Doubling of chromosomes in haploid embryos

Abstract

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 comprising:
 a) pollinating silks of a maize ear with an inducer line to produce at least one haploid embryo;   b) contacting said haploid embryo scutellum side up on a medium comprising with a chromosome doubling agent to produce at least one doubled haploid embryo cell;   c) generating a doubled haploid maize plant from said doubled haploid embryo cell, and;   d) wherein step (b) occurs 4-21 days after step (a).   
     
     
         2 . The method of  claim 1  wherein the 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 embryo of step (b) 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 step (b) the haploid embryo is in contact with the doubling agent for 4 hours to 48 hours. 
     
     
         11 - 18 . (canceled) 
     
     
         19 . A method of obtaining a doubled haploid maize plant comprising contacting a haploid embryo scutellum side up on a non-callus forming medium comprising a chromosome doubling agent to produce a doubled haploid embryo and generating a doubled haploid maize plant from said doubled haploid embryo. 
     
     
         20 . A method of obtaining a doubled haploid maize embryo comprising contacting a haploid embryo scutellum side up on a non-callus forming medium comprising a chromosome doubling agent to produce a doubled haploid embryo. 
     
     
         21 . (canceled)