Engineered intraspecific hybrid lethality enables rapid haploid isolation and cytoplasm conversion in tobacco

Haploid plants can improve the efficiency of cultivar development by shortening breeding cycles and increasing genetic gain per unit of time. The ability to produce androgenic haploids from seed can also permit development of approaches for rapid conversion of female parents of new F₁ hybrids to cytoplasmic male sterility (CMS). A method for rapid and routine cyto-conversion does not currently exist for tobacco (Nicotiana tabacum L.). In this research, we used gene editing, genetic engineering, and interspecific hybridization to develop an approach for identification of gynogenic and androgenic tobacco haploids produced from seed using a novel N. tabacum × N. tabacum intraspecific lethality system. An established genetic stock was observed to produce viable progeny after self-pollination. Progeny produced from crosses with normal N. tabacum genotypes exhibited greater than 99.7% intraspecific lethality, however. Amongst surviving seedlings were haploids believed to be produced via spontaneous parthenogenesis. The frequency of verified gynogenic haploids was observed to be approximately 1 in 5000 seeds, while the frequency of verified androgenic haploids was lower, approximately 1 in 50,000 seeds. Because of the fecundity of tobacco (∼2500 seeds per pollination), these frequencies are high enough for use in practical plant breeding. A CMS version of the established genetic stock was demonstrated to be useful for converting an elite inbred line to CMS via androgenic haploidy.