High temperature increases haploid induction efficiency

Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type, whereas the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor, Kinetochore Null2 (KNL2), can be used as a haploid inducer when pollinated by wild-type. We discovered that the short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction by 10-fold. We have also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the GMO approach at any stage. Furthermore, the cenh3-4 mutant functions as a haploid inducer in response to short heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid inducer lines and showed that exposure of mutants of centromeric proteins to high temperatures strongly increases their haploid induction efficiency.