Molecular Plant
                                                                                                                                          Editor’s Highlights
                  Next-Generation Crop Breeding Methods
                  Different from conventional labor-intensive and time-consuming           restricting direct utilization of genome editing tools for crop
                  breeding approaches such as genetic cross and mutation                   breeding.
                  screening, new breeding technologies such as double haploid
                  (DH) and CRISPR/Cas-mediated genome editing can greatly                  Twostudies recently reported innovative, rapid breeding strate-
                  enhance breeding efficiency and accelerate crop improvement.              gies combining HI and CRISPR/Cas9-mediated genome editing
                  DH technology relies on an inducer line for haploid induction            by a single cross, which would accelerate breeding in a wide
                  (HI) when pollination onto elite varieties or inbred lines. DH lines     range of crops (Kelliher et al., 2019; Wang et al., 2019a). The
                  with desired traits are then obtained from haploids in a subse-          method reported by a Syngenta research group was termed
                  quent generation by natural or artificial chromosome doubling             HI-Edit (Kelliher et al., 2019). In their study, they first used
                  using mitotic inhibitors. Natural HI systems have been widely            CRISPR/Cas9 to edit MATL in a maize inbred line NP2222.
                  usedfor breeding in maize, wheat, and barley, while engineering          They found that pollens of created matl plants expressing Cas9
                  of HI systems is also feasible by manipulating CENTROMERIC               and guide RNA (gRNA) targeting MATL could further edit
                  HISTONE3 (CENH3) in dicots or mutating MATL/PLA1/NLD in                  maternal MATL after fertilization with another commercial
                  rice and maize (Ravi and Chan, 2010; Kelliher et al., 2017; Liu          inbred to produce haploids with new matl mutations but without
                  et al., 2017; Dong et al., 2018). The CRISPR/Cas9 system and             the male parent genome, suggesting that matl-mediated HI, like
                  its variants can directly modify trait-associated genes in a short       CENH3-induced HI, caused post-fertilization male genome
                  period and have become powerful tools for breeding in many               elimination to form haploids. They then applied the HI-Edit strat-
                  crops nowadays. However, many elite crop varieties and inbred            egy to edit four maize yield-associated genes (VLHP1, VLHP2,
                  lines are recalcitrant to genetic transformation and regeneration,       ZmGW2-1, and ZmGW2-2). Individual Cas9-gRNA constructs
                  Figure 1. The Process and Principle of HI-Edit or IMGE for Crop Breeding.
                                                                                           Published by the Molecular Plant Shanghai Editorial Office in association with
                                                                                           Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
                  470    Molecular Plant 12, 470–471, April 2019 ª The Author 2019.
              Editor’s Highlights                                                                                               Molecular Plant
              were transformed into NP222, and Cas9-positive plants were             reduce unwanted editing during vegetative development using
              crossed with native matl HI line. F2 individuals carrying both         gamete-specific promoters and improve the efficiency of both
              matl mutation and Cas9 transgene were identified and pollinated         genomeediting and HI (including induced by intergeneric cross)
              onto the ears of several field-grown inbred lines (Figure 1).           in these approaches may be the focus of future research. With
              Analyses of the editing efficiency for each gene showed that            the development of these highly promising approaches and
              HI-Edit produced over 3% edited haploids in total from five             further improvements, we trust a new era of crop breeding is
              of six inbred lines. In an independent study, Wang et al. (2019a)      coming of age.
              reported   a similar approach combining genome editing
              with matl-mediated HI for maize breeding, called Haploid-                                   Juanying Ye and Xiaofeng Cui*
              Inducer Mediated Genome Editing (IMGE). They tested three
              development-related genes and obtained Cas9/matl-free edited                                                               Molecular Plant
              haploids for two genes in B73 background, with a higher effi-                            *Correspondence: Xiaofeng Cui (xiaofeng@sibs.ac.cn)
                                                                                                                  https://doi.org/10.1016/j.molp.2019.03.007
              ciency (4.1%) for LIGULELESS1 (LG1), disruption of which
              greatly reduced leaf angle due to the lack of leaf auricle and         REFERENCES
              ligules. Notably, they obtained DH seeds from three lg1 haploids       Dong, L., Li, L., Liu, C., Liu, C., Geng, S., Li, X., Huang, C., Mao, L.,
              resulted from natural chromosome doubling, demonstrating the             Chen, S., and Xie, C. (2018). Genome editing and double-
              feasibility of HI-Edit/IMGE for maize breeding.                          fluorescence proteins enable robust maternal haploid induction and
                                                                                       identification in maize. Mol. Plant 11:1214–1217.
              SincetherearenoclearorthologsofMATLindicots,Kelliheretal.              Kelliher, T., Starr, D., Richbourg, L., Chintamanani, S., Delzer, B.,
              (2019) established an HI-Edit approach in Arabidopsis using the          Nuccio, M.L., Green, J., Chen, Z., McCuiston, J., Wang, W., et al.
              CENH3 HI system. They created a transgenic Col-0 line with               (2017). MATRILINEAL, a sperm-specific phospholipase, triggers
              native AtCENH3 replaced by ZmCENH3 transgene. After this                 maize haploid induction. Nature 542:105–109.
              line was further transformed with a Cas9-gRNA construct target-        Kelliher,T.,Starr,D.,Su,X.,Tang,G.,Chen,Z.,Carter,J.,Wittich,P.E.,
              ing GLABROUS1 (GL1) that controls trichome development,                  Dong,S.,Green,J.,Burch,E.,etal.(2019).One-stepgenomeediting
              they pollinated wild-type Ler pollens onto flowers of created gl1         of elite crop germplasm during haploid induction. Nat. Biotechnol.
              mutant for HI-Editing. Analyses of haploid progenies showed              37:287–292.
              high maternal HI-Edit efficiency (16.9%) and faithful inheritance       Khanday, I., Skinner, D., Yang, B., Mercier, R., and Sundaresan, V.
              ofeditedlociinArabidopsis,providingapotentialHI-Editstrategy             (2019). A male-expressed rice embryogenic trigger redirected for
              for dicot crops. In addition, they demonstrated the success of           asexual propagation through seeds. Nature 565:91–95.
              HI-Edit in wheat by employing a natural HI phenomenon, i.e.,           Liu,C.,Li,X.,Meng,D.,Zhong,Y.,Chen,C.,Dong,X.,Xu,X.,Chen,B.,
              intergeneric cross of wheat with maize pollens could induce              Li, W., Li, L., et al. (2017). A 4-bp insertion at ZmPLA1 encoding a
              wheat haploid, and found that Cas9 expression in maize driven            putative phospholipase a generates haploid induction in maize. Mol.
              by pollen-specific promoters may enhance HI-Edit efficiency                Plant 10:520–522.
              in wheat.                                                              Ravi, M., and Chan, S.W. (2010). Haploid plants produced by
                                                                                       centromere-mediated genome elimination. Nature 464:615–618.
              The HI-Edit/IMGE methods highlighted here might enable wide-           Wang, B., Zhu, L., Zhao, B., Zhao, Y., Zheng, Z., Li, Y., Sun, J., and
              spread applications of genome editing technologies in many               Wang, H. (2019a). Development of a haploid-inducer mediated
              commercial crop varieties. Together with two other recently re-          genome editing (IMGE) system for accelerating maize breeding. Mol.
              ported approaches that combine CRISPR/Cas9-created MiMe-                 Plant 12. https://doi.org/10.1016/j.molp.2019.03.006.
              mediated apomeiosis with either ectopic expression of BBM1             Wang,C.,Liu,Q.,Shen,Y.,Hua,Y.,Wang,J.,Lin,J.,Wu,M.,Sun,T.,
              or knockout of MATL for clonal propagation of hybrids                    Cheng,Z.,Mercier,R.,etal.(2019b).Clonalseedsfromhybridriceby
              (Khanday et al., 2019; Wang et al., 2019b), these likely                 simultaneous genome engineering of meiosis and fertilization genes.
              constitute part of next-generation crop breeding methods. To             Nat. Biotechnol. 37:283–286.
                                                                                      Molecular Plant 12, 470–471, April 2019 ª The Author 2019.   471