Methods and devices involving oil matrices

Abstract

Methods for preserving viability of plant tissues such as plant embryos are provided herein. Also included are methods for storing genomic DNA and/or molecular marker assay materials in an oil bilayer as part of a high-throughput molecular characterization system. Moreover, plant embryos may be treated while in an oil matrix. The treatment may include chromosome doubling, Agrobacterium -mediated transformation, or herbicide selection as part of an embryo rescue process.

We claim: 
     
         1 . A method of storing genomic DNA, said method comprising placing the genomic DNA, immersed in an aqueous solution, between two oils, wherein one of the oils is more dense than water and the other is less dense than water. 
     
     
         2 . The method of  claim 1 , wherein said genomic DNA is stored under light or dark conditions. 
     
     
         3 . The method of  claim 1 , wherein said genomic DNA is stored at a temperature between room temperature and −25 degrees Celsius. 
     
     
         4 . The method of  claim 1 , further comprising removing an aliquot of said genomic as part of an automated process to perform a molecular analysis. 
     
     
         5 . A method of storing molecular marker assay materials, said method comprising placing the molecular marker assay materials, immersed in an aqueous solution, between two oils, wherein one of the oils is more dense than water and the other is less dense than water. 
     
     
         6 . The method of  claim 5 , wherein said molecular marker assay materials comprise primers and probes. 
     
     
         7 . The method of  claim 5 , wherein said molecular marker assay materials are stored under light or dark conditions. 
     
     
         8 . The method of  claim 5 , wherein said molecular marker assay materials are stored at a temperature between room temperature and −25 degrees Celsius. 
     
     
         9 . The method of  claim 5 , further comprising removing an aliquot of said molecular marker assay materials as part of an automated process to perform a molecular analysis. 
     
     
         10 . A method of treating one or more plant embryos with a doubling agent, said method comprising:
 a. placing doubling media between two oils, wherein one of the oils is more dense than water and the other is less dense than water;   b. placing one or more plant embryos in the doubling media ;   c. selecting one or more plant embryos; and   d. transferring the selected plant embryos to media for germination or storage.   
     
     
         11 . The method of  claim 10 , wherein said one or more plant embryos are haploid embryos. 
     
     
         12 . The method of  claim 10 , wherein between steps (c) and (d) cellular material is collected from the one or more plant embryos; DNA is obtained from the cellular material; and genotypic information is obtained from the one or more plant embryos. 
     
     
         13 . The method of  claim 12 , wherein said plant embryos are selected based on genotypic information. 
     
     
         14 . A method of selecting one or more plant embryos during doubled haploid production, said method comprising:
 a. placing doubling media between two oils, wherein one of the oils is more dense than water and the other is less dense than water;   b. placing one or more plant embryos in the doubling media and   c. selecting one or more plant embryos for germination or storage.   
     
     
         15 . The method of  claim 14 , wherein between steps (b) and (c) cellular material is collected from the one or more plant embryos; DNA is obtained from the cellular material; and genotypic information is obtained from the one or more plant embryos. 
     
     
         16 . The method of  claim 15 , wherein said one or more plant embryos are selected based on genotypic information. 
     
     
         17 . The method of  claim 16 , further comprising transferring the selected plant embryos to media for germination or storage. 
     
     
         18 . The method of  claim 14 , wherein said doubling media comprises an anti- microtubule agent. 
     
     
         19 . The method of  claim 14 , wherein said doubling media comprises colchicine, pronamide, dithipyr, am iprophosmethyl or trifluralin. 
     
     
         20 . The method of  claim 14 , wherein said one or more plant embryos are haploid. 
     
     
         21 . The method of  claim 14 , wherein said one or more plant embryos in step (c) are haploid maize embryos produced by a cross between a male inducer line and a female line of interest. 
     
     
         22 . The method of  claim 21 , wherein said male inducer line contains a marker gene that is expressed in embryo tissue. 
     
     
         23 . The method of  claim 22 , wherein said marker gene expresses anthocyanin pigments. 
     
     
         24 . The method of  claim 23 , wherein plant embryos that are white are selected to be transferred to media for germination or storage. 
     
     
         25 . The method of  claim 24 , wherein selection of the white plant embryos is performed using a camera or other imaging device. 
     
     
         26 . The method of  claim 24 , wherein expression of anthocyanin is enhanced by aeration of the doubling media. 
     
     
         27 . The method of  claim 24 , wherein expression of anthocyanin is enhanced by placing the plant embryos in a hypotonic doubling media comprising perfluorodecalin. 
     
     
         28 . The method of  claim 24 , further comprising germinating or storing the selected plant embryos. 
     
     
         29 . A method of transforming plant tissue, said method comprising:
 a. forming a suspension comprising  Agrobacterium tumefaciens , wherein said  Agrobacterium tumefaciens  comprises within its genome a recombinant DNA construct comprising one or more genes of interest and a selectable marker gene, between two oils, wherein one of the oils is more dense than water and the other is less dense than water;   b. placing the plant tissue in said suspension;   c. removing the plant tissue from said suspension and cultivating the plant tissue in media;   d. resting the plant tissue in media; and   e. placing the plant tissue in contact with media comprising a selection agent corresponding to the selectable marker gene.   
     
     
         30 . The method of  claim 29 , further comprising regenerating a plant from said plant tissue. 
     
     
         31 . The method of  claim 29 , wherein the plant tissue is a plant embryo or plant callus. 
     
     
         32 . The method of  claim 29 , wherein the plant tissue is stored in said suspension for up to one hour. 
     
     
         33 . The method of  claim 29 , wherein step (d) comprises resting the plant tissue in media in the dark at a temperature of about 28 degrees Celsius for a period of up to  14  days. 
     
     
         34 . The method of  claim 29 , wherein said media comprising a selection agent corresponding to the selectable marker gene is located between two oils, wherein one of the oils is more dense than water and the other is less dense than water. 
     
     
         35 . A method comprising incubating plant tissue in a solution between two oils, wherein one of the oils is more dense than water and the other is less dense than water, the solution comprising a selection agent. 
     
     
         36 . The method of  claim 35 , wherein the selection agent is glyphosate, glufosinate, bialaphos, hygromycin B, kanamycin, paromomycin, mannose, phosphinothricin, butafenacil, or R-haloxyfop. 
     
     
         37 . The method of  claim 35 , further comprising the step of selecting plant tissue that remains viable after said incubating step. 
     
     
         38 . The method of  claim 35  wherein the plant tissue is a plant embryo or plant callus.