Preparation and use of plant embryo explants for transformation

Abstract

The present invention relates to excision of explant material comprising meristematic tissue from seeds, and storage of such material prior to subsequent use in plant tissue culture and genetic transformation. Methods for tissue preparation, storage, and transformation are disclosed, as is transformable meristem tissue produced by such methods, and apparati for tissue preparation.

1 . (canceled) 
     
     
         2 . The explant of  claim 41 , wherein the explant comprises a substantially intact seed modified to expose at least a first transformable cell of the seed. 
     
     
         3 . The explant of  claim 2 , wherein preparing the explant comprises drilling at least a first aperture in the seed. 
     
     
         4 . The explant of  claim 2 , wherein preparing the explant comprises nicking the seed. 
     
     
         5 . The explant of  claim 2 , wherein the substantially intact seed comprises a meristem of the seed. 
     
     
         6 . The explant of  claim 1 , wherein the method further comprises the step of:
 (c) transforming at least a first cell of the explant with a selected DNA.   
     
     
         7 . The explant of  claim 6 , wherein the method further comprises the step of:
 (d) regenerating a transgenic plant from said cell, wherein the plant is stably transformed with the selected DNA.   
     
     
         8 . The explant of  claim 7 , wherein the method further comprises storing the explant prior to step (d) under conditions wherein the explant does not germinate. 
     
     
         9 . The explant of  claim 1 , wherein said seed and/or explant is dehydrated prior to or after preparing the explant. 
     
     
         10 . The explant of  claim 7 , wherein the moisture content of the explant is increased prior to or concurrently with step (d). 
     
     
         11 . The explant of  claim 41 , wherein preparing the explant is carried out in the absence of a liquid. 
     
     
         12 . The explant of  claim 41 , wherein the seed or explant comprises an internal moisture content of from about 3% to about 25%. 
     
     
         13 . The explant of  claim 12 , wherein the seed or explant comprises an internal moisture content of from about 4% to about 16%. 
     
     
         14 . The explant of  claim 8 , wherein the explant is stored at a temperature of between about −80° C. and about 60° C. 
     
     
         15 . The explant of  claim 8 , wherein the explant is dehydrated prior to storing. 
     
     
         16 . The explant of  claim 41 , wherein the method is further defined as comprising preparing a plurality of explants from a plurality of seeds and selecting one or more explant exhibiting at least a first characteristic indicative of the ability to transform the explant and/or regenerate a plant from the explant. 
     
     
         17 . The explant of  claim 16 , wherein the characteristic is selected from the group consisting of color, size, shape, density, proximate analysis, carbohydrate content, protein content, fat content, fibre (ash) content, moisture content, viability, germplasm, intactness, pathogen presence, and optical characteristics. 
     
     
         18 . The explant of  claim 6 , wherein the method is further defined as comprising priming the plant seed and/or the explant prior to or concurrently with step (c). 
     
     
         19 . The explant of  claim 18 , wherein priming the seed comprises contacting the seed with an aqueous solution. 
     
     
         20 . The explant of  claim 19 , wherein the aqueous solution comprises a disinfectant. 
     
     
         21 . The explant of  claim 20 , wherein the aqueous solution comprises bleach or alcohol. 
     
     
         22 . The explant of  claim 41 , wherein the plant seed is a soybean, canola, corn, cotton, onion, pepper, tomato, cucurbit, rice, or wheat seed. 
     
     
         23 . The explant of  claim 41 , wherein preparing the explant is automated. 
     
     
         24 . The explant of  claim 23 , wherein preparing the explant comprises an automated process in which the plant seed is oriented as it passes through a mechanical separator to provide a substantially uniform output of regenerable meristematic plant tissue. 
     
     
         25 . The explant of  claim 6 , wherein step (c) is carried out by bacterially-mediated transformation or microprojectile bombardment. 
     
     
         26 . The explant of  claim 7 , wherein the transgenic plant is regenerated without producing a callus tissue culture. 
     
     
         27 . The explant of  claim 7 , wherein the method is further defined as comprising disinfecting the plant seed and/or the explant prior to step (d). 
     
     
         28 . The explant of  claim 27 , wherein the method is further defined as comprising disinfecting the explant. 
     
     
         29 . The explant of  claim 27 , wherein disinfecting comprises application of a disinfectant selected from the group consisting of bleach, alcohol, ozone, chlorine gas, ultraviolet light, temperatures of −20° C. or lower, and exposure to a temperature higher than 50° C. 
     
     
         30 . The explant of  claim 29 , wherein disinfecting comprises application of a nystatin and/or thiabendazole. 
     
     
         31 . The explant of  claim 8 , wherein storing the explant is carried out for from about 1. hour to about 2 years prior to step (c). 
     
     
         32 . The explant of  claim 8 , wherein storing the explant is carried out for from about 1 hour to about 24 hours. 
     
     
         33 . The explant of  claim 7 , wherein the method is carried out without manipulating the development of the explant with a plant growth regulator. 
     
     
         34 . The explant of  claim 7 , wherein step (d) comprises culturing the explant in the presence of a selective agent at about 35° C. 
     
     
         35 . The explant of  claim 7 , wherein step (d) comprises culturing the explant under lighting conditions that allow for normal plastid development. 
     
     
         36 . A regenerable explant competent for genetic transformation produced by a method comprising:
 (a) obtaining an explant from a plant seed; and   (b) dehydrating the plant seed and/or explant prior to, concurrently with or after said step (a) to obtain an explant that does not germinate and remains viable and competent for genetic transformation.   
     
     
         37 . The explant of  claim 36 , wherein the method further comprises the steps of:
 (c) hydrating said explant; and   (d) regenerating a plant from the explant.   
     
     
         38 . The explant of  claim 37 , wherein the method further comprises the step of:
 (e) transforming at least a first cell of the explant with a selected DNA prior to the step of regenerating the plant from the explant, wherein the plant is stably transformed with the selected DNA.   
     
     
         39 . The explant of  claim 37 , wherein hydrating said explant is performed under dark conditions. 
     
     
         40 . The explant of  claim 7 , wherein at least a portion of the regenerating is performed at a temperature of up to about 40° C. 
     
     
         41 . A regenerable explant competent for genetic transformation produced by a method comprising the steps of:
 (a) obtaining a plant seed; and   (b) preparing a regenerable explant from the plant seed under conditions wherein the explant does not germinate and remains viable and competent for genetic transformation.   
     
     
         42 .- 45 . (canceled)