Tomato (Solanum lycopersicon) is the second largest vegetable crop and the largest fruit crop (Costa and Heuvelink, 2018). Progress in tomato breeding has been achieved by classical breeding, introgression of traits found in related wild Solanum species and exploiting heterosis in F₁ hybrid crosses (Lin et al., 2014). These approaches require the development of inbred lines to reduce or largely eliminate heterozygosity. Classically, multiple rounds of selfing or backcrossing are used to generate inbred lines (Gale, 1980), but homozygous lines can also be obtained in a single generation using doubled haploid (DH) technology (Jacquier et al., 2020). However, tomato is highly recalcitrant for haploid induction (HI) (Seguí-Simarro, 2010).

Summary: In vivo haploid induction has been extended from maize to monocotyledonous plants like rice, wheat, millet and dicotyledonous plants such as tomato, rapeseed, tobacco and cabbage. Accurate identification of haploids is a crucial step of doubled haploid technology, where a useful identification marker is very pivotal. R1‐nj is an extensively used visual marker for haploid identification in maize. RFP and eGFP have been shown to be feasible in identifying haploid. However, these methods are either limited to specific species, or require specific equipment. It still lacks an efficient visual marker that is practical across different crop species. In this study, we introduced the RUBY reporter, a betalain biosynthesis system, into maize and tomato haploid inducers as a new marker for haploid identification. Results showed that expression of RUBY could result in deep betalain pigmentation in maize embryos as early as 10 days after pollination, and enabled 100% accuracy of immature haploid embryo identification. Further investigation in tomato revealed that the new marker led to deep red pigmentation in radicles and haploids can be identified easily and accurately. The results demonstrated that the RUBY reporter is a background‐independent and efficient marker for haploid identification and would be promising in doubled haploid breeding across different crop species.

SUMMARY: Because of their high efficiency during chromosome doubling, immature haploid maize (Zea mays L.) embryos are useful for doubled haploid production. The R1‐nj marker is commonly used in doubled haploid breeding and has improved the efficiency of haploid identification. However, its effectiveness is limited by genetic background and environmental factors. We addressed this technical challenge by developing an efficient and accurate haploid embryo identification marker through co‐expression of two transcription factor genes (ZmC1 and ZmR2) driven by the embryo–aleurone‐specific bidirectional promoter P_ZmBD1; these factors can activate anthocyanin biosynthesis in the embryo and aleurone layer during early seed development. We developed a new haploid inducer, Maize Anthocyanin Gene InduCer 1 (MAGIC1), by introducing the transgenes into the haploid inducer line CAU6. MAGIC1 could identify haploids at 12 days after pollination, which is nine days earlier than CAU6. Importantly, MAGIC1 increased haploid identification accuracy to 99.1%, compared with 88.3% for CAU6. In addition, MAGIC1 could effectively overcome the inhibition of anthocyanin synthesis in some germplasms. Furthermore, an upgraded anthocyanin marker was developed from ZmC1 and ZmR2 to generate MAGIC2, which could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. This haploid identification system is more efficient and accurate than the conventional R1‐nj‐based method, and it simplifies the haploid identification process. Therefore, this system provides technical support for large‐scale doubled haploid line production.