Particle bombardment transformation of Brassica

Abstract

The invention involves methods and materials related to the transformation of Brassica by particle bombardment. Specifically, the invention provides methods of preparing non-embryo Brassica tissue such that Brassica cells are capable of being cultured, transformed by particle bombardment, and regenerated into plants. In addition, this invention provides stably transformed Brassica cells as well as their progeny. This invention also provides methods of culturing Brassica tissue with liquid medium such that transformed Brassica cells are identified and regenerated into transformed Brassica plants.

What is claimed is:  
     
         1 . A method of producing transformed Brassica cells, said method comprising the steps of: 
 (a) bombarding cells prepared from non-embryo Brassica tissue with nucleic acid-coated microprojectiles to form a bombarded cell preparation; and    (b) identifying cells of said bombarded preparation that have been transformed with said nucleic acid.    
     
     
         2 . The method of  claim 1 , wherein said cells prepared from non-embryo Brassica tissue are diploid.  
     
     
         3 . The method of  claim 1 , wherein said non-embryo Brassica tissue is from a Brassica seedling.  
     
     
         4 . The method of  claim 3 , wherein said seedling is a 4-6 day old sterile seedling.  
     
     
         5 . The method of  claim 1 , wherein said cells being bombarded are prepared from non-embryo Brassica tissue by cutting said tissue into at least one piece and contacting said at least one piece with induction medium.  
     
     
         6 . The method of  claim 1 , wherein said cells being bombarded are prepared from non-embryo Brassica tissue by cutting a hypocotyl from a seedling in a generally transverse orientation to form at least one hypocotyl piece, slicing said at least one hypocotyl piece longitudinally, and contacting at least one of said longitudinal slices with induction medium.  
     
     
         7 . The method of  claim 1 , wherein said cells being bombarded are prepared from non-embryo Brassica tissue by macerating said tissue into a cellular slurry.  
     
     
         8 . The method of  claim 1 , wherein said cells being bombarded are prepared from non-embryo Brassica tissue by macerating the upper portion of a seedling in induction medium to form a cellular slurry, said upper portion comprising the shoot tip, cotyledons, and 10-50 percent of the hypocotyl.  
     
     
         9 . The method of  claim 7 , wherein said cellular slurry is enriched for cellular matter having a size of about 46 microns to about 230 microns.  
     
     
         10 . The method of  claim 1 , wherein said nucleic acid encodes a polypeptide or regulates the expression of a polypeptide.  
     
     
         11 . The method of  claim 1 , wherein said nucleic acid comprises an anti-sense molecule.  
     
     
         12 . The method of  claim 1 , wherein said nucleic acid comprises a ribozyme.  
     
     
         13 . The method of  claim 1 , wherein said nucleic acid comprises an anti-sense oligonucleotide.  
     
     
         14 . The method of  claim 1 , wherein said Brassica tissue is from a Brassica species selected from the group consisting of  Brassica napus, Brassicajuncea, Brassica carinata, Brassica nigra, Brassica oleracea,  and  Brassica campestris.    
     
     
         15 . The method of  claim 1 , wherein said identification comprises culturing said bombarded cells on a floatation device in contact with a liquid selection medium, and selecting those cells that survive in the presence of said selection medium.  
     
     
         16 . The method of  claim 1 , wherein said method further comprises the step of regenerating a Brassica plant from said identified cells.  
     
     
         17 . The method of  claim 16 , wherein said regeneration comprises culturing said identified cells on a floatation device in contact with liquid regeneration medium.  
     
     
         18 . Brassica cells and progeny thereof produced according to the method of  claim 1 .  
     
     
         19 . A method of culturing Brassica tissue, said method comprising culturing said tissue on a floatation device in the presence of liquid selection medium or in the presence of liquid regeneration medium.