Plant Science

Murashige-Skoog medium solutions have been used in a variety of plant plate growth assays, yet most research uses Arabidopsis thaliana as the study organism. For larger seeds such as maize (Zea mays), most protocols employ a paper towel roll method for experiments, which often involves wrapping maize seedlings in wet, sterile germination paper. What the paper towel roll method lacks, however, is the ability to image the roots over time without risk of contamination. Here, we describe a sterile plate growth assay that contains Murashige-Skoog medium to grow seedlings starting two days after germination. This protocol uses a section of a paper towel roll method to achieve uniform germination of maize seedlings, which are sterilely transferred onto large acrylic plates for the duration of the experiment. The media can undergo modification to include an assortment of plant hormones, exogenous sugars, and other chemicals. The acrylic plates allow researchers to freely image the plate without disturbing the seedlings and control the environment in which the seedlings are grown, such as modifications in temperature and light. Additionally, the protocol is widely adaptable for use with other cereal crops.

Key features

• Builds upon plate growth methods routinely used for Arabidopsis seedlings but that are inadequate for maize.

• Real-time photographic analysis of seedlings up to two weeks following germination.

• Allows for testing of various growth conditions involving an assortment of additives and/or modification of environmental conditions.

• Samples are able to be collected for genotype screening.

Graphical overview

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC₂(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC₂(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors.

Key features

• Non-invasive method to relatively quantify membrane potential in plant roots.

• Method suitable for imaging seedlings root in agar or liquid medium.

• Straightforward quantification.

Histological stains are useful tools for characterizing cell shape, arrangement and the material they are made from. Stains can be used individually or simultaneously to mark different cell structures or polymers within the same cells, and to visualize them in different colors. Histological stains can be combined with genetically-encoded fluorescent proteins, which are useful for understanding of plant development. To visualize suberin lamellae by fluorescent microscopy, we improved a histological staining procedure with the dyes Fluorol Yellow 088 and aniline blue. In the complex plant organs such as roots, suberin lamellae are deposited deep within the root on the endodermal cell wall. Our procedure yields reliable and detailed images that can be used to determine the suberin pattern in root cells. The main advantage of this protocol is its efficiency, the detailed visualization of suberin localization it generates in the root, and the possibility of returning to the confocal images to analyze and re-evaluate data if necessary.

Cannabis seed germination is an important process for growers and researchers alike. Many biotechnological applications require a reliable sterile method for seed germination. This protocol outlines a seed germination procedure for Cannabis sativa using a hydrogen peroxide (H₂O₂) solution as liquid germination media. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in an H₂O₂ solution of different concentrations; 1% H₂O₂ solution showed the fastest and the most efficient germination. This protocol also exhibited high germination efficiency for very old cannabis seeds with lower viability. Overall, this protocol demonstrates superior germination compared to water control and reduces the risk of contamination, making it suitable for tissue culture and other sensitive applications.

Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, virus-induced gene silencing (VIGS) and Agrobacterium tumefaciens-mediated transformation in fruit have been widely used as the transient expression approaches. Unlike VIGS, the latter one can be utilized not only for gene knock-down, but also for overexpression and knock-out. Here, we show the procedures of transiently expressing the 35S::FveMYB10 construct into fruit of the white-fruited F. vesca accession Yellow Wonder. As a master regulator of anthocyanin production, overexpressing FveMYB10 will cause fruit coloration, which was observed at one week post infiltration. We also exhibit the previous results of knocking down Reduced Anthocyanin in Petioles (RAP), encoding an anthocyanin transporter, by RNAi in fruit of the strawberry cultivar ‘Sweet Charlie’. Overall, Agrobacterium-mediated transient transformation in strawberry fruit is a quick and versatile approach for studying gene functions in fruit ripening.

Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the tissue. Here we describe a protocol to utilize micro-computed tomography (micro-CT) scanning to visualize vascular networks in the maize stem. The protocol covers sample fixation and staining with contrasting reagents, data acquisition using micro-CT, reconstructing three-dimensional (3D) models of stem inner structures and extraction of vascular networks from the model. This protocol can be easily applied to various types of species and organs/tissues.

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).

Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic analyses. It allows continuous imaging at fine spatiotemporal resolution and disturbs roots minimally. Examples are provided of how the procedure and materials can be adapted for 3D monitoring and of how environmental stress may be manipulated for experimental purposes.

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana UBIQUITIN10 (UBQ10)-promoter and guide RNA (gRNA) expression cassettes driven by the A. thaliana U6-promoter for efficient multiplex editing in Arabidopsis (Yan et al., 2016). Here, we describe a step-by-step protocol to cost-efficiently generate the binary vector containing multiple gRNAs and the Cas9 nuclease based on classic cloning procedure.

Electro-fusion system with isolated gametes has been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. In this protocol, procedures for isolation of rice gametes, electro-fusion of gametes, and culture of the produced zygotes are described.