Doubled haploid cells, embryos and plants

Abstract

Methods for producing homozygous plants, seeds, and plant cells are provided. Also provided are methods of transformation.

1 . A method of transformation comprising stably transforming a plant cell with a polynucleotide of interest and treating the transformed cell with a chromosome doubling agent, wherein the plant cell comprises a haploid somatic cell. 
     
     
         2 . The method of  claim 1  wherein the plant cell is a maize cell. 
     
     
         3 . The method of  claim 1  further comprising providing a growth stimulation protein. 
     
     
         4 . The method of  claim 3  wherein the growth stimulation protein is RepA or Lec1. 
     
     
         5 . The method of  claim 1  wherein the plant cell is a haploid embryo cell produced by crossing a selected female parent with a male inducer line. 
     
     
         6 . The method of  claim 1  wherein the inducer line contains a scorable marker gene operably linked to an embryo-preferred promoter. 
     
     
         7 . The method of  claim 1  further comprising regenerating a homozygous plant from the homozygous plant cell. 
     
     
         8 . A method for producing a homozygous plant comprising:
 a) stably transforming a plant cell with a polynucleotide of interest, wherein the plant cell comprises a haploid somatic cell;   b) treating the transformed cell with a chromosome doubling agent; and   c) growing the treated cell to produce a homozygous plant.   
     
     
         9 . The method of  claim 8  wherein the plant cell is a maize cell. 
     
     
         10 . The method of  claim 8  further comprising providing a growth stimulation protein. 
     
     
         11 . The method of  claim 10  wherein the growth stimulation protein is RepA or Lec1. 
     
     
         12 . The method of  claim 8  wherein the plant cell is a haploid embryo cell produced by crossing a selected female parent with a male inducer line. 
     
     
         13 . The method of  claim 12  wherein the inducer line contains a scorable marker gene operably linked to an embryo-preferred promoter. 
     
     
         14 . The method of  claim 8  wherein the polynucleotide of interest is replaced with a second polynucleotide of interest. 
     
     
         15 . A method of producing a stably transformed double haploid maize plant comprising a) pollinating a maize plant with an inducer line to form a haploid maize embryo and a diploid maize embryo, wherein said inducer line comprises a visual marker that is expressed in said double haploid embryo within 15 days after pollination, b) isolating said haploid maize embryo, c) stably transforming a haploid maize embryo cell with a polynucleotide of interest to form a transformed maize cell, d) growing transformed callus from the transformed maize cell, e) treating the transformed callus with a chromosome doubling agent to produce a stably transformed double haploid maize cell and f) regenerating said double haploid maize cell into a double haploid maize plant. 
     
     
         16 . The method of  claim 15  further comprising providing a growth stimulation protein wherein said growth stimulation protein is provided through the expression of a transgene stably integrated into said maize cell of step (c) or wherein said growth stimulation protein is provided through an expression cassette during said transforming of step (c). 
     
     
         17 . The method of  claim 16  wherein the growth stimulation protein is RepA or Lec1.