Abstract: To investigate the chromosome doubling and double haploid rapid propagation technique of ovule haploid plants, six developmental stages of leaves from ovule haploid plants of male sterile tobacco hybrid KRK26 were selected, and their main vein segments were used as explants to double the chromosomes of ovule haploid plants by the cell culture doubling method. The results showed that chromosome doubling of ovule haploid plants by somatic cell culture was superior to the colchicine soaking method. Among leaves at different developmental stages, the chromosome doubling frequency of regenerated plants induced by the main vein segment of leaves, which remained on the plant for 21 to 28 days after full unfolding, was the highest, reaching 50.15%-54.06%. At the same developmental stage, the chromosome doubling frequency of regenerated plants induced by the main vein segments of leaves was higher than that induced by the mesophyll tissue fragments of leaves; that induced by the main vein segment from the base of the main vein to about 1/4 of the base of the main vein in leaves was the highest, reaching 81.79%. The transplantation survival rate of the double haploid seedlings selected by flow cytometry identification could reach 100% after hydroponic cultivation for about 14 days. An integrated technology system for chromosome doubling and rapid propagation of ovule haploid plants was established.