Induction of pollen embryo and chromosome doubling in tobacco (Nicotiana tabacum L.)

Abstract

Anther culture is an effective breeding method for improving desirable traits, since it allows fast achievement of geneticallyhomozygous lines. The present studies were carried out to evaluate the response of flue-cured tobacco anthers for high frequency pollenembryo induction in different culture media, and the effects of colchicine treatment on tobacco chromosome doubling. The resultsshowed that different media caused significant changes in pollen embryo induction, in which the most obvious change was found in Bmedium with induction rates of 23.28%, 26.64%, and 28.07% for TMK-12 × Jiyan 5, K326 × Shangkang 70, and NC71 × Jingyehuang,respectively. In addition, shoot tips of the haploid aseptic seedlings cultivated from anthers were treated with 4 g/L colchicine for 48 h,and the ploidy levels were then checked by using flow cytometry and the number of chloroplasts in stomatal guard cells. The resultssuggested that colchicine treatment not only doubled the chromosomes of sterile haploid seedlings, but also caused some sterile haploidseedlings to die. The survival rate of TMK-12 × Jiyan 5, K326 × Shuangkang 70, and NC71 × Jingyehuang seedlings were 33.29%,35.11%, and 17.61%, respectively. Similarly, TMK-12 × Jiyan 5 and K326 × Shuangkang 70 also showed a relatively high chromosomedoubling rate compared with NC71 × Jingyehuang; their chromosome doubling rates were 21.53%, 18.54%, and 7.98%, respectively.In general, our work provides a theoretical basis for accelerating the breeding and improvement of new flue-cured tobacco varieties.