Method and apparatus for substantially isolating plant tissues

Abstract

The present invention discloses and claims methods and devices for the rapid mechanical isolation of monocot plant tissues suitable for transformation or tissue culture. The invention includes mechanical devices for substantially isolating target plant tissues for use as transformable explants, and propagation of transgenic plants and plant tissues.

1 . A method of providing monocot embryos suitable for transformation or tissue culture, comprising:
 (a) providing monocot seeds containing immature embryos having an opening in the pericarp of said seeds; and   (b) applying force to said seeds sufficient to extract said immature embryos from said seeds, wherein said extracted immature embryos comprise embryos or pieces of embryos suitable for genetic transformation or tissue culture.   
     
     
         2 . The method of  claim 1 , further comprising separating said extracted immature embryos from associated non-embryo tissue, wherein said separated extracted immature embryos comprise embryos suitable for tissue culture. 
     
     
         3 . The method of  claim 1 , wherein said monocot is in the family Poaceae. 
     
     
         4 . The method of  claim 1 , wherein said monocot is a  Zea  species. 
     
     
         5 . The method of  claim 4 , wherein said multiple monocot seeds comprise multiple corn kernels on at least one corn ear. 
     
     
         6 . The method of  claim 1 , wherein said embryos suitable for tissue culture comprise intact embryos. 
     
     
         7 . The method of  claim 1 , wherein said embryos suitable for tissue culture comprise partial embryos. 
     
     
         8 . The method of  claim 1 , wherein said tissue culture comprises transformation. 
     
     
         9 . The method of  claim 1 , wherein said tissue culture comprises regeneration. 
     
     
         10 . The method of  claim 9 , wherein said regeneration results in callus formation. 
     
     
         11 . The method of  claim 9 , wherein said regeneration results in at least one fertile plant. 
     
     
         12 . The method of  claim 1 , wherein said force is mechanically applied force. 
     
     
         13 . The method of  claim 1 , wherein said force comprises one or more force selected from fluid jet positive pressure, liquid jet positive pressure, mechanical positive pressure, negative pressure, centrifugal force, linear acceleration, linear deceleration, fluid shear, fluid turbulent flow, and fluid laminar flow. 
     
     
         14 . The method of  claim 1 , wherein said separating comprises use of at least one of size-exclusion, hydrophobic separation, and density differentials. 
     
     
         15 . An apparatus for preparing multiple corn embryos suitable for tissue culture, comprising at least one aperture for guiding a fluid stream, wherein said fluid stream contacts kernels on said corn ear and extracts embryos from said kernels, said extracted embryos being suitable for transformation or tissue culture. 
     
     
         16 . The apparatus of  claim 15 , further comprising a means for moving at least one corn ear relative to said fluid stream. 
     
     
         17 . The apparatus of  claim 15 , further comprising at least one separator for separating embryos from non-embryo tissues, wherein said separated embryos comprise at least some corn embryos suitable for tissue culture. 
     
     
         18 . The apparatus of  claim 16 , wherein said means for moving at least one corn ear relative to said fluid stream rotates said at least one corn ear and said at least one aperture relative to each other. 
     
     
         19 . The apparatus of  claim 16 , wherein said means for moving at least one corn ear relative to said fluid stream moves said fluid stream along the longitudinal axis of said at least one corn ear. 
     
     
         20 . The apparatus of  claim 15 , wherein said fluid stream is a liquid stream. 
     
     
         21 . The apparatus of  claim 15 , wherein said fluid stream is a gas stream. 
     
     
         22 . The apparatus of  claim 17 , wherein said at least one separator comprises a size-exclusion device. 
     
     
         23 . The apparatus of  claim 17 , wherein said at least one separator separates said embryos from said non-embryo tissues by hydrophobicity. 
     
     
         24 . The apparatus of  claim 17 , wherein said at least one separator separates said embryos from said non-embryo tissues by density differentials. 
     
     
         25 . An apparatus for mechanically extracting multiple corn embryos suitable for tissue culture from at least one immature corn ear, comprising at least one component selected from:
 a) at least one solid surface for applying mechanical positive pressure to the exterior of kernels on said at least one immature corn ear;   b) at least one aperture for guiding a fluid flow, wherein said fluid flow contacts kernels on said at least one immature corn ear; and   c) at least one aperture for applying negative fluid pressure, wherein said negative fluid pressure contacts kernels on said at least one immature corn ear; and   wherein said at least one component applies force to said kernels sufficient to extract embryos from said kernels, said extracted embryos comprising multiple immature embryos suitable for tissue culture.   
     
     
         26 . A transformed monocot plant, produced by the steps comprising:
 a) providing at least one transformable explant derived from said embryos suitable for tissue culture of  claim 1 ;   b) introducing a heterologous nucleic acid molecule into said at least one transformable explant, resulting in transformed tissue;   c) growing a transformed monocot plant from said transformed tissue.   
     
     
         27 . A transformed progeny monocot plant that is derived from the transformed monocot plant of  claim 26 .