RNA in situ hybridization is a method for visualizing spatiotemporal transcript accumulation in cells and tissues. The method provides clear resolution, is highly sensitive and specific, and can uncover gradients of transcript accumulation within a histologically-intact tissue, which is not possible currently with other methods for transcript detection. RNA in situ hybridization, however, is not a quantitative approach for gene expression. Protocols for RNA in situ hybridization have numerous steps that can span several days of work, complicating troubleshooting procedures. Here, we build on previously published RNA in situ hybridization protocols optimized for paraffin-embedded and sectioned maize tissue (Jackson, 1991; Long et al., 1996 ; Javelle et al., 2011) by providing additional measures for optimized transcript detection.

Graphic abstract:


Workflow for RNA in situ hybridization

Hydroponic approaches have been a boon for root research by facilitating root-feeding studies as well as secretion analysis. Results have aided our understanding of root-shoot signaling, transport, hormone function and more. However, existing approaches are often restricted to small plants and seedlings by prohibitive cost or availability of experimental substrates. In addition to this, research on the hormone, strigolactone (SL) has been constrained in species like maize by a lack of specific assays for quantifying responses. Here a low-volume hydroponic approach was developed for growing maize plants to the 3-leaf stage (about 2.5 weeks and 12 cm tall at the 2^nd leaf collar) using 4 plants with 1 L of aerated media. This protocol also allowed development of an assay for root-to-shoot impacts of the SL analog, rac-GR24, which has a prominent, easily-quantifiable effect on outgrowth of lateral buds on maize shoots. Advantages of the protocol include the cost-effective ease and precision of its capacity for quantifying root-to-shoot SL responses. In addition, the low-volume hydroponic approach can be readily adapted for continuous or pulse-type root-feeding studies, root-labeling experiments, and/or secretion sampling in maize and other large, C4 grasses like sorghum, sugarcane, and miscanthus.

Brachypodium distachyon is a model grass closely related to wheat and barley. New resources and methods are being developed and Brachypodium is becoming a popular model for developmental biology, cell wall biology, (phylo) genomics, and genome evolution (Scholthof et al., 2018). Sterilization of seeds and seedling growth on plates is the first step to start working with Brachypodium. This is a cheap and straightforward protocol that was successfully used before (Raissig et al., 2016 and 2017).

The protocol provides fully detailed steps for preparing microscopic slides of acrylamide-embedded maize meiotic cells. This method is particularly useful for examining chromatin structure and chromosome arrangement without destroying the three-dimensional organization of the nucleus.

Histology incorporates microtechnique, the preparation of living material for observation and microscopy to study the anatomy of cells, tissues and organs. Interpreting histological patterns in a specimen may help to correlate form and function at the cellular, tissue and organ levels. Many stain options and staining methods (“schedules”) for plant tissues have been described previously (Johansen, 1940; Jensen, 1962; O’Brien and McCully, 1981; Sylvester and Ruzin, 1994; Ruzin, 1999). Here, we present a straightforward, inexpensive protocol that uses Toluidine Blue O (TBO) to stain maize tissue. TBO is a general metachromatic stain that can be used on paraffin-embedded histological sections, as well as fixed, hand-sectioned material. The value of TBO as a stain is the inherent metachromasia, whereby the dye is altered by pH resulting in violet blue color variants. This property is particularly useful even in sectioned material for identifying acidic components of plant cells. We demonstrate the utility of TBO staining for cellular-, tissue- and organ-level studies using maize seedling root, shoot apex and mature leaf (Figure 2).

This protocol combines a classical chromosome spreading technique with 3-dimensional image reconstruction for visualization of bivalent configurations during meiosis to aid chiasma counting.

This protocol describes a quick method for immunolocalization of meiotic proteins in maize. It includes a fixation step that allows for long-term storage of material and provides good preservation of chromatin structure.

Maize is an important model organism for understanding plant traits essential for proper growth and germination. One type of growth, skotomorphogenesis, occurs in the absence of light. Seedlings grown in the absence of light exhibit dramatic differences in stem and leaf development compared to light-grown plants. Dark-growth conditions require the use of highly controlled plant growth environments. Here, we provide step-by-step instructions for creating a soilless and dark plant growth environment for maize using half-strength Murashige and Skoog media solidified with agar in clear boxes that are covered in aluminum foil. The benefits of this protocol are that it does not require special dark-growth conditions and the growth media can be easily and uniformly supplemented with hormones or other chemicals.

Zea mays (maize) is an important model organism for studying monocot growth and development. Genotyping maize in the greenhouse or field can be time consuming and costly. Here, we describe a method to remove or chip a small amount of the endosperm from a maize kernel to genotype the kernel prior to planting. The seed chip is removed with a razor blade for DNA extraction and subsequent genotyping. When done correctly, seeds germinate normally and the kernel genotype can be determined before planting thus saving time, money, and field space.

Maize is one of the most important crop species and serves also as a model plant for grass research. A major bottleneck in maize research is stable transformation, which is both time and cost consuming, but also a technical challenge for most labs due to limited access to sufficient and optimal plant growth facilities. However, many studies in maize cell biology, physiology and biochemistry don’t require stable transformed plants and can be accomplished by using transiently transformed suspension cultures. Here, we report a detailed protocol to establish Black Mexican Sweet (BMS) maize suspension cell cultures and transiently transform it via particle bombardment. We demonstrate how reliable subcellular protein localization data can be obtained within a very short time period and analyzed using open access software.