Methods to identify plant metabolites

#Plants are sources of drugs and other useful materials. These materials are the products of plant metabolism, which is the total collection of biochemical pathways within a plant. Genes encode the enzymes that function in plant metabolic pathways and regulatory genes, in turn, control the activity of metabolic pathways by directing the expression of entire sets of enzyme-encoding genes specific to a given pathway. It follows that methods to identify any genes that govern the production of such plant metabolites are critical to enable the manipulation of pathways for elevating product yield and for increasing the diversity of these substances. The present invention provides methods to identify, in a relatively efficient and reliable manner, plant-genetic material required for production of metabolites which would ordinarily be difficult to identify. The invention is the realization of the successful merging of two distinct methodologies: activation-tagging mutagenesis and high-throughput screening. The present unique combination of these methodologies results in the ability to assign metabolic functionality to plant genes involved in the production of biologically active molecules and to create a means of compound discovery based on the genetic capabilities of a plant or group of plants.

#This invention is therefore based on the following technical developments: 1) methodology which enables propagation and maintenance of mutagenized “microcallus” material in a “library” format in such a way as to permit large scale screening; 2) procedures to nondestructively sample the microcallus; 3) methodology to screen the microcallus sample in a miniaturized high throughput pharmacological screen.

#Therefore, the invention is in the field of plant molecular biology, in particular, genetic methods for production and identification of useful compounds.

#The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawing, which is given by way of illustration only, and thus is not limitative of the present invention.

#FIG. 1 shows a protoplast isolation and transformation flowchart.

#FIGS. 2A-B. FIG. 2A shows a photograph taken through a bright field microscope (40× magnification) of freshly isolated protoplasts from tobacco leaves two days after isolation. At seven days the protoplasts are co-cultivated with Agrobacterium tumefacians (GV3101) to conduct transformation resulting in the insertion of the activation-tagging T-DNA. FIG. 2B shows a photograph of a cluster of clonal cells after several steps of incubation, washing, antibiotic selection and embedding of the multiplied protoplasts. The clusters of clonal cells become macroscopic (referred to as “microcalli”) as shown in the dish in the right of the figure. Diameter of the petri dish shown is 150 mm.

#The present invention provides a method for identifying plant genetic material whose actions cause increased production of a metabolite or metabolites of interest in plant cells, said method comprising causing random or directed integration into the plant genome in plant protoplasts of at least one enhancer-containing T-DNA genetic element harboring sequences to enable bacterial replication and selection; growing said protoplasts to the stage of callus cultures; sampling said callus cultures in such a manner as to retain viability of said clonal cultures; analyzing said samples to identify the callus cultures producing metabolite or metabolites of interest; and isolating and identifying the plant genetic material, the action of which has been stimulated by the enhancer-containing T-DNA genetic element in the sampled, identified callus cultures.

#Preferred is a method wherein the plant genetic material, which is identified, is a plant gene whose action causes a plant cell to produce an increased amount of a metabolite or metabolites of interest. More preferred is a method wherein the plant genetic material, which is identified, is a regulatory gene. Also preferred is a method wherein the analysis of callus cultures detects the production of metabolites of interest having pharmacological properties. Most preferred is a method wherein said metabolites are detected via at least one radioligand displacement assay.

#Methods of the present invention may also comprise the further step of propagating at least one callus culture producing said metabolite or metabolites. Most preferred are methods, which involve a tobacco plant and wherein said enhancer sequence is a plant viral enhancer sequence.