An Optimized Culture System of Haploid Plants from Unfertilized Ovules of Male-Sterile Tobacco

Haploid breeding is an important method for efficient modern breeding of crops. Compared to the natural doubling, doubling induction of tobacco anther derived haploids often leads to the deterioration of yield and quality, limited utilization value and the doubling low frequency. Obtaining lines similar to the conventional selfing with no deterioration of yield and quality is not an easy task. Therefore, in view of that, the haploid induction of unfertilized ovule in vitro was studied using the tobacco male-sterile hybrid KRK26. The ovules of five developmental stages were selected and cultured on five optimized media based on H, HW and N6. The results showed that the more mature the embryo sac was, the worse the induction effect of ovule proliferation was under the test scope. The ovules which developed earlier, i.e. the corolla of the flower was about to be exposed from the calyx stage to the time when the corolla was twice as long as the calyx, are recommended to be used for culture. Among the five optimized media, the medium CM1 based on H medium had the best induction effect on embryoids, and adjusting the agar concentration to 1% on the basis of CM1 was beneficial to inhibit the vitrification of embryoids. The embryoids were transferred to H medium without hormones, which could avoid the browning of embryoids, and facilitate the induction of regenerated buds. The regenerated buds were transferred to H medium or N6 medium supplemented with 10 mg/L IAA, which was beneficial to the rapid rooting of regenerated buds and obtaining a large number of regenerated plants. The haploid regenerated plants were identified based on Flow cytometry test. In this study, an optimized culture system was established to induce haploid plants from unfertilized ovule of male sterile tobacco.

Author(s): Muna Alariqi, Chen Luo, Cao Jinglin and Jin Shuangxia

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