We are grateful to Garth Ilsley for his support in establishing the culture system. We acknowledge Ritsuko Suyama&#8217;s and Sylvain Guillot&#8217;s contributions to early sampling and species identification efforts. Special thanks are due to Hiroki Nishida, Takeshi Onuma, and Tatsuya Omotezako for their generous support and guidance throughout, including initial establishment of the local culturing system and sharing animals and microalgal culture. We also thank Daniel Chourrout, Jean-Marie Bouquet, Anne Aasjord, Cristian Ca&#241;estro, and Alfonso Ferr&#225;ndez-Rold&#225;n for sharing their expertise on sampling and culturing. Jai Denton, Charles Plessy, and Jeffrey Jolly provided invaluable feedback on the manuscript. Charlotte West formulated a generalized equation for algal calculation. Finally, we thank OIST for funding, Mary Collins and the OIST Fieldwork Safety Committee for advice on safe sampling procedures, the staff of OIST machine shop for the construction of culturing and sampling equipment, and Koichi Toda for delivering seawater.

The author has nothing to declare.

To facilitate flexibility in the establishment of O. dioica culture, it is important to understand the animals&#8217; natural habitat. Seasonal data provides information about the ranges of physical parameters, which can be used to guide laboratory culturing conditions. It also helps in understanding seasonal fluctuations in the abundance of animals. In Okinawa, O. dioica is most reliably found from June to October. However, in Tokyo bay, populations peak in February and October41. Although culturing of O. dioica is often reported at 20 &#176;C or lower27,28,29, Okinawan O. dioica shows better survival at temperatures above 20 &#176;C; this might be explained by the fact the minimum surface seawater temperature in Okinawa is ~20 &#176;C (Figure 6). The abundance of O. dioica might also be influenced by phytoplankton blooms42 and predator abundance43,44. Regardless of where O. dioica are collected, understanding the seasonality of local populations maximizes the chance of sampling and culturing success.
Given the appropriate season and location, net sampling is an effective way of collecting large numbers of Oikopleura with minimal effort. Plankton nets with smaller mesh size (60-70 &#181;m) may also be used to collect all stages of the animals. Fully mature animals are rarely found in the net, perhaps due to their fragility at the end of the lifecycle. Therefore, species identification followed by sampling is achieved by microscopic observation of subchordal cells. Mature individuals usually appear one or two days post-sampling as animals continue to grow in the laboratory. Although net sampling is efficient, alternative sampling methods might be necessary in different circumstances. For example, net sampling near urban areas can collect large numbers of phytoplankton, making it difficult to isolate Oikopleura. In such cases, simple bucket sampling to collect surface seawater or boat sampling from areas beyond the harbor are recommended. The results showed that the gradual change in salinity due to consecutive days of rain did not affect the abundance of O. dioica; however, shore sampling immediately after extreme weather events such as tropical cyclones should be avoided. These events cause sudden and drastic biogeochemical changes in a sheltered body of water45,46. The stormwater runoff may carry pollutants, sediments, and excess nutrients, which increase turbidity and lower water quality47. Filter-feeding plankton, such as Oikopleura, may be especially susceptible to these changes due to their mode of feeding and limited mobility. In such a circumstance, we recommend postponing sampling for a few days until the local conditions return to normal.
The introduction of a multi-step filter system is essential to maintain small, filter-feeding organisms such as O. dioica. Using poorly filtered seawater (for example, a 25 &#181;m mesh in the previous culture system), the culture was often unstable especially during summer, potentially due to the higher abundance of phytoplankton. Although some phytoplankton are beneficial to O. dioica growth, others produce biotoxins that can cause abnormal development of O. dioica embryos48. In addition, a high concentration of diatoms such as Chaetoceros spp. are potentially harmful to O. dioica growth as they can possess long setae which can clog the house and prevent efficient feeding49. We frequently observed houses of small animals being clogged by C. calcitrans setae; therefore, we now feed C. calcitrans only to animals at Day 2 and older (Table 3).
Although it was not a problem here, small scale long-term culturing of O. dioica can experience sudden drops in population size due to a genetic bottleneck; in such cases, Mart&#237;-Solans et al.29 recommend adding new wild individuals to the culture every 20 generations.
The Oikopleura culture system is flexible. A stable culture can be established within a week. Long-term culturing of O. dioica is possible on a modest budget with non-specialist equipment. The daily effort required for the maintenance of 5-10 beakers of Oikopleura is generally less than 2 hours with 2 people. O. dioica can also be maintained in artificial seawater, which is beneficial to those without access to natural seawater28. Long-term storage of algal food is possible using solid-culture and cryopreservation29. Moreover, O. dioica sperm can be cryopreserved, and remain viable for more than a year50. All of these factors mean that cultures can be easily re-established. Finally, past experience with accidental culturing of Pleurobrachia sp. may suggest that the culturing system developed for Oikopleura could potentially be extended to a broader community of fragile pelagic organisms.
O. dioica continues to provide powerful insights into various biological fields. An understanding of local seasonality, a meticulous culture system, and a few dedicated individuals allow effective culture to be established with little effort. Oikopleura culture system provides the baseline resources to investigate a wide range of biological fields relating to ecology, development, genomics, and evolution of this unique marine chordate.

Model organisms have been instrumental in addressing many biological questions including those relating to development, genetics, and physiology. Moreover, additional model organisms facilitate new discoveries and therefore are crucial to achieve a greater understanding of nature1,2. Marine zooplankton are diverse groups of organisms that play an important role in ocean ecosystems3,4,5,6. Despite their abundance and ecological importance, gelatinous organisms such as planktonic tunicates are often under-represented in plankton biodiversity studies because their transparency and fragility make field collection and identification challenging7,8. Adapted sampling techniques and laboratory culturing allow closer observation of the animals in vitro, which has furthered the knowledge in the biology of planktonic tunicates9,10,11,12.
Larvaceans (Appendicularians) are a class of free-swimming marine tunicates comprising around 70 described species worldwide8,13. As they are one of the most abundant groups within zooplankton communities14,15,16,17, larvaceans represent a primary food source for larger planktonic organisms such as fish larvae18,19. Unlike ascidians-the sessile tunicates-larvaceans retain a tadpole-like morphology and remain planktonic throughout their lives20. Each animal lives inside a self-built, intricate filter-feeding structure known as a house. They accumulate particulates in their houses by creating water currents through the undulating motion of their tails21. Clogged houses are discarded throughout the day, some of which form carbon aggregates and eventually sink to the seabed22; thus, larvaceans play a major role in global carbon flux23. Most species are reported to live in the pelagic zone within the upper 100 m of the water column13; however, the giant larvacean Bathochordaeus is known to inhabit the depths of 300 m24. A study on Bathochordaeus in Monterey Bay, California revealed that the animals also serve as a biological vector of microplastics, suggesting a potential importance in understanding the role of appendicularians in the vertical transport and distribution of microplastics in the oceans25.
Oikopleura dioica, a species of larvacean, has attracted attention in recent years as a model organism due to several remarkable characteristics. It is commonly reported throughout the world&#8217;s oceans. It is especially abundant in coastal waters26, which allows easy sampling from the shore. Long-term, stable culturing is possible with both natural and artificial seawater27,28,29. Temperature dependent generation times are as short as 4-9 days in laboratory conditions. It has high fecundity with each female capable of producing &#62;300 eggs throughout the year. As a tunicate, it occupies an important phylogenetic position for understanding chordate evolution30,31. At 70 Mb, O. dioica has the smallest identified genome amongst all chordates32. Among larvaceans, O. dioica is the only described non-hermaphroditic species thus far33.
The first successful O. dioica culture with laboratory grown microalgae was reported by Paffenho&#776;fer34. The original culture protocol using synchronous motors and paddles was developed by Fenaux and Gorsky35 and later adopted by multiple laboratories. More recently, Fujii et al.36 reported O. dioica culturing in artificial seawater, a robust culture system and field collection were described by Bouquet et al.27 and an optimized protocol for a simplified, affordable system was reported by Marti-Solans et al.29. Aside from the traditional Oikopleura culture system, a newly reported design with a double tube rearing tank also has the potential to culture Oikopleura sp.37.
We present a detailed protocol for initiating an O. dioica monoculture based on a combination of protocols developed by major Oikopleura research groups at the Sars International Centre for Marine Molecular Biology27, the University of Barcelona29, Osaka University28, and our own observations. In previously published culture protocols, detailed information regarding the composition of algal media, shore sampling techniques, and Oikopleura identification were only roughly described, leaving a lot of ambiguity. Here, with the aid of visual information in the video protocol, we have assembled all the critical information needed to set up an O. dioica culture from the ground up in a straightforward, step-by-step manner. We describe how to distinguish O. dioica from another commonly reported species, O. longicauda, which is one of the most challenging steps. Although the existing culture systems are applicable for the cultivation of O. dioica worldwide, we highlight the importance of protocol adjustment based on local environmental conditions. The presented information combines widely published data as well as knowledge gained through experience. The current protocol is ideally suited for researchers interested in establishing a culture from scratch.

<table><tbody><tr><td>Activated charcoal</td><td>Sigma</td><td>C2764-2.5KG</td><td></td></tr><tr><td>Alluminum pulley</td><td>Rainbow Products</td><td>10604-10607</td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4501-100MG</td><td></td></tr><tr><td>Boric acid</td><td>Wako</td><td>021-02195</td><td></td></tr><tr><td>Cobalamin (B12)</td><td>Sigma</td><td>V2876-100MG</td><td></td></tr><tr><td>Cobalt(II) chloride hexahydrate</td><td>Wako</td><td>036-03682</td><td></td></tr><tr><td>Copper(II) sulfate pentahydrate</td><td>Wako</td><td>039-04412</td><td></td></tr><tr><td>Disodium edetate hydrate</td><td>Wako</td><td>044-29525</td><td></td></tr><tr><td>Hexaammonium heptamolybdate tetrahydrate</td><td>Wako</td><td>019-03212</td><td></td></tr><tr><td>Hexagon wrench</td><td>Anex</td><td>No.6600</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Wako</td><td>080-01066</td><td></td></tr><tr><td>Iron(III) chloride hexahydrate</td><td>Wako</td><td>091-00872</td><td></td></tr><tr><td>Jebao programmable auto dosing pump</td><td>Jebao</td><td>DP-4</td><td></td></tr><tr><td>Magnet pump</td><td>REI-SEA</td><td>RMD-201</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Wako</td><td>134-15302</td><td></td></tr><tr><td>Polypropylene wound cartridge filter</td><td>Advantec</td><td>TCW-10N-PPS</td><td></td></tr><tr><td></td><td></td><td>TCW-5N-PPS</td><td></td></tr><tr><td></td><td></td><td>TCW-1N-PPS</td><td></td></tr><tr><td>Screwless terminal block</td><td>SATO PARTS</td><td>SL4500</td><td></td></tr><tr><td>Simple plankton net</td><td>RIGO, Japan</td><td>5512-C</td><td></td></tr><tr><td>Sodium metasilicate</td><td>Sigma</td><td>307815-1KG</td><td></td></tr><tr><td>Sodium nitrate</td><td>Wako</td><td>195-02545</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>MP Biomedicals</td><td>194740</td><td></td></tr><tr><td>Streptomycin sulfate salt</td><td>Sigma</td><td>S6501-25G</td><td></td></tr><tr><td>Synchronous electric motor</td><td>Servo</td><td>D5N6Z15M</td><td></td></tr><tr><td>Thiamin hydrochloride</td><td>Wako</td><td>201-00852</td><td></td></tr><tr><td>UV sterilizer</td><td>Iwaki</td><td>UVF-1000</td><td></td></tr><tr><td>Zinc chloride</td><td>MP Biomedicals</td><td>194858</td><td></td></tr></tbody></table>

streamlined sampling and cultivation of the pelagic cosmopolitan larvacean, oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

Watch this Scientific Journal Video about Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica at JoVE.com

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact ...

streamlined-sampling-cultivation-pelagic-cosmopolitan-larvacean

Research

JoVE Journal

Environment

8.8K Views.  Okinawa Institute of Science and Technology Graduate University. Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

Video: Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Developmental Biology

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion annually. The global decline of giant kelp forests due to climate-driven ecological stressors underscores the need for innovative restoration strategies. An emerging restoration technique known as &#39;green gravel&#39; aims to seed young kelps over large areas without extensive underwater labor and represents a promising restoration tool due to cost-effectiveness and scalability. This video article illustrates a protocol and tools for culturing giant kelp, Macrocystis pyrifera. It also provides a resource for further studies to address the successes and limitations of this method in field settings. We outline field and laboratory-based methods for collecting reproductive tissue, sporulating, inoculating, rearing, maintaining, and monitoring substrates seeded with early life stages using the &#39;green gravel&#39;&#160;technique. The protocol simplifies and centralizes current restoration practices in this field to support researchers, managers, and stakeholders in meeting kelp conservation objectives.

This protocol describes the field collection and regular laboratory maintenance of substrates seeded with canopy-forming giant kelp for use in restoration trials to address the success and limitations of the 'green gravel' technique in field settings.

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion ...

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Presented here is ovarian tissue oocyte-in vitro maturation (OTO-IVM), an accessible technique within a medical assisted reproduction (MAR) laboratory offering realistic additional fertility preservation options to patients who need ovarian tissue cryopreservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Bioengineering

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies&#8212;microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

We describe protocols to prepare oocysts and purify sporozoites for studying infection of human intestinal and airway organoids by Cryptosporidium parvum. We demonstrate the procedures for microinjection of parasites into the intestinal organoid lumen and immunostaining of organoids. Finally, we describe the isolation of generated oocysts from the organoids.

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an ...

Immunology and Infection

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Biology

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Summary

Explore More Videos

Chapters in this video

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Establishment of a Clonal Culture of Unicellular Conjugating Algae

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants