Cold Spring Harbor protocols最新文献

The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limitations in explant availability. Indeed, although the most commonly used method, immature embryo transformation, has been improved through optimization of tissue culture media composition and selection methods, the approach is only applicable to a limited number of public genotypes, including B104 and Hi II. Recently, genotype-flexible methods have been developed using coexpression cassettes of morphogenic transcription factors (MTFs) Baby boom (Bbm) and Wushel2 (Wus2), which have enabled the successful transformation of many previously recalcitrant maize lines. This MTF-based transformation method has also allowed for the use of alternate explants, such as seedling leaf whorl, whose production is cost-effective and requires only minimum controlled growth space. In this review, we summarize recent advances in Agrobacterium-mediated maize transformation methods that use immature embryos or seedling leaf whorls as starting material.

Phage display is a versatile and effective platform for the identification and engineering of biologic-based therapeutics. Using standard molecular biology laboratory techniques, one can create a highly diverse and functional antibody phage-displayed library, and rapidly identify antibody fragments that bind to a target of interest with exquisite specificity and high affinity. Here, we discuss key aspects for the development of an antibody discovery strategy to harness the power of phage display technology to obtain molecules that can successfully be developed into therapeutics, including target validation, antibody design goals, and considerations for preparing and executing phage panning campaigns. Careful design and implementation of discovery campaigns-regardless of the target-provides the best chance of identifying desirable antibody fragments for further therapeutic development, so these principles can be applied to any new discovery project.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (V_H) scaffold and four distinct variable light (V_L) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a V_L-linker-V_H orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.

Conventional maize transformation has largely relied on immature embryos as explants, and is thus often hampered by the limited access to high-quality immature embryos year-round. Here, we present a detailed protocol using seedling leaf whorls as alternative explants for tropical maize inbred transformation. This approach involves the use of a cassette that drives the expression of the morphogenic transcription factors (MTFs) Baby boom (Bbm) and Wuschel2 (Wus2), which have been shown to greatly enhance transformation efficiency. We outline here the steps required for the preparation of seedling leaf whorl explants and subsequent Agrobacterium infection, and describe the tissue culture regimen that results in transgenic plant regeneration. Because constitutive expression of Bbm and Wus2 prevents normal plant regeneration and the production of fertile plants, the cassette containing these genes must be excised. As such, we include the steps for the Cre/loxP-mediated excision of the MTF gene cassette. The protocol outlines a year-round, more affordable, and efficient approach for carrying out maize transformation for crop improvement.

Phage display technology is enabled by genetic fusion of a foreign protein domain to a phage coat protein, without interfering with the phage's ability to replicate by infecting bacterial host cells. The displayed domain is exposed on the phage particle (virion) surface, where it can interact with molecules or other substances in the surrounding medium; in this regard, it acts like a normal protein. However, it possesses a superpower that is unavailable to ordinary proteins: It is easily replicated in great abundance because it is attached to a replicating virion whose genome includes its coding sequence. The main way this technology is exploited is construction of huge phage display "libraries," comprising billions of phage clones, each displaying a different protein domain, and each represented by thousands, millions, or billions of genetically identical virions-all mixed together in a single vessel. Surface display allows exceedingly rare virions whose displayed protein domains happen to bind a user-defined molecule or other substance-generically called the "selector"-to be isolated from such libraries by an affinity selection process. The yield of selector-binding virions is much too low to be of practical use, but their number is readily increased by many orders of magnitude by propagating the virions in host bacteria in culture. This overview is a critical review of recent developments of this technology. It does not review the entire arena of contemporary phage display; there is special emphasis on phage display's most prominent application, phage antibodies, in which the displayed domain is an antibody domain, and the selector is an antigen of interest.

The mechanical properties of individual roots and entire root systems play key roles in essential root functions such as water and nutrient acquisition, defense against soil microorganisms, and plant anchorage. However, relatively few studies have quantified the mechanics (e.g., stiffness and strength) of individual and entire root systems, or explored the link between root mechanics and root functions. This limitation is likely due to a lack of standardized methods for quantifying root mechanical properties, and has created a gap in our understanding of how root mechanical traits contribute to root functions. To date, most of our knowledge comes from studies in maize, where mechanical failure (i.e., root lodging) has detrimental impacts on crop yield. Here, we review the importance of root mechanics for maize production and discuss methods used to measure individual and entire root system mechanics.

Root lodging, the agronomic term for plant mechanical failure, causes yield loss in crops, including maize. Brace roots can provide structural support and assist in preventing root lodging. While the mechanics of brace roots (e.g., stiffness and strength) can play a role in their ability to prevent root lodging, there has been limited characterization of individual brace root mechanical properties. Methods to quantify root mechanics can thus be useful for characterizing maize mechanical traits and breeding new varieties with improved root anchorage and lodging resistance. Here, we describe a protocol for evaluating mechanical properties of maize brace roots. Specifically, we outline the steps necessary to perform three-point bend mechanical testing of maize brace roots using an Instron Universal Testing Stand. We describe root preparation, instrument setup, method establishment, testing, and data analysis. While we exemplify the protocol using maize brace roots, the approach can be adapted for assessing the mechanics of other plants or root types.

Root lodging due to wind is common in maize production worldwide, and can reduce photosynthetic capacity as well as nutrient uptake, resulting in significant yield loss and seed quality reduction. Lodging also causes harvesting problems, and ultimately increases production costs. Evaluating maize resistance to lodging is thus important for both breeders and researchers, to optimize agricultural practices, enhance breeding strategies, and ultimately develop new maize varieties with improved resilience. Here, we describe a novel procedure to accurately and quantitatively assess the resistance of maize plants to root lodging in the field. In this approach, users measure mechanical properties of maize root systems and estimate the magnitude of the wind force acting on the maize plants to ultimately derive an antilodging index, a measure that thus considers the balance between internal and external forces acting on the plants in the field. The procedure, which focuses on the plant as a whole and not only on the root system, has been successfully used to evaluate lodging resistance throughout the entire growth period, from the V8 growth stage to plant maturity, in different maize genotypes. We also compare the procedure to others in the literature, and discuss its applicability for assessing crop root lodging resistance in breeding and cultivation programs.

Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.

Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.