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We determined the chronic effects of dehydration on the photochemical efficiency of a cultivated brown alga, Undaria pinnatifida (Alariaceae, Laminariales), in young sporophytes as cultivated sporelings. The effective quantum yields of photosystem II (ΔF/Fm′) at 50% humidity decreased markedly after 20 min of emersion and dropped almost zero after 60 min of emersion; the values did not restore even after subsequent 1-day immersion. The decreasing values coincided with a decrease in absolute water content of less than 40%. However, under 99% humidity up to 5-day emersion, the ΔF/Fm′ well remained and the last state exhibited a similar level to the initial value after a 48-h emersion at 20 °C and after 72 h of emersion at 10 °C, suggesting that the thalli were not truly dehydrated under saturated humidity and that photosynthetic activity was maintained for several days even without immersion in seawater. In addition, the subsequent growth of young sporophytes exposed to transportation storage stress featuring (1) immersed in seawater with aeration (ST1), (2) those without aeration (ST2), and (3) wrapped in paper towels moistened with seawater (ST3) revealed that the sporophytes exposed at lower temperature exhibited a higher subsequent growth than those at a higher temperature. The subsequent growth of ST3 was lower than ST1; however, the values of ST3 were higher than those of ST2 more than 12 °C, associating with poor seawater quality without aeration. These results indicated that the maintenance of moisture in the alga at low temperatures might be essential for the transportation of the sporelings.

As the largest genus of Halymeniaceae, Grateloupia has been widely reported. Here, we observed the life history and early development of Grateloupia constricata Li et Ding and investigated the effects of temperature, irradiance, and photoperiod on the discoid crust and sporeling development of G. constricata under laboratory conditions. We observed that the type of carpospore development was “mediate discal type”. The life history included homotypic gametophyte (haploid), carposporophyte (diploid), and tetrasporophyte (diploid), with typical isomorphic alternation of generations. The results of double factorial analysis showed that both single factorial effects and interaction among temperature, photoperiod, and irradiance were obviously significant on the discoid crust and sporeling development. Furthermore, we found that the optimum combination of condition for the early growth and development of G. constricata was temperature 20°C, irradiance 80 μmol photons/(m2·s) and photoperiod 16L:8D. This study provides the theoretical basis and technical support for the conservation of the Grateloupia germplasm, artificial breeding, large-scale cultivation and sustainable development.

Isoetes L. is a nearly cosmopolitan rhizomorphic lycopsid genus fundamental to disentangling plant evolution because of its singular position in the Plantae kingdom. A threatened Amazonian species recently described is I. cangae, inhabiting an oligotrophic upland lake over an iron-rich region facing substantial environmental alterations. In this study, we aim to optimize a reproduction protocol that can assist in the conservation efforts of Isoetes species in tropical areas. We observed an average of 126 megaspores or 318,000 microspores per sporangia in field-collected plants. We obtained an elevated percentage of sporelings (63%) from an in vitro mixed culture of mega- and microspores. In addition, we recovered a large number of sporelings around adult plants after cultivating them over quartzite sand and distilled water. The megaspore nutrient reserve ensured longstanding sporeling growth and development in these environmental conditions. However, the sporeling growth rate increased when they were planted over their original lake sediments or over an organic substrate, with nonsignificant differences between the substrates. An increase in temperature also increased the sporeling growth rate. Together with the very low sporeling mortality, these results suggest that I. cangae is a resilient species able to grow and colonize environments with low trophic characteristics with a range of substrates and temperature regimes. The protocols established in this study could be useful for the ex situ propagation and growth of a large number of sporelings, representing critical steps towards the population rescue and conservation of tropical Isoetes species.

We describe a simple and efficient plastid transformation method for the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the method described here, a few plastid transformants are obtained from a single bombardment of sporelings. Homoplasmic transformants of thalli are obtained immediately after primary selection.

Iso&euml, tes sabatina is an aquatic quillwort endemic to Italy. It is one of the rarest quillworts in Europe, and is critically endangered due to restricted range and to the continuous decline of both population and habitat quality. This study aims to develop an optimized protocol to reproduce and grow I. sabatina sporelings. Mature and immature megaspores were mixed with mature microspores to evaluate the influence of the developmental stage on germination and sporeling development. Two substrates, distilled water and water-agar medium, were tested for germination and sporeling emergence, and three substrates, sand, lake sediment and water-agar, were tested for transplants. A high percentage of megaspore germination (a total of 79.1%) was obtained in both substrates, higher for mature than immature spores. A total of 351 sporelings were produced in distilled water and water-agar cultures, with similar percentages (64.5% and 69.6%, respectively). The development stage of the megaspores affected both germination and sporeling development. Sporeling emergence showed significantly higher percentages in mature megaspores than immature ones (69.6% vs. 11.6%, respectively), with 85% of germinated spores developing sporelings. Only transplants over water-agar medium were successful. This protocol could be useful for the propagation of sporelings as the key step towards the planning of in situ actions to save this Mediterranean quillwort from extinction.

The giant kelp farming industry in China, with an annual production of 1.4 million t (dry weight, DW) in a farming area of 130,564 ha, depends on a safe and constant supply of sporelings each year that are nurtured in ca. 20 commercial hatchery enterprises in Shandong and Fujian provinces in the north and south, respectively. Extension of the kelp growing period in the sea relies on the use of the so-called summer sporeling. Its production is based on using recirculated cold water (5–10 °C) to allow gametogenesis to happen and thereafter the young sporophytes to grow in summer when the ambient seawater temperature (above 20 °C) is too high. This allows sporophytic sporelings to be transferred to open water in early autumn, 2 months earlier than naturally occurring plants in coastal populations. Although historically commercially viable and successful, there have been few reports to describe this hatchery process. In this case study, the entire process of a commercial hatchery manipulation in the 2015 production season at Lvshun, Dalian, northern China, is presented, including pre-treatment of parental plants, control of spore release, and control and adjustment of solar irradiance and temperature at different developmental stages of the plants as well as the nutrient supply regime over the entire course. Analyses of the data and process in this report is expected to shed light on future optimization of the technique as well as providing useful clues for other farmed kelp species in other regions of the world.

The endemic red seaweed Sarcothalia atropurpurea is a potentially valuable source of carrageenan, but no information is available on growing this species as a crop in New Zealand. Tetraspores released from wild-harvested reproductive thalli were settled onto strings and grown initially in tanks under two different day-lengths. Seeded strings were transplanted to open water at 2, 6 and 10 weeks after spore release. Gametophytic biomass was harvested twice by pruning plants approximately six and nine months after spore release. Sporeling length was correlated with day length during tank culture but these differences were not apparent after a period of open-water culture. Spores cultured with medium day-length for 6–10 weeks before transfer to open water grew into harvestable plants that contained similar amounts and types of carrageenan to that from wild material. These results demonstrate for the first time that aquaculture of gametophytic S. atropurpurea from tetraspores is possible.

In bryophytes, establishment can occur by a sexual or asexual process, but the production of spores enables colonization of a wider range of habitats and substrates than can asexual propagules. Successful germination is critical for establishment in a new environment. This paper addresses germination and sporeling development in Frullania ericoides, a leafy liverwort species. Fresh spores were inoculated in vitro in different culture strengths of Knop’s nutrient solution (one-fourth strength, half strength, full strength, one and a half strength and double strength), in order to evaluate the effects of this solution on spore germination and on the development of external protonema. On the first assessment, spore germination was observed at all the concentrations. Germination was endosporic, with cell division and proliferation, resulting in a globular protonema, within the spore wall. Beginning at the fourth week, the development of tightly concave primordial leaves was observed in all but the double-strength medium. Throughout the period of study, the treatments with lower concentrations exhibited external protonema with greater lengths. The double-strength treatment was statistically different from other treatments in at least two parameters. The results of this study demonstrate the potential of in vitro culture techniques for bryophyte spore studies and germplasm preservation.

The liverwort species, Marchantia polymorpha, shows environment-dependent morphological plasticity throughout its life cycle. Thalli, representing the predominant body form throughout most of this bryophyte's life cycle, grow with repeated dichotomous branching at the apex and develop horizontally under sufficient light intensity. Spores, after germination, produce a mass of cells, called sporelings, which then grow into thalli. Both thalli and sporelings, if grown under weak light conditions, form narrow shapes, and their apices grow toward the light source. These phototropic responses are specific to blue light and dependent on the blue-light receptor phototropin. This chapter provides several basic procedures, along with some tips, for designing and performing experiments with M. polymorpha to observe their phototropic responses, as well as methods for observing the localization of the phototropin "Mpphot" with a fluorescent protein tag.

The composition and diversity of phycospheric microbes are critical to the health of algae. Pyropia yezoensis is a popular edible seaweed in Asia and yellow spot disease (YSD) is a serious disease during its conchocelis sporeling culture period. We studied the changes of the bacterial flora in the phycosphere and the surrounding seawater of P. yezoensis conchocelis during a YSD outbreak and treatment. Four types of algal conditions were studied 1) healthy (no previous YSD); 2) YSD; 3) YSD being treated with ClO2 and 4) recovery (14 d after treatment). In the YSD samples, the microbial diversity was significantly decreased. The abundance of bacteria that were reported having algal dissolving and degrading functions (Kangiella, Alteromonas, Kordiimona, Tenacibaculum and Winogradskyella) was significantly increased, and some beneficial bacteria (Ruegeria and Bdellovibrio) had significantly decreased populations (P

Takakia lepidozioides is a rare and potentially relictual species of a genus that diverged early in the evolution history of mosses. Plants of Takakia grow very slowly in the wild and are almost too small (only ∼ 5 mm high) to be genetically manipulated. We collected Takakia plants with mature spore-bearing capsules from the Nyingchi region at about 4,000 meters elevation in the Tibetan Plateau. We analyzed the pH and inorganic elements of the soil below the population to then optimize the composition of the Beneck medium that is generally suitable for moss culture. We explored the effects of several factors on spore germination and gametophyte growth and established the conditions for efficient spore germination and rapid gametophyte propagation. The characterization of spore germination and early gametophyte development revealed that the sporeling of Takakia is thalloid as in Sphagnum and Andreaea. These results provide the foundation to facilitate genetic, physiological and biochemical research with T. lepidozioides and also developmental studies, which may contribute to our understanding of the morphological transformations in the early diversification of mosses.

Gracilariopsis lemaneiformis is an economically important red alga worldwide, and is currently cultured by vegetative propagation. However, information related to the practice of spore culturing in red alga, especially for this species, is limited. Here, we describe a successful pilot study that used tetraspores and carpospores for the industrial cultivation of G. lemaneiformis in Jiaozhou Bay, Qingdao, China. Morphological observation and culture studies of G. lemaneiformis spores were carried out in the laboratory and field. Related spore parameters of daily output, germination rate, survival number, and growth rate were determined. The results showed that the maximum daily output occurred on the 6th day for tetraspores, with a value of 5.71 ± 0.68 × 105 g− 1 fresh weight, and on the 4th day for carpospores, with the value of 1876 ± 153 carpospores per cystocarp. During early culturing, the development and growth of tetraspores and carpospores were very similar. Inoculation density variation had significant effects on germination rate, survival number, and growth rate, the highest values of which were recorded at medium density, high density, and medium density inoculation, respectively. After 3 weeks in the field, the tetrasporelings and carposporelings were ~ 3 cm in length. Different heights below sea level also had a notable effect on sporeling growth, and the optimal condition was 0.5 m below sea level. After further culturing, the sporelings showed fast adaptation to the sea with average growth rates of 6.21 ± 0.48% day− 1 for tetrasporophytes and 4.21 ± 0.36% day− 1 for gametophytes. This study has demonstrated that the mass cultivation of G. lemaneiformis using tetraspores and carpospores under controlled culture conditions is feasible, and has also provided valuable information for improving the only available culture method (vegetative propagation) for G. lemaneiformis in the field.

] Muller, J. 2014. Moosflora des Parks Sanssouci in Potsdam. Verhandlungen des Botanischen Vereins von Berlin und Brandenburg 147: 157–184. Muster, C., G. Gaudig, M. Krebs & H. Joosten. 2015. Sphagnum farming: the promised land for peat bog species? Biodiversity and Conservation 24(8): 1989–2009. Na, Y., S. R. Manchester, C. Sun & S. Zhang. 2015. The Middle Jurassic palynology of the Daohugou area, Inner Mongolia, China, and its implications for palaeobiology and palaeogeography. Palynology 39(2): 270–287. Nelson, G., P. W. Sweeney, L. E. Wallace, R. K. Rabeler, D. Allard, H. Brown, J. R. Carter, M. W. Denslow, E. R. Ellwood, C. C. Germain-Aubrey, E. Gilbert, E. Gillespie, L. R. Goertzen, B. S. Legler, D. B. Marchant, T. D. Marsico, A. B. Morris, Z. E. Murrell, M. Nazaire, C. Neefus, S. Oberreiter, D. Paul, B. R. Ruhfel, T. Sasek, J. Shaw, P. S. Soltis, K. Watson, A. Weeks & A. R. Mast. 2015. Digitization workflows for flat sheets and packets of plants, algae, and fungi. Applications in Plant Sciences 3(9): 1500065 [1–9]. Nelson, J. M. 2015. [Abstract] Interactions of the liverwort Marchantia polymorpha with its fungal endophytes. Page 72. In: Botany 2015: Science and Plants for People. Abstracts. July 25–29, 2015, Edmonton Alberta Canada. Nemeth, Cs. 2008. Contributions to the bryophyte flora of SuriBakonyalja, Bakony and Vertes Mts (NW Hungary). Flora Pannonica 6: 79–87. [In Hungarian with English abstract.] Nemeth, Cs. 2009. Data on the distribution of Dicranum tauricum Sapjegin in Hungary. Flora Pannonica 7: 51–55. [In Hungarian with English abstract.] Nilsson, S. 2015. Rapport fran Projekt Skanes mossor varen 2015. Myrinia 25(1): 56–60. [In Swedish with English summary.] Nishihama, R., K. Ishizaki, M. Hosaka, Y. Matsuda, A. Kubota & T. Kohchi. 2015. Phytochrome-mediated regulation of cell division and growth during regeneration and sporeling development in the liverwort Marchantia polymorpha. Journal of Plant Research 128(3): 407–421. Norden, B., A. Gardiennet, J.-P. Priou & P. Dobbeler. 2015. Bryocentria hypothallina (Hypocreales) — a new species on Metzgeria furcata. Ascomycete.org 7(4): 121–124. Novotný, I. & R. Mudrova. 2015. Ecology and distribution of the rare copper moss Streptocolea atrata (Bryopsida, Grimmiaceae) in the Czech Republic. Acta Musei Silesiae, Scientiae Naturales 64(1): 23–26. Nydahl, A. C., C. K. King, J. Wasley, D. F. Jolley & S. A. Robinson. 2015. Toxicity of fuel-contaminated soil to Antarctic moss and terrestrial algae. Environmental Toxicology and Chemistry 34(9): 2004–2012. Buck & Atwood: Recent literature on bryophytes 101

Sporeling and tissue propagation of agarophytic seaweed, Gracilaria fisheri, aimed to investigate the optimal shading colour for the preservation. This study was conducted under different shading colours indoor and outdoor experiments. The indoor experiment: tissues and sporelings of the seaweed were cultured in plastic chambers under fluorescent lights and were wrapped with white, green, blue and red colour PVC sheets of light intensity of 20 ± 1 μmol m−2 s−1. The outdoor tissues were cultured in the plastic tanks under different shadings of Saran sheets: white, green, blue and black covering the hoop-houses with light intensity of 110 ± 10 μmol m−2 s−1. The results showed that different shading colours strongly influenced growth of G. fisheri sporelings and tissues but little effect on pigmentation. After 40 days, the highest growth rates of G. fisheri tissues and sporelings were found under red light with 1.44 and 1.89% day−1 respectively. There was no significant difference (P > 0.05) on pigment content of tissues indoor, whereas it was found that chlorophyll concentration of the sporelings under green light with 416.8 μg g−1 fresh weight was significantly higher (P

Pyropia is an important cultivated seaweed in Northeast Asia. Yellow spot disease (YSD) commonly occurs in conchocelis sporeling cultures of Pyropia and causes huge economic losses. In this study, the isolated strain 117-T6 was identified as a pathogenic bacterium of YSD in Pyropia by Koch's postulates. Based on the morphological, physiological, and biochemical characteristics and multi-locus analysis of house-keeping gene sequences, this gram-negative bacterium was named as Vibrio mediterranei 117-T6. V. mediterranei 117-T6, including its intracellular and the extracellular extracts, caused both free-living conchocelis (FLC) and shell-borne conchocelis (SBC) to exhibit YSD symptoms within 12 days, indicating that the bacteria might attack Pyropia by secreting certain compounds that can be transported into algal cells, breaking down their structure and leading to death. During the infection, four stages were observed. 1) Prophase: a large number of pathogenic bacteria gathered around the algae. The conchocelis was in healthy condition at that time. 2) Early stage: organelle edges of Pyropia were blurred, mitochondria and chloroplasts became fluffy and unraveled, autophagosomes appeared. 3) Middle stage: cellular structures and contents of the algae kept being lost and degraded, cell walls became thin. 4) Late stage: the conchocelis bleached completely, and as the cell wall disintegrated gradually, the bacteria took over the remains of Pyropia. Finally, the whole algal cell disappeared, which may be the reason for shell bleaching. Our results show that YSD caused by V. mediterranei 117-T6 is a severe disease of algal disintegration caused by bacterial secretion, with a short onset and severe symptoms.

Undaria pinnatifida is an annual brown kelp growing naturally in coastal areas as a major primary producer in temperate regions and is cultivated on an industrial scale. Kelps have a heteromorphic life cycle characterized by a macroscopic sporophyte and microscopic sexual gametophytes. The sex-dependent effects of different environmental factors on the growth and maturation characteristics of the gametophyte stage were investigated using response surface methodology. Gametophytes were taken from three sites in Japan: Iwate Prefecture, Tokushima Prefecture, and Kagoshima Prefecture in order to confirm the sexual differences in three independent lines. Optimum temperature and light intensity were higher for males (20.7&ndash, 20.9 &deg, C and 28.6&ndash, 33.7 &micro, mol m&minus, 2 s&minus, 1, respectively) than females (16.5&ndash, 19.8 &deg, C and 26.9&ndash, 32.5 &micro, 1), and maturity progressed more quickly in males than females. Optimum wavelengths of light for growth and maturation of the gametophytes were observed for both blue (400&ndash, 500 nm, &lambda, max 453 nm) and green (500&ndash, 600 nm, &lambda, max 525 nm) lights and were sex-independent. These characteristics were consistent among the three regional lines. Slower growth optima and progress of maturation could be important for female gametophytes to restrict fertilization and sporophyte germination to the lower water temperatures of autumn and winter, and suggest that the female gametophyte may be more sensitive to temperature than the male. The sexual differences in sensitivity to environmental factors improved the synchronicity of sporeling production.

The aquatic quillwort Isoetes lacustris L. survived five decades of severe acidification in Cerne Lake (Bohemian Forest, central Europe), but failed to reproduce. To detect which ontogenetic stage is more affected by water acidification and to identify water chemistry thresholds for successful recruitment, experiments were carried out on quillwort sporelings grown in the laboratory, using different pH (4–8) and aluminium (Al) concentrations (0–1000 μg/L). Growth-related symptoms and Al accumulations in tissues were compared with those observed in sporelings collected in the lake. In the laboratory, the threshold Al concentrations ≥100 μg/L and pH ≤ 5.0 reduced absorptive organs (macrogametophyte rhizoids, roots, and root hairs), so that the ratio of below-ground to above-ground sporeling biomass decreased to

Chimerism theoretically increases the genetic heterogeneity of coalescing organisms, which in turn may increase phenotypic variability in chimeras, allowing them greater tolerance to environmental changes when compared with non-coalescing individuals. In order to test this hypothesis, we compared abiotic stress tolerance between coalescing and non-coalescing organisms. The specific daily growth was compared using discs formed with 1, 5, and 20 Mazzaella laminarioides carpospores. These were cultivated under three different temperature (6, 12, and 25 °C) and salinity (10, 35, and 50 g L−1) conditions. Growth of the disc area was measured after 30 days of cultivation under controlled conditions. Sporeling survival similarly was calculated under each of these temperatures and salinities in order to record whether stress-resistant phenotypes were present. The results showed that, under stressful conditions, non-coalescing specimens experienced a significant reduction in the specific growth rate compared with those under non-stressful conditions. The reduction in growth falls off, however, as the number of coalescing spores increases. The cultivation of spore populations also indicates the presence of sporelings with the capacity to survive alone in stressful situations. The results suggest that the coalescing discs exhibit higher tolerance to environmental stressors than non-coalescing discs, allowing them to survive and grow under these conditions. This can be explained by an increase in phenotypic plasticity, resulting from greater genetic heterogeneity due to somatic fusion of a larger number of spores.

Notothylas vitalii is an endemic Brazilian hornwort species, easily identifi ed by the absence of pseudoelaters and columella, and the presence of yellow spores. Plant material was collected in Recife, Brazil, and the spores were sown onto Knop’s medium, germinating after thirty days only with the presence of light. Germination occurred outside the exospore, and only after the walls had separated into three or four sections did a globose sporeling initiate its development. Following longitudinal and transversal divisions, the initial loose mass of cells became a thalloid gametophyte, subsequently developing into a rosette-like juvenile thallus with fl attened lobes. Additional information concerning sporeling types in key genera of hornworts, such as and FoliocerosPhymatoceros, will be crucial for inferring the possible ancestral type and the evolution of this trait among hornworts. Our study supports the necessity of supplementary studies on sporeling development, combined with morphological and phylogenetic investigations, to help elucidate the evolution of the Anthocerotophyta and their distribution patterns.

Dolichomitriopsis diversiformis is an endemic and threatened moss in eastern Asia. In vitro culture and light microscopic observation were employed to study its developmental process from spore germination to the formation of young gametophyte, as well as effects of light and temperature on its spore germination and protonemal development. Microscopic observations revealed that its spores were positively photoblastic with exosporous germination, and sporelings were classified as the bryum-type. Light and dark conditions were compared to understand their effects on spore germination. In 24 h continuous illumination, all spores germinated in both 20 ℃ and 25 ℃. In contrast, in darkness at 20 ℃ from one to 30 days, spores did not germinate. However, when dark-cultured spores transferred to continuous light, they started to geminate in 48 hours. In addition, effects of 20 ℃ and 25 ℃ on sporeling were compared. In a continuous light condition, sporelings grew longer and developed more protonema branches in the 20 ℃ treatment than in the 25 ℃ treatment. It was interesting that a phenomenon of slow and hysteretic spore germination and protonematal development was observed in natural light and room temperature (5-10℃). These observations suggest that spore germination and sporeling development is regulated by light and temperature.

Light regulates various aspects of development throughout the life cycle of sessile land plants. Photoreceptors, such as the red (R) and far-red (FR) light receptors phytochromes, play pivotal roles in modulating developmental programs. Reflecting high developmental plasticity, plants can regenerate tissues, organs, and whole bodies from varieties of cells. Among land plants, bryophytes exhibit extraordinary competency of regeneration under hormone-free conditions. As an environmental factor, light plays critical roles in regeneration of bryophytes. However, how light regulates regeneration remains unknown. Here we show that using the liverwort Marchantia polymorpha, which contains a single phytochrome gene, the phytochrome regulates re-entry into the cell cycle and cell shape in newly regenerating tissues. Our morphological and cytological observations revealed that S-phase entry of G1-arrested epidermal cells around the midrib on the ventral surface of thallus explants was greatly retarded in the dark or under phytochrome-inactive R/FR cycle irradiation conditions, where, nevertheless, small, laterally narrow regenerants were eventually formed. Thus, consistent with earlier descriptions published over a century ago, light is not essential for, but exerts profound effects on regeneration in M. polymorpha. Ventral cells in regenerants grown under R/FR cycle conditions were longer and narrower than those under R cycle. Expression of a constitutively active mutant of M. polymorpha phytochrome allowed regeneration of well grown, widely expanded thalli even in the dark when sugar was supplied, further demonstrating that the phytochrome signal promotes cell proliferation, which is rate-limited by sucrose availability. Similar effects of R and FR irradiation on cell division and elongation were observed in sporelings as well. Thus, besides activation of photosynthesis, major roles of R in regeneration of M. polymorpha are to facilitate proliferation of rounder cells through the phytochrome by mechanisms that are likely to operate in the sporeling.