brid,. and Polytrichum formosum Hedw.) were exposed in sterile cultures to low concentrations of the bracken herbicide asulam, added to the solidified growth medium. Protonemata were either exposed for 24 hours and then transferred onto media without asulam, or exposed...
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... response curves, EC 50 values (g a.i. l 1 ), Wald 95% confidence intervals and R 2 values for each species at day 25 and when mean diameter of the control replicates was four times its mean initial size are shown in Figure 2. The EC 50 values calculated showed the protonemata of the three mosses to have different sensitivities to asulam, under the conditions tested. ...
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... We have little knowledge about the sensitivity of ferns, horsetails, lichens and mosses to herbicides. Nonetheless, some limited data does indicate that these taxa are indeed quite sensitive to exposure (47,(54)(55)(56)(57). ...
Over the past decades declining biodiversity has been a major concern across the world; however, preventing biodiversity declines in agricultural areas has been ineffective. Failure to adequately assess and properly regulate herbicide effects can have important implications for species richness and overall biodiversity within natural and semi-natural habitats adjacent to agricultural fields. While the use of agrochemicals including both fertilizers and pesticides has been considered of great concern for the observed biodiversity declines, this paper primarily focusses on herbicide effects on non-target terrestrial plants. Although not explicitly addressed in current pesticide regulations, environmental protection, in particular, should include considerations of the impacts on non-target species, biodiversity and ecosystems. However, current toxicity tests are not optimised in terms of the endpoints assessed, the species tested, or the importance of pesticide effects on community composition, ecosystem functioning and biodiversity and thus, do not adequately fulfil the requirements of the EU pesticide legislation for environmental protection.
... In these cases gametophores are adopted as explants, but the success rate of procedures is highly variable and contamination is rather common (Rowntree 2006). Rowntree et al. (2005) reported that pro-tonemata were sensitive to several compounds, but Gonzalez et al. (2006) successfully cultured protonemal explants of Splachnum ampullaceum in vitro. Asexual propagules were also used as explants, although they were mostly vulnerable to disinfectants. ...
Rhodobryum giganteum is a rare Chinese traditional medicinal moss species and has special therapy value in heart diseases. In the present study, we successfully induced gametophores and protonemata of R. giganteum in vitro from its new shoots, which were disinfected with 0.1% HgCl2, for eight minutes. We also tested the factors influencing the growth of its protonemata and found that: (1) modified Knop medium is able to facilitate the elongation of the protonemata in vitro and prolong their growth time in vitro, whereas MS medium is able to facilitate the branching of the protone mata, and the protonemata grew slowly in modified White medium, accompanied by the development of gametophores; (2) cutting of the protonemata increases protonemal proliferation in vitro; (3) 2,4-D facilitates the induction of protonemata from leaves at 0.5 mgl-1, but inhibits the induction and quickens the aging process of the protonemata at 2.0 mgl-1.
... The protocol was a four-stage process: (1) encapsulation in 3 % sodium alginate , solidified with 100 mM CaCl solution, (2) pretreatment for 2 weeks with 10 mM ABA and 50 g L 21 sucrose, (3) dried for 6 h followed by (4) rapid immersion and storage in LN. Ten protonematal plugs (approx. 1 mm diameter) were removed from the stock cultures, transferred onto standard medium and their growth monitored weekly for a total of 5 weeks by measuring plug diameter along two permanent transects marked on the base of the dishes with vernier callipers according to Rowntree et al. (2005) (control treatment). Forty protonematal plugs were each encapsulated in an alginate strip (for methods, see Burch and Wilkinson, 2002). ...
Successful cryopreservation of bryophytes is linked to intrinsic desiccation tolerance and survival can be enhanced by pre-treatment with abscisic acid (ABA) and sucrose. The pioneer moss Ditrichum plumbicola is naturally subjected to desiccation in the field but showed unexpectedly low survival of cryopreservation, as well as a poor response to pre-treatment. The effects of the cryopreservation protocol on protonemata of D. plumbicola were investigated in order to explore possible relationships between the production in vitro of cryopreservation-tolerant asexual propagules and the reproductive biology of D. plumbicola in nature.
Protonemata were prepared for cryopreservation using a four-step protocol involving encapsulation in sodium alginate, pre-treatment for 2 weeks with ABA and sucrose, desiccation for 6 h and rapid freezing in liquid nitrogen. After each stage, protonemata were prepared for light and electron microscopy and growth on standard medium was monitored. Further samples were prepared for light and electron microscopy at intervals over a 24-h period following removal from liquid nitrogen and re-hydration.
Pre-treatment with ABA and sucrose caused dramatic changes to the protonemata. Growth was arrested and propagules induced with pronounced morphological and cytological changes. Most cells died, but those that survived were characterized by thick, deeply pigmented walls, numerous small vacuoles and lipid droplets in their cytoplasm. Desiccation and cryopreservation elicited no dramatic cytological changes. Cells returned to their pre-dehydration and cryopreservation state within 2 h of re-hydration and/or removal from liquid nitrogen. Regeneration was normal once the ABA/sucrose stimulus was removed.
The ABA/sucrose pre-treatment induced the formation of highly desiccation- and cryopreservation-tolerant propagules from the protonemata of D. plumbicola. This parallels behaviour in the wild, where highly desiccation-tolerant rhizoids function as perennating organs allowing the moss to endure extreme environmental conditions. An involvement of endogenous ABA in the desiccation tolerance of D. plumbicola is suggested.
Herbicides are used extensively to control invasive plants in natural areas but, herbicide application often has undesired effects on non-target plants. Using a lab bioassay, we investigated the impacts of glyphosate and two bioherbicides (cinnamon plus clove oil, and pelargonic acid) on the establishment of bryophyte and fern propagules from an urban woodland park soil propagule bank. Mosses were slower to emerge and establish in soil treated with cinnamon plus clove oil than soil treated with pelargonic acid or glyphosate. Fern gametophyte establishment was negatively affected by glyphosate but not by the two bioherbicides. Results from this study confirmed those of a previous field study indicating that an herbicide containing pelargonic acid could be useful for controlling non-native plants in locations suspected of harboring high biodiversity in soil propagule banks.
Following a request from the European Food Safety Authority, the Panel on Plant Protection Products and their Residues developed an opinion on the science to support the development of a risk assessment scheme of plant (crop) protection products on non-target terrestrial plants (NTTPs). This scientific opinion is largely a literature review on the most up-to-date knowledge of factors influencing phytotoxicity testing and risk assessment of NTTPs. Specific protection goals (SPGs) were defined for off-field, in-field and endangered species. SPGs are closely linked to ecosystem services and functions, and include maintaining provision of water regulation, food web support, aesthetic values, genetic resources and biodiversity. Gaps were identified in standard guidelines currently used in lower tier testing (tier I/II). In these guidelines, tests are conducted at the seedling/juvenile stage using mostly annual crops, and effects are recorded at the juvenile/vegetative stage under greenhouse conditions with plants grown individually or in monoculture. Endpoints measured do not include the overall effect on the whole life cycle (germinating seeds, seedling, juvenile stages, flowering, and seed production and germinability). It is also noted that it is unknown whether the following groups of organisms are covered by the plant risk assessment as it is carried out now: ferns, mosses, liverworts, hornworts, horsetails, lichens or woody species. In terms of exposure, droplet drift is considered to be the most important factor for off-field emissions to non-target areas. Models are available to calculate loadings from spray drift. Higher tier assessment is not required if the risk based on the tier II level can be managed by risk mitigation measures. When required, higher tier tests should be conducted under more realistic conditions. They may include additional laboratory/greenhouse tests (e.g. to measure reproductive endpoints or species interactions), microcosms or field experiments with experimentally or already established species. Other issues were considered, including exposure to mixtures, adjuvants, co-formulants and metabolites. Recommendations for the improvement of current guidelines and the elaboration of new guidelines and risk assessment schemes are provided.
The value of ex situ plant collections for conservation is increasingly recognised. The Royal Botanic Gardens, Kew (RGB, Kew) has been pioneering methods for the development of such collections with the establishment of the Millennium Seed Bank and a project for the ex situ conservation of threatened UK bryophytes (mosses, liverworts, hornworts). A broad range of bryophytes are currently held in a tissue culture collection at RBG, Kew on sucrose-free ¼ or ½ Murashige & Skoog or Knops minimal medium. The efficacy of the novel sterilising agent Sodium dichloroisocyanurate (NaDCC) was tested on a range of taxa, utilising a variety of bryophytic initiation material. Concentrations of 1% (w/v) for 3min and 0.5% (w/v) for 2min, without the addition of detergent, were found to be successful in initiating cultures from sporophytes and leafy gametophores respectively. Initiation of cultures from wild-collected sporophytes was more successful than from wild-collected gametophores. However, pre-culturing of gametophore material was found to enhance success rate of procedures. Transferring visibly clean material away from contamination post-initiation was also determined to increase protocol success rate. The ability of the biocide Plant Preservative MixtureTM (PPMTM) to control fungal and bacterial contamination in bryophyte protonemal cultures was also tested, but not found to be suitable. However, results obtained indicated that contamination may confer resistance on the moss against the phytotoxic effects of the biocide. Methods developed have wider applicability for the establishment of in vitro collections of other threatened plants.
