Doubled haploid cells, embryos and plants
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.