Method of meristem excision and transformation

Abstract

The present invention relates to excision of explant material comprising meristematic tissue from cotton seeds. Methods for tissue preparation, storage, transformation, and selection or identification of transformed plants are disclosed, as are transformable meristem tissues and plants produced by such methods, and apparati for tissue preparation.

What is claimed is: 
     
         1 . A high-throughput method for producing transformed cotton plant tissue comprising:
 a) mechanically disrupting cotton seeds to obtain a plurality of cotton embryonic meristem explants; and   b) contacting the explants with a selected DNA sequence to obtain at least a first explant transformed with the selected DNA.   
     
     
         2 . The method of  claim 1 , wherein the explants are stored at a temperature of between 0-15° C. for between 1 hour and 7 days prior to step (b). 
     
     
         3 . The method of  claim 1 , further comprising the step of regenerating a transgenic cotton plant transformed with the selected DNA from at least the first explant. 
     
     
         4 . The method of  claim 3 , wherein regenerating the transgenic cotton plant does not comprise generating a callus culture from the explant. 
     
     
         5 . The method of  claim 3 , wherein the transgenic cotton plant arises from transformation of a meristem that results in transformation of germline tissue. 
     
     
         6 . The method of  claim 3 , wherein the resulting plant is non-chimeric. 
     
     
         7 . The method of  claim 3 , wherein the resulting plant is chimeric. 
     
     
         8 . The method of  claim 3 , further comprising screening the explants prior to contacting the explants with the selected DNA sequence to identify a fraction of explants amenable to transformation with the selected DNA and regeneration of a transgenic plant therefrom. 
     
     
         9 . The method of  claim 8 , wherein screening the explants comprises placing mechanically disrupted cotton seeds comprising the explants in an aqueous environment and selecting explants for contacting with the selected DNA based on buoyancy. 
     
     
         10 . The method of  claim 8 , wherein screening the explants comprises sieving mechanically disrupted cotton seeds to remove the explants from seed coat or cotyledon tissue. 
     
     
         11 . The method of  claim 8 , wherein screening the explants comprises enriching the fraction of explants amenable to transformation. 
     
     
         12 . The method of  claim 3 , wherein the selected DNA sequence encodes a selectable or screenable marker, or specifies an agronomic trait. 
     
     
         13 . The method of  claim 3 , wherein the explants of step (a) comprise a transgene. 
     
     
         14 . The method of  claim 12 , further comprising selecting or screening for an explant transformed with the selected DNA by contacting the explant with a selective agent, wherein the selectable marker confers tolerance to the selective agent. 
     
     
         15 . The method of  claim 3 , further defined as comprising regenerating chimeric transgenic plant tissue from the explant and selecting or screening for transgenic tissue from the plant tissue. 
     
     
         16 . The method of  claim 15 , wherein the transgenic cotton plant tissue arises from meristem transformation. 
     
     
         17 . The method of  claim 15 , further comprising regenerating a chimeric plant from the explant and selecting or screening for transgenic tissues from the plant. 
     
     
         18 . The method of  claim 15 , wherein selecting or screening for transgenic tissue comprises contacting the tissue with a selective agent or an agent that yields a screenable phenotype, selected from the group consisting of glufosinate, dicamba, glyphosate, spectinomycin, streptomycin, kanamycin, G418, paromomycin, imidazolinone, a substrate of GUS, and combinations thereof. 
     
     
         19 . The method of  claim 3 , wherein mechanically disrupting cotton seeds comprises passing the seeds through rollers that crush the seed. 
     
     
         20 . The method of  claim 19 , wherein the rollers comprise secondary grooves. 
     
     
         21 . The method of  claim 19 , wherein the rollers are comprised of stainless steel. 
     
     
         22 . The method of  claim 3 , wherein contacting the explants with a selected DNA sequence comprises contacting the explants with a recombinant  Rhizobium  or  Agrobacterium  spp. capable of transforming at least a first cell of the explant with the selected DNA. 
     
     
         23 . The method of  claim 22 , comprising contacting the explant with a recombinant  Agrobacterium  culture grown to an OD 660  of from about 0.0045 to about 1.4. 
     
     
         24 . The method of  claim 22 , comprising contacting the explant with an  Agrobacterium  culture wherein the pH in which the cotton meristematic tissue is contacted by the  Agrobacterium  cells is from about 5.0 to about 10.0. 
     
     
         25 . The method of  claim 22 , wherein the  Rhizobium  or  Agrobacterium  spp. are suspended in the presence of a selective agent active against an untransformed explant prior to contacting the explants with a recombinant  Rhizobium  or  Agrobacterium  spp. 
     
     
         26 . The method of  claim 3 , wherein contacting the explants with a selected DNA sequence comprises transforming the explants by microprojectile bombardment. 
     
     
         27 . The method of  claim 22 , wherein, following the contacting of explants with a selected DNA sequence, explants are grown in the presence of a selective agent at 35° C., or are grown under lighting conditions that allow for normal plastid development. 
     
     
         28 . The method of  claim 27 , wherein growth at 35° C. is performed for 1-7 days; the selective agent is selected from the group consisting of spectinomycin, streptomycin, kanamycin, glyphosate, glufosinate, hygromycin, and dicamba; or the explants are grown under a light intensity of ≧5 μEinsteins with about a 16 hour light/8 dark photoperiod. 
     
     
         29 . The method of  claim 22 , wherein the explants are grown in the presence of a fungicide prior to, during, or subsequent to step (b). 
     
     
         30 . The method of  claim 29 , wherein the explants are grown in the presence of a fungicide and DMSO. 
     
     
         31 . The method of  claim 30 , wherein the explants are grown in the presence of nystatin, thiabendazole, and DMSO. 
     
     
         32 . An apparatus for high-throughput generation of transformable plant tissue comprising spaced apart rollers comprising secondary grooves for applying a force to seeds passing through the rollers. 
     
     
         33 . The apparatus of  claim 32 , wherein the rollers are spaced from about 2.2 mm to about 3.5 mm apart, as measured by peak to valley of opposing rollers. 
     
     
         34 . The apparatus of  claim 32 , wherein the rollers are comprised of stainless steel. 
     
     
         35 . The apparatus of  claim 32 , further comprising a separator for separating cotton meristematic explants from seed coat or cotyledon tissue. 
     
     
         36 . The apparatus of  claim 35 , wherein the separator comprises a liquid container for determining the buoyancy of the transformable plant tissue. 
     
     
         37 . The apparatus of  claim 32 , further comprising a water and seed containment tray. 
     
     
         38 . The apparatus of  claim 37 , further comprising an automated mechanism for cleaning the apparatus, which controls one or more of the following cleaning steps: (a) applying a sanitizing solution (b) removing the sanitizing solution; and (c) storing the apparatus under sterile conditions.