Fatty acids (FAs) are carboxylic acids with long aliphatic chains that may be straight, branched and saturated or unsaturated. Most of the naturally occurring plant FAs contains an even number of carbon (C4-C24). FAs are used in food and pharmacological industries due to their nutritional importance. In addition, FAs are considered as a promising alternative for the production of biodiesel from terrestrial plant biomass. To establish commercial applications, more reliable analytical methods are needed for the identification, quantification, and composition determination of FAs. Here, we describe a relatively rapid and sensitive method for the extraction, identification, and quantification of FAs from a small quantity of plant tissue. The method includes steps of lipid extraction, conversion of lipid to fatty acid methyl esters (FAMEs) by transmethylation, identification and quantification of FAMEs using gas chromatography-mass spectrometry (GC-MS). In this protocol, an internal standard is added prior to GC-MS analysis. The amount of each FA is calculated from its peak area relative to the peak area of the internal standard.

The shoot apical meristem is the origin of bamboo wood. Its structure and morphology are important for maintaining the normal development of bamboo wood. However, the traditional method to describe the morphology of the shoot apical meristem in bamboo or other plants only depends on qualitative approaches. Here we present a protocol for precisely describing the morphology of bamboo shoot apical meristem, which is adapted from our recently published papers (Shi et al., 2015; Wei et al., 2017).

The shapes of chloroplasts and the architectures of internal thylakoid membranes are altered by growth and environmental changes (Lichtenthaler et al., 1981; Kutik, 1985; Terashima and Hikosaka, 1995). These morphological alterations proceed via transitional intermediates, during which dynamic and heterogeneous thylakoid membranes are observed in cells (Nozue et al., 2017). Light microscopy is useful for the detection of morphological differences in chloroplasts. The thylakoid architecture of such morphologically variable chloroplasts is confirmed by transmission electron microscopy (TEM). The method of monitoring structural variation by light microscopy in combination with electron microscopy is described.

Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation protocol using olive somatic embryos as explants (Torreblanca et al., 2010). Embryogenic cultures derived from radicles of matured zygotic embryos are infected with Agrobacterium tumefaciens, AGL1 strain, containing a binary plasmid with the gene of interest and the nptII selection gene. After a meticulous selection procedure, carried out using solid and liquid media supplemented with paromomycin, the putative transformed lines are established. A preliminary confirmation of their transgenic nature is carried out through PCR amplification. Afterwards, plants can be obtained through an efficient regeneration protocol, whose main characteristics are the use of a low-ionic-strength mineral formulation, a phase in liquid medium for synchronization of cultures and the use of semi-permeable cellulose acetate membranes for embryo maturation (Cerezo et al., 2011). Final confirmation of transgene insertion is carried out through Southern or Northern analysis using leaf samples of regenerated plants.

Silica cells are specialized leaf epidermal cells in grasses with almost the whole cell volume filled with solid silica. In sorghum, silica deposition in silica cells takes place in young, elongating leaves around the mid-length of the leaf. We developed a protocol for estimating the level of silica cell silicification in Sorghum bicolor leaves using in situ charring method (Kumar et al., 2017a). Here, we provide greater details on our protocol and method of image analysis. Although we based our protocol on sorghum, this protocol can be extended for estimating silica cell silicification level in any grass species.

Male gametes (spermatozoids) are the only motile cells produced during the life cycle of land plants. While absent from flowering and most cone-bearing plants, motile cells are found in less derived taxa, including bryophytes (mosses, liverworts and hornworts), pteridophytes (lycophytes and ferns) and some seed plants (Ginkgo and cycads). During development, these cells undergo profound changes that involve the production of a locomotory apparatus, unique microtubule (MT) arrays, and a series of special cell walls that are produced in sequence and are synchronized with cellular differentiation. Immunogold labeling in the transmission electron microscope (TEM) provides information on the exact location and potential function of macromolecules involved with this developmental process. Specifically, it is possible to localize epitopes to proteins that are associated with the cellular inclusions involved in MT production and function. Spermatogenesis in these plants is also ideal for examining the differential expression of carbohydrates and glycoproteins that comprise the extracellular matrixes associated with the dramatic architectural changes in gamete shape and locomotory apparatus development. Here we provide methodologies using monoclonal antibodies (MAbs) and immunogold labeling in the TEM to localize macromolecules that are integral to spermatozoid development.

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the ‘Hartig net’, where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor ³²Phosphate efflux from the fungus previously incubated with ³²P-orthophosphate.

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied ³²P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing ³²P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after ³²P supply in order to avoid overestimation of ³²P efflux into the medium.

Here we present a protocol that describes how to image the structure of the olive axillary bud meristem with a scanning electron microscope (SEM) in order to characterize its identity and developmental stage. Briefly, the specimen is fixed with glutaraldehyde, saturated with ethanol, dried in a critical point dryer (CPD) system, dissected, coated with a conducting material and imaged with a scanning electron microscopy (SEM).

The only motile cells produced in land plants are male gametes (spermatozoids), which are reduced to non-flagellated cells in flowering plants and most gymnosperms. Although a coiled architecture is universal, the complexity of land plant flagellated cells varies from biflagellated in bryophytes to thousands of flagella per gametes in the seed plants Ginkgo and cycads. This wide diversity in number of flagella is associated with vast differences in cell size and shape. Scanning electron microscopy (SEM) has played an important role in characterizing the external form, including cell shape and arrangement of flagella, across the varied motile gametes of land plants. Because of the size and scarcity of released swimming sperm, it is difficult to concentrate them and prepare them for observation in the SEM. Here we detail an SEM preparation technique that yields good preservation of sperms cells across plant groups.