@article{barco_dong_matsuba_crook_xu_zhang_zhang_carlin_potter_rigoulot_et al._2025, title={Development of efficient targeted insertion mediated by CRISPR-Cas12a and homology-directed repair in maize}, DOI={10.3389/fgeed.2025.1713347}, abstractNote={Targeted insertion (TIN) of transgenic trait cassettes has the potential to reduce timeline and cost for GM product development and commercialization. Precise genome engineering has made remarkable progress over the last several decades, particularly with the use of site-directed nucleases as core editing machinery. However, there are still many critical factors that can impact TIN efficiency including insertion site selection, nuclease optimization and expression, donor vector design, gene delivery, and stable event regeneration. Here, we develop workflows for target site sequence identification and gRNA screening for CRISPR-Cas12a system and demonstrate its successful application for TIN in maize with donor sequences up to 10 kilobase pairs (kb) in size. We first prioritize genomic regions for inserting transgenes <i>in silico</i> using bioinformatics tools and then test gRNA performance using a leaf protoplast transient assay. Despite its known low efficiency, we identify homology-directed repair (HDR) as the preferential pathway for directing targeted insertions of large sequences in immature embryos and demonstrate double-junction integrations at a rate of up to 4%. We further apply a molecular analysis workflow using large amplicon TaqMan assays and nanopore sequencing for streamlined identification and characterization of high-quality insertion events with intact large inserts. Analysis of TIN events across generations suggests that efficiency bottlenecks are not limited to donor targeted integration; attrition in efficiency also results from partial or additional donor insertion, chimerism, and close linkage with undesired sequence insertions such as those encoding the editing machinery. This work represents a major step forward in realizing the potential of precise genome engineering in maize for basic research and biotech trait development applications.}, journal={Frontiers in Genome Editing}, author={Barco, Brenden and Dong, Shujie and Matsuba, Yuki and Crook, Ashley and Xu, Ruiji and Zhang, Yingxiao and Zhang, Chengjin and Carlin, Ryan and Potter, Kevin and Rigoulot, Stephen B. and et al.}, year={2025}, month={Dec} }
 @article{lv_liang_bumann_mirleau thebaud_jin_li_paris_dan_li_cui_et al._2025, title={Haploid facultative parthenogenesis in sunflower sexual reproduction}, volume={641}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/s41586-025-08798-2}, DOI={10.1038/s41586-025-08798-2}, abstractNote={Flowering plant sexual reproduction requires double fertilization, yielding embryo and endosperm seed compartments: the latter supports embryo growth and seed germination. In an experiment to generate haploid embryos through inhibition of pollen phospholipase activity in sunflower (Helianthus annus), we serendipitously discovered that emasculated sunflowers spontaneously form parthenogenic haploid seed. Exploration of genetic, chemical and environmental factors demonstrated that a specific genotype background enabled high parthenogenesis and that full spectrum high-intensity light supplementation boosted parthenogenesis, yielding hundreds of haploid seeds per head. Induction of doubled haploid plants can greatly accelerate plant breeding efficiency; however, despite successful engineering of haploid induction in many crops, few reported systems are commercially scalable 1 . Here we report efficient methods of chemical emasculation and genome doubling to produce fertile plants and enable a scalable sunflower doubled haploid system.}, number={8063}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Lv, Jian and Liang, Dawei and Bumann, Eric and Mirleau Thebaud, Virginie and Jin, Huaibing and Li, Changbao and Paris, Clemence and Dan, Yinghui and Li, Chao and Cui, Ruijie and et al.}, year={2025}, month={Apr}, pages={732–739} }
 @article{nelson_ranney_liu_kelliher_duan_da_2025, title={Overcoming Recalcitrance: A Review of Regeneration Methods and Challenges in Roses}, url={https://doi.org/10.3390/plants14243797}, DOI={10.3390/plants14243797}, abstractNote={Roses (<i>Rosa</i> spp.) are among the most economically and ornamentally important floricultural crops worldwide, yet their improvement is constrained by inefficient breeding methods. Tissue culture regeneration based plant transformation and genome editing technologies provide innovative and increasingly effective approaches to surmount these longstanding challenges; however, rose tissue culture regeneration remains notoriously recalcitrant. Successful plant regeneration in roses depends on multiple factors, including genotype, explant source, physiological status, and the precise combination of plant growth regulators and culture conditions. Over the past three decades, numerous efforts have focused on optimizing rose organogenesis and somatic embryogenesis systems. Despite progress, low regeneration frequencies, strong genotype dependency continue to limit molecular breeding and genome editing application in rose. This review synthesizes current advances in <i>in vitro</i> regeneration methods for roses, emphasizing key determinants of morphogenic response, including explant selection, hormonal balance, media composition, light and temperature regimes, and the organic and inorganic additives. The underlying causes of recalcitrance are discussed in relation to tissue physiology, biochemical and molecular regulation of morphogenesis. Finally, strategies for overcoming regeneration barriers-such as the use of morphogenic regulators and <i>in planta</i> transformation-are highlighted as emerging avenues toward cultivar independent transformation and genome editing for rose.}, journal={Plants}, author={Nelson, Anna and Ranney, Thomas and Liu, Wusheng and Kelliher, Tim and Duan, Hui and Da, Kedong}, year={2025}, month={Dec} }
 @article{bortiri_selby_egger_tolhurst_dong_beam_meier_fabish_delaney_dunn_et al._2024, title={Cyto-swapping in maize by haploid induction with a cenh3 mutant}, volume={10}, ISSN={2055-0278}, url={http://dx.doi.org/10.1038/s41477-024-01630-1}, DOI={10.1038/s41477-024-01630-1}, abstractNote={Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.}, number={4}, journal={Nature Plants}, publisher={Springer Science and Business Media LLC}, author={Bortiri, Esteban and Selby, Rebecca and Egger, Rachel and Tolhurst, Lindsey and Dong, Shujie and Beam, Kayla and Meier, Kerry and Fabish, Jon and Delaney, Donna and Dunn, Mary and et al.}, year={2024}, month={Mar}, pages={567–571} }
 @article{delzer_liang_szwerdszarf_rodriguez_mardones_elumalai_johnson_nalapalli_egger_burch_et al._2024, title={Elite, transformable haploid inducers in maize}, volume={12}, ISSN={2214-5141}, url={http://dx.doi.org/10.1016/j.cj.2023.10.016}, DOI={10.1016/j.cj.2023.10.016}, abstractNote={The introduction of alleles into commercial crop breeding pipelines is both time consuming and costly. Two technologies that are disrupting traditional breeding processes are doubled haploid (DH) breeding and genome editing (GE). Recently, these techniques were combined into a GE trait delivery system called HI-Edit (Haploid Inducer-Edit) [1]. In HI-Edit, the pollen of a haploid inducer line is reprogrammed to deliver GE traits to any variety, obviating recurrent selection. For HI-Edit to operate at scale, an efficient transformable HI line is needed, but most maize varieties are recalcitrant to transformation, and haploid inducers are especially difficult to transform given their aberrant reproductive behaviors. Leveraging marker assisted selection and a three-tiered testing scheme, we report the development of new Iodent and Stiff Stalk maize germplasm that are transformable, have high haploid induction rates, and exhibit a robust, genetically-dominant anthocyanin native trait that may be used for rapid haploid identification. We show that transformation of these elite “HI-Edit” lines is enhanced using the BABYBOOM and WUSCHEL morphogenetic factors. Finally, we evaluate the HI-Edit performance of one of the lines against both Stiff Stalk and non-Stiff Stalk testers. The strategy and results of this study should facilitate the development of commercially scalable HI-Edit systems in diverse crops.}, number={1}, journal={The Crop Journal}, publisher={Elsevier BV}, author={Delzer, Brent and Liang, Dawei and Szwerdszarf, David and Rodriguez, Isadora and Mardones, Gonzalo and Elumalai, Sivamani and Johnson, Francine and Nalapalli, Samson and Egger, Rachel and Burch, Erin and et al.}, year={2024}, month={Feb}, pages={314–319} }
 @article{lv_yu_wei_gui_liu_liang_wang_zhou_carlin_rich_et al._2020, title={Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3}, volume={38}, ISSN={1087-0156 1546-1696}, url={http://dx.doi.org/10.1038/s41587-020-0728-4}, DOI={10.1038/s41587-020-0728-4}, abstractNote={New breeding technologies accelerate germplasm improvement and reduce the cost of goods in seed production 1-3 . Many such technologies could use in vivo paternal haploid induction (HI), which occurs when double fertilization precedes maternal (egg cell) genome loss. Engineering of the essential CENTROMERIC HISTONE (CENH3) gene induces paternal HI in Arabidopsis 4-6 . Despite conservation of CENH3 function across crops, CENH3-based HI has not been successful outside of the Arabidopsis model system 7 . Here we report a commercially operable paternal HI line in wheat with a ~7% HI rate, identified by screening genome-edited TaCENH3α-heteroallelic combinations. Unlike in Arabidopsis, edited alleles exhibited reduced transmission in female gametophytes, and heterozygous genotypes triggered higher HI rates than homozygous combinations. These developments might pave the way for the deployment of CENH3 HI technology in diverse crops.}, number={12}, journal={Nature Biotechnology}, publisher={Springer Science and Business Media LLC}, author={Lv, Jian and Yu, Kun and Wei, Juan and Gui, Huaping and Liu, Chunxia and Liang, Dawei and Wang, Yanli and Zhou, Hongju and Carlin, Ryan and Rich, Randy and et al.}, year={2020}, month={Nov}, pages={1397–1401} }
 @article{santeramo_howell_ji_yu_liu_kelliher_2019, title={DNA content equivalence in haploid and diploid maize leaves}, volume={251}, ISSN={0032-0935 1432-2048}, url={http://dx.doi.org/10.1007/s00425-019-03320-1}, DOI={10.1007/s00425-019-03320-1}, abstractNote={Abstract Main conclusion The qPCR assay developed to differentiate haploid and diploid maize leaf samples was unsuccessful due to DNA content difference. Haploid cells are packed more closely together with less cellular expansion. Abstract Increased ploidy content (> 2 N) directly correlates with increased cell size in plants, but few studies have examined cell morphology in plants with reduced ploidy (i.e., haploids). To pioneer a scalable new ploidy test, we compared DNA content and cellular morphology of haploid and diploid maize leaves. The amount of genomic DNA recovered from standardized leaf-punch samples was equivalent between these two ploidy types, while both epidermal and mesophyll cell types were smaller in haploid plants. Pavement cells had a substantially smaller size than mesophyll cells, and this effect was more pronounced in the abaxial epidermis. Interveinal distance and guard cell size were significantly reduced in haploids, but the cell percentage comprising stomata did not change. These results confirm the direct correlation between ploidy content and cell size in plants, and suggest that reduced cell expansion predominantly explains DNA content equivalence between haploid and diploid samples, confounding efforts to develop a haploid detection method using DNA content.}, number={1}, journal={Planta}, publisher={Springer Science and Business Media LLC}, author={Santeramo, D. and Howell, J. and Ji, Y. and Yu, W. and Liu, W. and Kelliher, T.}, year={2019}, month={Dec} }
 @book{kelliher_delzer_chintamanani_skibbe_chen_starr_wendeborn_2019, title={Haploid induction compositions and methods for use therefor}, number={WO2017087682A1}, author={Kelliher, T. and Delzer, B. and Chintamanani, S. and Skibbe, D.S. and Chen, Z. and Starr, D. and Wendeborn, S.}, year={2019} }
 @article{kelliher_starr_su_tang_chen_carter_wittich_dong_green_burch_et al._2019, title={One-step genome editing of elite crop germplasm during haploid induction}, url={https://doi.org/10.1038/s41587-019-0038-x}, DOI={10.1038/s41587-019-0038-x}, abstractNote={Genome editing using CRISPR-Cas9 works efficiently in plant cells 1 , but delivery of genome-editing machinery into the vast majority of crop varieties is not possible using established methods 2 . We co-opted the aberrant reproductive process of haploid induction (HI) 3-6  to induce edits in nascent seeds of diverse monocot and dicot species. Our method, named HI-Edit, enables direct genomic modification of commercial crop varieties. HI-Edit was tested in field and sweet corn using a native haploid-inducer line 4  and extended to dicots using an engineered CENH3 HI system 7 . We also recovered edited wheat embryos using Cas9 delivered by maize pollen. Our data indicate that a transient hybrid state precedes uniparental chromosome elimination in maize HI. Edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery. Therefore, edited plants could be used in trait testing and directly integrated into commercial variety development.}, journal={Nature Biotechnology}, author={Kelliher, Timothy and Starr, Dakota and Su, Xiujuan and Tang, Guozhu and Chen, Zhongying and Carter, Jared and Wittich, Peter E. and Dong, Shujie and Green, Julie and Burch, Erin and et al.}, year={2019}, month={Mar} }
 @misc{que_chen_kelliher_skibbe_dong_chilton_2019, title={Plant DNA Repair Pathways and Their Applications in Genome Engineering}, ISBN={9781493989904 9781493989911}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-4939-8991-1_1}, DOI={10.1007/978-1-4939-8991-1_1}, abstractNote={Remarkable progress in the development of technologies for sequence-specific modification of primary DNA sequences has enabled the precise engineering of crops with novel characteristics. These programmable sequence-specific modifiers include site-directed nucleases (SDNs) and base editors (BEs). Currently, these genome editing machineries can be targeted to specific chromosomal locations to induce sequence changes. However, the sequence mutation outcomes are often greatly influenced by the type of DNA damage being generated, the status of host DNA repair machinery, and the presence and structure of DNA repair donor molecule. The outcome of sequence modification from repair of DNA double-strand breaks (DSBs) is often uncontrollable, resulting in unpredictable sequence insertions or deletions of various sizes. For base editing, the precision of intended edits is much higher, but the efficiency can vary greatly depending on the type of BE used or the activity of the endogenous DNA repair systems. This article will briefly review the possible DNA repair pathways present in the plant cells commonly used for generating edited variants for genome engineering applications. We will discuss the potential use of DNA repair mechanisms for developing and improving methodologies to enhance genome engineering efficiency and to direct DNA repair processes toward the desired outcomes.}, journal={Methods in Molecular Biology}, publisher={Springer New York}, author={Que, Qiudeng and Chen, Zhongying and Kelliher, Tim and Skibbe, David and Dong, Shujie and Chilton, Mary-Dell}, year={2019}, pages={3–24} }
 @book{kelliher_que_2019, title={Simultaneous editing and haploid induction}, number={WO2018102816A1}, author={Kelliher, T.Que and Que, Q.}, year={2019} }
 @article{yao_zhang_liu_liu_wang_liang_liu_sahoo_kelliher_2018, title={OsMATL mutation induces haploid seed formation in indica rice}, url={https://doi.org/10.1038/s41477-018-0193-y}, DOI={10.1038/s41477-018-0193-y}, abstractNote={Intraspecific haploid induction in maize (Zea mays) is triggered by a native frameshift mutation in MATRILINEAL (MATL), which encodes a pollen-specific phospholipase. To develop a haploid inducer in rice (Oryza sativa), we generated an allelic series in the putative ZmMATL orthologue, OsMATL, and found that knockout mutations led to a reduced seed set and a 2-6% haploid induction rate. This demonstrates MATL functional conservation and represents a major advance for rice breeding.}, journal={Nature Plants}, author={Yao, Li and Zhang, Ya and Liu, Chunxia and Liu, Yubo and Wang, Yanli and Liang, Dawei and Liu, Juntao and Sahoo, Gayatri and Kelliher, Timothy}, year={2018}, month={Jul} }
 @article{kelliher_starr_richbourg_chintamanani_delzer_nuccio_green_chen_mccuiston_wang_et al._2017, title={MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction}, volume={542}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/nature20827}, DOI={10.1038/nature20827}, abstractNote={Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.}, number={7639}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Kelliher, Timothy and Starr, Dakota and Richbourg, Lee and Chintamanani, Satya and Delzer, Brent and Nuccio, Michael L. and Green, Julie and Chen, Zhongying and McCuiston, Jamie and Wang, Wenling and et al.}, year={2017}, month={Jan}, pages={105–109} }
 @article{kelliher_starr_wang_mccuiston_zhong_nuccio_martin_2016, title={Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize}, volume={7}, ISSN={1664-462X}, url={http://dx.doi.org/10.3389/fpls.2016.00414}, DOI={10.3389/fpls.2016.00414}, abstractNote={Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3 (-∕-) and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3 (-∕-) lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.}, journal={Frontiers in Plant Science}, publisher={Frontiers Media SA}, author={Kelliher, Timothy and Starr, Dakota and Wang, Wenling and McCuiston, Jamie and Zhong, Heng and Nuccio, Michael L. and Martin, Barry}, year={2016}, month={Mar} }
 @article{kelliher_walbot_2014, title={Maize germinal cell initials accommodate hypoxia and precociously express meiotic genes}, volume={77}, ISSN={0960-7412 1365-313X}, url={http://dx.doi.org/10.1111/tpj.12414}, DOI={10.1111/tpj.12414}, abstractNote={Summary In flowering plants, anthers are the site of de novo germinal cell specification, male meiosis, and pollen development. Atypically, anthers lack a meristem. Instead, both germinal and somatic cell types differentiate from floral stem cells packed into anther lobes. To better understand anther cell fate specification and to provide a resource for the reproductive biology community, we isolated cohorts of germinal and somatic initials from maize anthers within 36 h of fate acquisition, identifying 815 specific and 1714 significantly enriched germinal transcripts, plus 2439 specific and 2112 significantly enriched somatic transcripts. To clarify transcripts involved in cell differentiation, we contrasted these profiles to anther primordia prior to fate specification and to msca1 anthers arrested in the first step of fate specification and hence lacking normal cell types. The refined cell‐specific profiles demonstrated that both germinal and somatic cell populations differentiate quickly and express unique transcription factor sets; a subset of transcript localizations was validated by in situ hybridization. Surprisingly, germinal initials starting 5 days of mitotic divisions were enriched significantly in >100 transcripts classified in meiotic processes that included recombination and synapsis, along with gene sets involved in RNA metabolism, redox homeostasis, and cytoplasmic ATP generation. Enrichment of meiotic‐specific genes in germinal initials challenges current dogma that the mitotic to meiotic transition occurs later in development during pre‐meiotic S phase. Expression of cytoplasmic energy generation genes suggests that male germinal cells accommodate hypoxia by diverting carbon away from mitochondrial respiration into alternative pathways that avoid producing reactive oxygen species ( ROS ).}, number={4}, journal={The Plant Journal}, publisher={Wiley}, author={Kelliher, Timothy and Walbot, Virginia}, year={2014}, month={Jan}, pages={639–652} }
 @article{que_elumalai_li_zhong_nalapalli_schweiner_fei_nuccio_kelliher_gu_et al._2014, title={Maize transformation technology development for commercial event generation}, volume={5}, ISSN={1664-462X}, url={http://dx.doi.org/10.3389/fpls.2014.00379}, DOI={10.3389/fpls.2014.00379}, abstractNote={Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.}, journal={Frontiers in Plant Science}, publisher={Frontiers Media SA}, author={Que, Qiudeng and Elumalai, Sivamani and Li, Xianggan and Zhong, Heng and Nalapalli, Samson and Schweiner, Michael and Fei, Xiaoyin and Nuccio, Michael and Kelliher, Timothy and Gu, Weining and et al.}, year={2014}, month={Aug} }
 @book{kelliher_walbot_2014, title={Method for modulating the number of archesporial cells in a developing anther}, number={WO2013123051A1}, author={Kelliher, T. and Walbot, V.}, year={2014}, month={Jul} }
 @article{zhang_egger_kelliher_morrow_fernandes_nan_walbot_2014, title={Transcriptomes and Proteomes Define Gene Expression Progression in Pre-meiotic Maize Anthers}, volume={4}, ISSN={2160-1836}, url={http://dx.doi.org/10.1534/g3.113.009738}, DOI={10.1534/g3.113.009738}, abstractNote={Abstract Plants lack a germ line; consequently, during reproduction adult somatic cells within flowers must switch from mitotic proliferation to meiosis. In maize (Zea mays L.) anthers, hypoxic conditions in the developing tassel trigger pre-meiotic competence in the column of pluripotent progenitor cells in the center of anther lobes, and within 24 hr these newly specified germinal cells have patterned their surrounding neighbors to differentiate as the first somatic niche cells. Transcriptomes were analyzed by microarray hybridization in carefully staged whole anthers during initial specification events, after the separation of germinal and somatic lineages, during the subsequent rapid mitotic proliferation phase, and during final pre-meiotic germinal and somatic cell differentiation. Maize anthers exhibit a highly complex transcriptome constituting nearly three-quarters of annotated maize genes, and expression patterns are dynamic. Laser microdissection was applied to begin assigning transcripts to tissue and cell types and for comparison to transcriptomes of mutants defective in cell fate specification. Whole anther proteomes were analyzed at three developmental stages by mass spectrometric peptide sequencing using size-fractionated proteins to evaluate the timing of protein accumulation relative to transcript abundance. New insights include early and sustained expression of meiosis-associated genes (77.5% of well-annotated meiosis genes are constitutively active in 0.15 mm anthers), an extremely large change in transcript abundances and types a few days before meiosis (including a class of 1340 transcripts absent specifically at 0.4 mm), and the relative disparity between transcript abundance and protein abundance at any one developmental stage (based on 1303 protein-to-transcript comparisons).}, number={6}, journal={G3 Genes|Genomes|Genetics}, publisher={Oxford University Press (OUP)}, author={Zhang, Han and Egger, Rachel L and Kelliher, Timothy and Morrow, Darren and Fernandes, John and Nan, Guo-Ling and Walbot, Virginia}, year={2014}, month={Jun}, pages={993–1010} }
 @article{kelliher_egger_zhang_walbot_2014, title={Unresolved issues in pre-meiotic anther development}, volume={5}, ISSN={1664-462X}, url={http://dx.doi.org/10.3389/fpls.2014.00347}, DOI={10.3389/fpls.2014.00347}, abstractNote={Compared to the diversity of other floral organs, the steps in anther ontogeny, final cell types, and overall organ shape are remarkably conserved among Angiosperms. Defects in pre-meiotic anthers that alter cellular composition or function typically result in male-sterility. Given the ease of identifying male-sterile mutants, dozens of genes with key roles in early anther development have been identified and cloned in model species, ordered by time of action and spatiotemporal expression, and used to propose explanatory models for critical steps in cell fate specification. Despite rapid progress, fundamental issues in anther development remain unresolved, and it is unclear if insights from one species can be applied to others. Here we construct a comparison of Arabidopsis, rice, and maize immature anthers to pinpoint distinctions in developmental pace. We analyze the mechanisms by which archesporial (pre-meiotic) cells are specified distinct from the soma, discuss what constitutes meiotic preparation, and review what is known about the secondary parietal layer and its terminal periclinal division that generates the tapetal and middle layers. Finally, roles for small RNAs are examined, focusing on the grass-specific phasiRNAs.}, journal={Frontiers in Plant Science}, publisher={Frontiers Media SA}, author={Kelliher, Timothy and Egger, Rachel L. and Zhang, Han and Walbot, Virginia}, year={2014}, month={Jul} }
 @article{moon_skibbe_timofejeva_wang_kelliher_kremling_walbot_cande_2013, title={Regulation of cell divisions and differentiation by <scp>MALE STERILITY</scp>32 is required for anther development in maize}, volume={76}, ISSN={0960-7412 1365-313X}, url={http://dx.doi.org/10.1111/tpj.12318}, DOI={10.1111/tpj.12318}, abstractNote={Summary Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 ( ms32 ) encodes a basic helix–loop–helix ( bHLH ) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre‐meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32‐ref mac1‐1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1 . Altogether, our results show that MS 32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified.}, number={4}, journal={The Plant Journal}, publisher={Wiley}, author={Moon, Jihyun and Skibbe, David and Timofejeva, Ljudmilla and Wang, Chung‐Ju Rachel and Kelliher, Timothy and Kremling, Karl and Walbot, Virginia and Cande, William Zacheus}, year={2013}, month={Oct}, pages={592–602} }
 @article{gao_kelliher_nguyen_walbot_2013, title={Ustilago maydis</i> reprograms cell proliferation in maize anthers}, volume={75}, ISSN={0960-7412 1365-313X}, url={http://dx.doi.org/10.1111/tpj.12270}, DOI={10.1111/tpj.12270}, abstractNote={Summary The basidiomycete U stilago maydis is a ubiquitous pathogen of maize ( Z ea mays ), one of the world's most important cereal crops. Infection by this smut fungus triggers tumor formation in aerial plant parts within which the fungus sporulates. Using confocal microscopy to track U . maydis infection on corn anthers for 7 days post‐injection, we found that U . maydis is located on the epidermis during the first 2 days, and has reached all anther lobe cell types by 3 days post‐injection. Fungal infection alters cell‐fate specification events, cell division patterns, host cell expansion and host cell senescence, depending on the developmental stage and cell type. Fungal effects on tassel and plant growth were also quantified. Transcriptome profiling using a dual organism microarray identified thousands of anther genes affected by fungal infection at 3 days post‐injection during the cell‐fate specification and rapid cell proliferation phases of anther development. In total, 4147 (17%) of anther‐expressed genes were altered by infection, 2018 fungal genes were expressed in anthers, and 206 fungal secretome genes may be anther‐specific. The results confirm that U . maydis deploys distinct genes to cause disease in specific maize organs, and suggest mechanisms by which the host plant is manipulated to generate a tumor.}, number={6}, journal={The Plant Journal}, publisher={Wiley}, author={Gao, Li and Kelliher, Timothy and Nguyen, Linda and Walbot, Virginia}, year={2013}, month={Aug}, pages={903–914} }
 @article{kelliher_walbot_2012, title={Hypoxia Triggers Meiotic Fate Acquisition in Maize}, volume={337}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.1220080}, DOI={10.1126/science.1220080}, abstractNote={Redox Status Incites Gametogenesis Germ cells differ from somatic cells in their chromosomal complement, being haploid rather than diploid. In animals, the germ cells are generally produced by a separate lineage set aside early in development. Plants, however, lack a reserved germ cell lineage. Kelliher and Walbot (p. 345 ; see the Perspective by Whipple ) now show that, in maize, the key signal for germ cell production is hypoxia, which triggers differentiation of anther germ cells from a generalized field of progenitors. The specializing germ cells then induce differentiation of supportive somatic cells.}, number={6092}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Kelliher, Timothy and Walbot, Virginia}, year={2012}, month={Jul}, pages={345–348} }
 @article{wang_nan_kelliher_timofejeva_vernoud_golubovskaya_harper_egger_walbot_cande_2012, title={Maize
                    <i>multiple archesporial cells 1
                    (
                    <i>mac1
                    ), an ortholog of rice
                    <i>TDL1A
                    , modulates cell proliferation and identity in early anther development}, volume={139}, ISSN={1477-9129 0950-1991}, url={http://dx.doi.org/10.1242/dev.077891}, DOI={10.1242/dev.077891}, abstractNote={To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.}, number={14}, journal={Development}, publisher={The Company of Biologists}, author={Wang, Chung-Ju Rachel and Nan, Guo-Ling and Kelliher, Timothy and Timofejeva, Ljudmilla and Vernoud, Vanessa and Golubovskaya, Inna N. and Harper, Lisa and Egger, Rachel and Walbot, Virginia and Cande, W. Zacheus}, year={2012}, month={Jul}, pages={2594–2603} }
 @article{kelliher_walbot_2011, title={Emergence and patterning of the five cell types of the Zea mays anther locule}, volume={350}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/j.ydbio.2010.11.005}, DOI={10.1016/j.ydbio.2010.11.005}, abstractNote={One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants, the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis; however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize (Zea mays) anther microsporangium, the somatic tissues consist of four concentric cell layers that surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild-type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over 30 days of wild-type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2'-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain.}, number={1}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Kelliher, Timothy and Walbot, Virginia}, year={2011}, month={Feb}, pages={32–49} }