Direct and continuous root alone or root/shoot production from transgenic events derived from green regenerative tissues and its applications

Abstract

The present invention provides assays and methods for efficiently testing a polynucleotide of interest for a phenotype in a root. In some embodiments, the assays and methods include regenerating green tissue that is transgenic for at least one polynucleotide of interest into one or more transgenic plantlets that have at least one transgenic root. Further provided are methods of making a root assay by contacting green tissue with a first rooting medium to produce a plantlet and a plurality of roots. Additionally provided are methods of assaying for insecticidal activity on a live root. Accordingly provided herein is a substantially contamination-free, root bioassay. Further provided are methods of identifying a promoter having activity in a root.

1 . A method for efficiently testing a polynucleotide of interest for a phenotype in roots comprising:
 a) regenerating green tissue that is transgenic for at least one polynucleotide of interest into one or more transgenic plantlets, wherein the one or more transgenic plantlets comprise at least one transgenic root; and   b) determining at least one phenotype of the transgenic root.   
     
     
         2 . The method of  claim 1 , further comprising subjecting the transgenic root to a pest or pathogen. 
     
     
         3 . The method of  claim 1 , wherein the phenotype is increased root size, increased overall root mass, altered root architecture, increased expression level of mRNA or protein, increased biochemical content, increased tolerance to stress, increased resistance to a pest, increased resistance to an insecticide, increased yield, or increased nitrogen use efficiency as compared to the corresponding phenotype of a control, wherein the polynucleotide of interest has not been introduced into the control. 
     
     
         4 . The method of  claim 1 , wherein the at least one transgenic root is isolated from the plantlet prior to determining the at least one phenotype. 
     
     
         5 . The method of  claim 1 , further comprising producing a transgenic plant from the green tissue. 
     
     
         6 . The method of  claim 1 , further comprising growing the transgenic plantlet into a transgenic plant. 
     
     
         7 . The method of  claim 1 , further comprising rooting the plantlet on medium. 
     
     
         8 . The method of  claim 7 , wherein the medium is medium gelled with agar or an agar substitute. 
     
     
         9 . The method of  claim 1 , wherein the plantlet is a monocot plantlet. 
     
     
         10 . The method of  claim 1 , wherein the green tissue is obtained by transforming an explant from a monocot and subjecting the explant to green tissue initiation medium for a time and under conditions sufficient to initiate growth from the explant, thereby producing green tissue. 
     
     
         11 . The method of  claim 10 , wherein the explant comprises an embryo, green tissue, callus, leaf, meristem, seedling, seed, stem, shoot, node, leaf base, or root. 
     
     
         12 . The method of  claim 1 , further comprising regenerating transgenic green tissue into one or more transgenic plantlets by contacting the green tissue with a rooting medium that induces root formation for a time and under conditions sufficient to initiate root growth from the green tissue, thereby producing a plantlet. 
     
     
         13 . The method of  claim 1 , wherein determining at least one phenotype of the transgenic root occurs under sterile conditions. 
     
     
         14 . A method of making a root assay comprising:
 a) contacting green tissue with a first rooting medium gelled with agar or an agar substitute to produce a plantlet having at least one transgenic root;   b) removing the root from the medium; and   c) contacting the root with a second rooting medium in the absence of agar or an agar substitute to produce a plurality of roots.   
     
     
         15 . The method of  claim 14 , further comprising regenerating a plurality of transgenic plantlets from the green tissue. 
     
     
         16 . The method of  claim 14 , placing the at least one transgenic root of the plantlet in a culture dish. 
     
     
         17 . The method of  claim 14 , contacting the roots with the second rooting medium, wherein the second rooting medium comprises a biocide. 
     
     
         18 . The method of  claim 14 , contacting the roots with the second rooting medium using an absorbent material. 
     
     
         19 . The method of  claim 14 , wherein the green tissue is obtained by transforming an explant from a monocot and subjecting the explant to green tissue initiation medium for a time and under conditions sufficient to initiate growth from the explant, thereby producing green tissue. 
     
     
         20 . The method of  claim 19 , wherein the explant comprises an embryo, green tissue, callus, leaf, meristem, seedling, seed, stem, shoot, node, leaf base, or root. 
     
     
         21 . The method of  claim 14 , further comprising regenerating transgenic green tissue into one or more transgenic plantlets by contacting the green tissue with a first rooting medium that induces root formation for a time and under conditions sufficient to initiate root growth from the green tissue, thereby producing a plantlet. 
     
     
         22 . The method of  claim 14 , wherein the roots are prepared under substantially sterile conditions. 
     
     
         23 . The method of  claim 14 , further comprising subjecting the root to a chemical, pest, or pathogen. 
     
     
         24 . The method of  claim 23 , further comprising sterilizing the pest prior to contacting the root. 
     
     
         25 . The method of  claim 23 , further comprising feeding the pest prior to infesting the pest on the transgenic roots. 
     
     
         26 . The method of  claim 14 , wherein the assay is produced within about 4 to 14 days. 
     
     
         27 . The method of  claim 14 , further comprising growing the transgenic plantlets into plants. 
     
     
         28 . The method of  claim 27 , further comprising obtaining transgenic seeds from the transgenic plants. 
     
     
         29 . A method of assaying for insecticidal activity on a live root comprising:
 a) regenerating green tissue into one or more plantlets comprising at least one live root;   b) contacting the at least one root of the plantlet with a rooting medium;   c) exposing the root to one or more pests to infest the medium for infestation; wherein the medium and pest are substantially contamination-free; and   d) determining a phenotype of the root.   
     
     
         30 . The method of  claim 29 , further comprising regenerating green tissue transgenic for a polynucleotide of interest into one or more transgenic plantlets comprising at least one live transgenic root. 
     
     
         31 . The method of  claim 29 , wherein the live root is assayed for endogenous resistance to root damage by the pest. 
     
     
         32 . The method of  claim 29 , further comprising contacting the green tissue with rooting medium to induce root formation. 
     
     
         33 . The method of  claim 29 , wherein the rooting medium comprises a biocide. 
     
     
         34 . The method of  claim 29 , contacting the root with the rooting medium using an absorbent material. 
     
     
         35 . The method of  claim 29 , determining the phenotype of the root by scoring the root for damage as compared to the phenotype of the control. 
     
     
         36 . The method of  claim 29 , further comprising sterilizing the pest prior to contacting the root. 
     
     
         37 . The method of  claim 29 , further comprising feeding the pest prior to infesting the pests on the transgenic roots. 
     
     
         38 . The method of  claim 29 , wherein the pest is from the order of lepidoptera, homoptera, heteroptera, or coleoptera. 
     
     
         39 . The method of  claim 29 , wherein the pest is of a developmental stage comprising an egg, larva, instar, or adult. 
     
     
         40 . The method of  claim 29 , further comprising determining a phenotype of the pest. 
     
     
         41 . The method of  claim 40 , wherein the phenotype is growth or mortality of the pest. 
     
     
         42 . The method of  claim 41 , further comprising determining the phenotype by scoring the pest for mortality or stunted growth. 
     
     
         43 . The method of  claim 42 , wherein more than half of the pests are dead or have stunted growth indicates the polynucleotide of interest is effective for controlling pest infestation or damage to the root or combinations thereof. 
     
     
         44 . The method of  claim 29 , comprising observing the stem area above a crown, wherein the stem area darkens from pest damage as compared to a control indicates that the root is not effective in controlling pest infestation. 
     
     
         45 . The method of  claim 29 , wherein the green tissue is obtained by transforming an explant from a monocot and subjecting the explant to green tissue initiation medium for a time and under conditions sufficient to initiate growth from the explant, thereby producing green tissue. 
     
     
         46 . The method of  claim 45 , wherein the explant comprises an embryo, green tissue, callus, leaf, meristem, seedling, seed, stem, shoot, node, leaf base, or root. 
     
     
         47 . The method of  claim 29 , further comprising regenerating transgenic green tissue into one or more transgenic plantlets by contacting the green tissue with a first rooting medium that induces root formation for a time and under conditions sufficient to initiate root growth from the green tissue, thereby producing a plantlet. 
     
     
         48 . The method of  claim 29 , further comprising producing a plurality of transgenic plantlets from the green tissue. 
     
     
         49 . The method of  claim 29 , further comprising producing a plurality of roots from the green tissue. 
     
     
         50 . The method of  claim 29 , further comprising placing the at least one root of the plantlet in a culture dish. 
     
     
         51 . The method of  claim 29 , wherein the plantlet has had its leaves removed. 
     
     
         52 . The method of  claim 29 , wherein the root is intact. 
     
     
         53 . The method of  claim 29 , wherein the assay is completed within about 4 to 14 days. 
     
     
         54 . The method of  claim 29 , further comprising regenerating transgenic plantlets from the green tissue from which a root has been assayed for its phenotype. 
     
     
         55 . A substantially contamination-free, root bioassay comprising:
 a monocot plantlet, wherein the plantlet comprises at least one live root in a culture dish, and wherein the dish comprises a rooting medium in contact with the root.   
     
     
         56 . The assay of  claim 55 , wherein the plantlet and root are transgenic for a polynucleotide of interest. 
     
     
         57 . The assay of  claim 55 , further comprising a plantlet having a plurality of roots. 
     
     
         58 . The assay of  claim 55 , further comprising a plurality of plantlets obtained from the green tissue. 
     
     
         59 . The assay of  claim 55 , wherein the rooting medium comprises a biocide. 
     
     
         60 . The assay of  claim 55 , wherein the assay comprises a means for bringing the root into contact with the rooting medium. 
     
     
         61 . The assay of  claim 55 , wherein the culture dish comprises a substantially sterile absorbent material that contacts the rooting medium and the roots. 
     
     
         62 . The assay of  claim 55 , wherein the assay comprises a sterilized pest, pathogen, or chemical. 
     
     
         63 . The assay of  claim 55 , wherein the pest is from the order of lepidoptera, homoptera, heteroptera, or coleoptera. 
     
     
         64 . The assay of  claim 55 , wherein the pest is of a developmental stage comprising an egg, larva, instar, or adult. 
     
     
         65 . The assay of  claim 62 , wherein the chemical is a pesticide, insecticide, fungicide, or bactericide. 
     
     
         66 . The assay of  claim 62 , wherein the root is transgenic for a Bt gene and wherein the pest is western corn root worm (WCRW). 
     
     
         67 . A method of identifying a promoter having activity in the root comprising:
 a) regenerating green tissue transgenic for a promoter of interest operably linked to a polynucleotide into one or more stably transformed transgenic plantlets, wherein the plantlets comprise at least one transgenic root; and   b) determining whether the polynucleotide is expressed in root cells of the plantlet.   
     
     
         68 . The method of  claim 67 , determining whether the polynucleotide is expressed preferentially in root cells of the plantlet. 
     
     
         69 . The method of  claim 67 , comprising determining whether the polynucleotide is expressed preferentially in root cells of the plantlet as compared to expression in cells of non-root tissues of the plantlet; wherein increased expression of the polynucleotide in root cells in comparison to expression of the polynucleotide in non-root cells indicates that the promoter is preferentially expressed in root cells. 
     
     
         70 . The method of  claim 67 , wherein the at least one transgenic root is isolated from the plantlet prior to determining the expression of the polynucleotide. 
     
     
         71 . The method of  claim 67 , further comprising producing a transgenic plant from the green tissue. 
     
     
         72 . The method of  claim 67 , further comprising growing the transgenic plantlet into a transgenic plant 
     
     
         73 . The method of  claim 67 , further comprising rooting the plantlet on medium. 
     
     
         74 . The method of  claim 73 , further comprising rooting the plantlet on medium gelled with agar or an agar substitute. 
     
     
         75 . The method of  claim 67 , wherein the plantlet is a monocot plantlet. 
     
     
         76 . The method of  claim 67 , wherein the green tissue is obtained by transforming an explant from a monocot and subjecting the explant to green tissue initiation medium for a time and under conditions sufficient to initiate growth from the explant, thereby producing green tissue. 
     
     
         77 . The method of  claim 76 , wherein the explant comprises an embryo, green tissue, callus, leaf, meristem, seedling, seed, stem, shoot, node, leaf base, or root. 
     
     
         78 . The method of  claim 67 , regenerating transgenic green tissue into one or more transgenic plantlets by contacting the green tissue with a rooting medium that induces root formation for a time and under conditions sufficient to initiate root growth from the green tissue, thereby producing a plantlet. 
     
     
         79 . The method of  claim 67 , wherein the polynucleotide encodes a marker polypeptide.