Plant Genetics Protocols

For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable. Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used. Includes metaphase arresting reagents. - [Read Accumulation and Fixation of Plant Metaphase Chromosomes Protocol]

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Protocol describing Agrobacterium mediated gene transfer via hypocotyls. - [Read Agrobacterium-Mediated Gene Transfer via Hypocotyls Protocol]

Virus-induced gene silencing (VIGS) uses a virus to deliver a sequence from a gene of interest into a host plant. The virus carrying the fragment of the gene of interest must be capable of replication if dsRNA is to be produced. One or two leaves are inoculated with Agrobacterium strains carrying the VIGS vector possessing the gene fragment. The virus then replicates and spreads throughout the plant, mediating silencing. - [Read Delivery of dsRNA into Plants by VIGS Methodology]

Microsatellite markers, also referred to as STMS (SequenceTagged Microsatellite Sites) or STR (Short Tandem Repeats) are widely used as molecular markers for intraspecific genotyping, molecular mapping and breeding purposes. The method described is an efficient,fast and relatively inexpensive way to obtain microsatellite markers without post-cloning selection methods. So far, the method has been successful in onion (Allium cepa L.), a plant with a large genome and for pathogenic fungi. - [Read Enrichment for Microsatellite Sequences in Onion (Allium cepa L.) Protocol]

Protocol for the fixation of plant metaphase chromosomes. - [Read Fixation of Plant Metaphase Chromosomes Protocol]

To accurately predict the activity of a transgene it is critical to understand its location and dynamics in the 3-D interphase nucleus. Developed in situ methods to visualize transgenes (including single copy genes) & their transcripts during interphase from different tissues & plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis and extend characterization to the interphase nucleus - [Read In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei]

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. - [Read Isolation of Plant Transcription Factors Using a Modified Yeast One-Hybrid System]

Protocol describing on how to perform crosses in plants. - [Read Plant Crosses Protocol]

Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

Protocol for the preparation of plant chromosomes using squash. - [Read Squash Preparation of Plant Chromosomes Protocol]

This procedure, which uses a root transformation protocol, provides a rapid method for assessing gene expression in Arabidopsis roots. It is useful for testing promoter:reporter gene constructs, for expressing genes, the overexpression of which is lethal in whole plants, and for transforming the roots of plants that are recalcitrant to conventional transformation techniques. The protocol has been used successfully with Ws, No-0, and RLD ecotypes. - [Read Transgene Expression in Regenerated Roots]

The study of transient gene expression provides a useful complement to the study of stably transformed plants. Transient assays offer a quick method of testing the effects of genes, using either phenotypic, molecular, or biochemical readouts. Transient assays based on Agrobacterium-mediated transformation of leaf explants have been described for other plant species, but it is not known how well these assays work in Arabidopsis. - [Read Transient Expression in Protoplasts]