Reviewer
Adam Idoine
  • Post-Doc, Carnegie Institution
Research fields
  • Molecular Biology
Mating and Tetrad Dissection in Chlamydomonas
Author:  Frej Tulin, date: 04/05/2019, view: 5013, Q&A: 0
The ability to carry out controlled genetic crosses has contributed to making the unicellular green alga Chlamydomonas reinhardtii an important model organism for the green plant kingdom (Merchant et al., 2007). This protocol describes a simple and reliable method for crossing Chlamydomonas strains of opposite mating types and dissecting out the meiotic progeny for genetic analyses. The protocol should apply in general to motile strains with functional flagella but has only been routinely used with strains in the CC-124/125 background. A key aspect is access to a microscope equipped with a microdissection needle for manipulation of individual cells with high accuracy. For an extended description of the mating process, the reader is referred to the excellent article by Dutcher (1995). For variations on this procedure, see Note 1.
Agrobacterium-mediated Transformation of Nicotiana tabacum cv. Xanthi Leaf Explants
Authors:  Ana Lilia Rosales-Campos and Abel Gutiérrez-Ortega, date: 01/20/2019, view: 11138, Q&A: 0
Plant transformation has become an important technology for the large-scale production of a number of goods, ranging from vaccines and pharmaceutical products to biofuel. A plant that is frequently used for this purpose is Nicotiana tabacum due to several advantages: it can be easily regenerated, its genome is completely sequenced and genetic transformation is straightforward. We describe here the most common method for tobacco genetic transformation, which involves Agrobacterium tumefaciens. Many plasmid vectors are compatible with this bacterium, allowing the transformation of a wide range of plant species, as well as the insertion of more than two genes of interest in the plant genome. Furthermore, the capability to transfer a single copy DNA fragment to its host reduces the probability to switch off the gene or genes of interest. Here, we present a simple and reliable methodology to transform tobacco leaf explants using Agrobacterium tumefaciens to insert two genes of interest.
Growth of Chlamydomonas reinhardtii under Circadian Conditions
Authors:  Volker Wagner and Maria Mittag, date: 08/20/2018, view: 5500, Q&A: 0
The green biflagellate unicellular alga Chlamydomonas reinhardtii serves as a model to study fundamental biological processes such as the structure and function of flagella or light-driven processes including photosynthesis, its behavioral responses, life cycle and circadian clock. Light-dark, as well as temperature cycles, are major Zeitgebers to entrain the algal circadian clock. In C. reinhardtii, several processes are under circadian control and many clock-controlled genes and/or proteins have been found in the past decades as well as components of the endogenous oscillator. Here, we describe a protocol for the growth of C. reinhardtii for the synchronization and analysis of its circadian clock.
Micropropagation of Prickly Pear by Axillary Shoot Proliferation
A protocol for the axillary bud proliferation of prickly pear (Opuntia; Cactaceae) is presented. This genus is widely used as a crop in the arid and semi-arid areas of the globe worldwide, providing numerous benefits for human and animal consumption. In vitro culture for axillary bud proliferation is of great use to obtain a large quantity of plants in a short period of time, with potential uses in production and for the preservation of endangered species of the Opuntia genus.

The optimal medium for Opuntia in vitro culture consists of Murashige and Skoog medium (MS) and L2 vitamins. To increase the yield of the axillary bud proliferation, we recommend the addition of plant growth regulators (PGRs). This work suggests a 15 d incubation in the medium with 2.2 mg/L of benzyl aminopurine (BA) after which the explants are transferred to the medium without PGRs. We explain as well how to adapt the plant to ex vitro conditions.
Phenol-based Extraction of RNA from Chlamydomonas reinhardtii
Authors:  Emanuel Sanz-Luque and Amaury de Montaigu, date: 07/05/2018, view: 5928, Q&A: 0
RNA extraction is a basic procedure in molecular biology and a wide variety of commercial kits are available. Some of these kits have been successfully used in Chlamydomonas. However, in some cases RNA quality and quantity may be dramatically reduced depending on the strains and/or the conditions where cells were grown or treated. Phenol-based protocols are the most robust methods to obtain both high quantity and quality RNA from any strain of this alga grown in any condition. Here, we describe an easy and cheap protocol using phenol but avoiding the acute toxicity of guanidine isothiocyanate present in the commercial phenol-based mixtures.
Total RNA Extraction from Dinoflagellate Symbiodinium Cells
Author:  Tingting Xiang, date: 06/05/2018, view: 4415, Q&A: 2
Dinoflagellates are unicellular algae that can have photosynthetic or nonphotosynthetic lifestyles. Dinoflagellates in the genus Symbiodinium can enter endosymbiotic associations with corals, providing the metabolic basis for the highly productive and biologically diverse coral-reef ecosystems (Hoegh-Guldberg, 1999), as well as with other cnidarians, including sea anemones and jellyfish, and non-cnidarian hosts (Trench, 1993; Lobban et al., 2002; Mordret et al., 2016).

Here, I describe a protocol for isolating total RNA from Symbiodinium cells.
Isolation of Genomic DNA from Chlamydomonas reinhardtii
Author:  Tyler M Wittkopp, date: 05/05/2018, view: 13415, Q&A: 0
Chlamydomonas reinhardtii is a soil-dwelling eukaryotic green alga that is widely used as a laboratory model organism for research on photosynthesis, ciliary biology, lipid metabolism and many other aspects of cell biology and physiology. With sequenced nuclear, chloroplast and mitochondrial genomes, Chlamydomonas is also an excellent organism for genetics and genomics research. This protocol describes the isolation of genomic DNA from Chlamydomonas using a standard phenol:chloroform extraction method followed by ethanol precipitation. The protocol requires minimal lab materials, takes approximately 4 h to complete, and can also be used for isolation of genomic DNA from other eukaryotic green algae.
Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation
Author:  Tyler M Wittkopp, date: 05/05/2018, view: 9657, Q&A: 1
The unicellular green alga Chlamydomonas reinhardtii is an important model organism for studying photosynthesis, acclimation to abiotic stress, cilia biology, and many other biological processes. Many molecular biology tools exist for interrogating gene function including the ability to easily transform the nuclear genome of Chlamydomonas. While technical advances such as TALENs, ZFNs and CRISPR are making it easier to precisely edit the nuclear genome, the efficiency of such methods in Chlamydomonas is at present very low. In contrast, random insertion by nuclear transformation tends to be a much more efficient process. This protocol describes a method for transformation of the Chlamydomonas nuclear genome by electroporation. The protocol requires at least 3 days of work and generally results in the appearance of small colonies within 1-2 weeks.
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