Doubling of chromosomes in haploid embryos
Methods for producing doubled haploid corn ( Zea mays ) plants, seeds, and plant cells are provided.
1. A method of obtaining a doubled haploid maize plant, said method comprising:
(a) pollinating silks of a maize ear with a maize inducer line to produce at least one diploid maize embryo and at least one haploid maize embryo;
(b) isolating said haploid maize embryo between 4-21 days after step (a), wherein said at least one haploid maize embryo is distinguished from the diploid maize embryos via expression of a marker;
(c) contacting said haploid maize embryo with a chromosome doubling agent to produce at least one doubled haploid maize embryo cell;
(d) culturing said doubled haploid maize embryo cell on a non-callus promoting medium; and
(e) generating a doubled haploid maize plant from said doubled haploid maize embryo cell.
2. The method of claim 1 , wherein the maize inducer line contains a marker gene that is expressed in embryo tissue.
3. The method of claim 2 , wherein said marker gene is R-nj.
4. The method of claim 2 , wherein said marker gene is GFP.
5. The method of claim 2 , wherein said marker gene is expressed 4 or more days after pollination.
6. The method of claim 2 , wherein said marker gene expresses anthocyanin pigments.
7. The method of claim 1 , wherein the haploid maize embryo of step (c) is 0.4 mm to 3 mm in length.
8. The method of claim 1 , wherein the chromosome doubling agent comprises an anti-microtubule agent.
9. The method of claim 1 , wherein said chromosomal doubling agent is selected from the group consisting of colchicine, pronamide, dithipyr, and trifluralin.
10. The method of claim 1 , wherein in step (c) the haploid embryo is in contact with the doubling agent for 4 hours to 48 hours.