Neofunctionalization is a rare fate of gene duplication, classically defined as the acquisition of novel functions that potentiate the emergence of new traits. Rather than evolving to function autonomously, neofunctionalized genes may also remain embedded within their ancestral regulatory networks, potentially reshaping the genetic trajectories through which phenotypic change occurs. Testing this hypothesis, we leveraged a pan-genetic platform comprising ten Solanaceae species and show that a paralog of the flowering hormone florigen neofunctionalized into a flowering antagonist and was repeatedly selected during crop domestication and adaptation of wild plants across 50 million years of evolution. Independent selection of cis-regulatory and coding mutations in SELF-PRUNING 5G (SP5G) enabled rapid flowering in the wild ancestor of domesticated tomato from Central America as well as major and indigenous eggplant crop lineages domesticated in Asia and Africa. We further found that cis-regulatory sequence changes reduced SP5G expression and flowering time in wild species native to distinct environments in the Americas and Australia, relationships that we validated by genome editing. Together with similar patterns observed across diverse species and developmental networks, we propose that antagonistic neofunctionalized paralogs create evolutionary contingencies that channel adaptive trajectories across plant lineages.

Introduction:Virus-induced gene editing (VIGE) provides a powerful alternative to conventional plant genome engineering by enabling in planta delivery of genome-editing reagents without repeated use of tissue culture. Here, we establish Tobacco rattle virus (TRV)-mediated VIGE as an efficient system for somatic and heritable genome editing in groundcherry (Physalis grisea). Methods:Using Cas9-expressing plants, we targeted the visual marker gene PHYTOENE DESATURASE (PDS) and the domestication gene CLAVATA1 (CLV1) via TRV-mediated delivery of guide RNAs. Editing efficiencies were evaluated in somatic tissues and across progeny to assess heritability. Results:Targeting of PDS resulted in somatic editing frequencies of 80-95% and consistent recovery of heritable edits, with all infected plants (n = 5) producing edited progeny, including fully albino seedlings carrying frameshift mutations in all alleles. The primary Cas9-expressing line was unexpectedly tetraploid, likely due to genome duplication during tissue culture. Despite this, VIGE efficiently generated mono-, bi-, tri-, and tetra-allelic mutations, demonstrating robust editing across four alleles simultaneously. Targeting of CLV1 achieved somatic editing frequencies of up to 73%, with 60% of T0 plants producing heritable edits. Edited plants exhibited increased floral organ number and multilocular fruits, consistent with CLV1 loss-of-function phenotypes. Conclusion:These results demonstrate that VIGE enables rapid, efficient, and heritable genome editing in groundcherry, even in a tetraploid context, highlighting its potential to accelerate genetic improvement and de novo domestication of underutilized crops.

Solanum retroflexum is cultivated for both its fruit and leaves in Africa, China, India, and Indonesia. However, it is still considered an underutilized crop due to limited genetic and genomic resources to support improvement. To enable gene function studies for future improvement by new breeding technologies, this study established efficient plant regeneration and Agrobacterium tumefaciens–mediated transformation approaches. Cotyledon and hypocotyl explants were used to test plant regeneration on a Murashige and Skoog salts-based medium supplemented with 0.5 mg L−1 zeatin. Both explant types showed 100

Fruit abscission is an agronomically important trait that would benefit from a deeper molecular understanding. Despite a prominent, deleterious, fruit drop phenotype, fruit abscission has yet to be characterized in Physalis grisea (groundcherry). Here we established a stage-resolved timeline of P. grisea pedicel abscission zone (AZ) development to expand the general knowledge of fruit abscission. We integrated microscopic imaging of the AZ, hormone (auxin and ethylene) applications, detachment force measurements, and gene expression analysis of AZ cells across maturation to connect the role of putative regulators to cell development and separation. A strong correlation between AZ development, hormone sensitivity, and force detachment was observed. RNA-seq showed upregulation of pathways involved in cell division/expansion early in AZ development, hormone signaling and transcriptional reprogramming at the middle stage, and cell wall degradation and protective barrier genes late in the abscission process. Furthermore, MADS-box transcription factors such as the P. grisea orthologs of JOINTLESS and MACROCALYX are co-expressed during AZ differentiation, suggesting involvement in the formation of AZ cells. These results provide a molecular and cellular framework for P. grisea fruit abscission, suggesting that key regulatory features of fruit abscission are shared within the Solanaceae. Characterization of fruit abscission in P. grisea is essential for understanding this trait to guide improvements needed for its adoption as a specialty crop in the United States.

Societal Impact Statement Goldenberry ( Physalis peruviana ) produces sweet, nutritionally rich berries, yet like many minor crops, is cultivated in limited geographical regions and has not been a focus of breeding programs for trait enhancement. Leveraging knowledge of plant architecture‐related traits from related species, we used CRISPR/Cas9‐mediated gene editing to generate a compact ideotype to advance future breeding efforts and agricultural production. Goldenberry growers will benefit from these compact versions because it optimizes per plot yield, facilitating larger scale production to meet rising consumer popularity and demand.

This report provides an overview of the content and data collected from the “Successes, Challenges, and Opportunities Plant Transformation Research in Africa” panel discussion. Organized by PlantGENE, this event brought together scientists and stakeholders across the globe to examine the complex challenges and emerging opportunities in plant transformation research in laboratories across Africa. The discussion, rooted in insights from a panel of six leading scientists, highlights critical issues including restrictive regulatory environments, prohibitive costs, and the inconsistent availability of essential research materials. Additionally, the pervasive “brain drain” phenomenon, where skilled researchers leave the continent for better opportunities, exacerbates the difficulties faced by African scientists. Despite these challenges, the report also identifies significant advancements, particularly in the growing recognition of African leadership within universities and national agricultural research systems (NARS). These institutions, supported by highly skilled faculty and motivated graduate students, are producing high-quality research that contributes to global scientific knowledge. The panelists emphasized the necessity of creating an environment that encourages African scientists to remain on the continent and address local challenges through innovative research. Strengthening intra-African networks and fostering collaborations with the global scientific community are proposed as essential strategies to achieve this. This report underscores the critical need for substantial investments from both global and African organizations, working with African governments, to support these efforts. Furthermore, it calls for science-based decision-making and fair regulatory frameworks to align with unique opportunities and risks associated with technological advancements in Africa. This paper details the observations of six panelists and analyzes the results of attendee surveys in order to document these challenges and opportunities while advocating for sustained investment and strategic partnerships to build a thriving bioeconomy across Africa.

Zea mays L. (maize) transformation is important for crop improvement and advancing plant genetics. One of the most commonly used methods is Agrobacterium-mediated transformation of immature maize embryos. However, this approach is often inefficient and requires costly greenhouse infrastructure for the production of high-quality ears. Moreover, immature embryo tissue culture response exhibits seasonal variation even under optimal greenhouse conditions. Recently, a novel approach using seedling-leaf whorl explants, combined with morphogenic genes BABY BOOM (Bbm) and WUSCHEL2 (Wus2) and an Agrobacterium ternary vector system, has emerged as a promising alternative. In this study, the leaf whorl-based transformation method was evaluated on two maize lines, PHR03 and B73, across two different institutions. Additionally, the performance of two helper plasmids, the proprietary PHP71539 and the publicly available pKL2299, was compared. Importantly, the study also utilized a maize drought-inducible Rab17 promoter to activate the Cre/loxP system, enabling the excision of the Bbm and Wus2 cassettes to mitigate negative effects on plant development in regenerated shoots. The comparative analysis revealed no significant differences in transformation efficiency or usable transgenic events between the two helper plasmids. These findings confirm the successful use of the publicly available pKL2299 plasmid for maize leaf whorl-based transformation. This approach offers a faster, more accessible, and cost-effective method that does not require high-quality greenhouse conditions typically required for explant preparation using immature embryos.

Pan-genomics and genome-editing technologies are revolutionizing breeding of global crops1,2. A transformative opportunity lies in exchanging genotype-to-phenotype knowledge between major crops (that is, those cultivated globally) and indigenous crops (that is, those locally cultivated within a circumscribed area)3-5 to enhance our food system. However, species-specific genetic variants and their interactions with desirable natural or engineered mutations pose barriers to achieving predictable phenotypic effects, even between related crops6,7. Here, by establishing a pan-genome of the crop-rich genus Solanum8 and integrating functional genomics and pan-genetics, we show that gene duplication and subsequent paralogue diversification are major obstacles to genotype-to-phenotype predictability. Despite broad conservation of gene macrosynteny among chromosome-scale references for 22 species, including 13 indigenous crops, thousands of gene duplications, particularly within key domestication gene families, exhibited dynamic trajectories in sequence, expression and function. By augmenting our pan-genome with African eggplant cultivars9 and applying quantitative genetics and genome editing, we dissected an intricate history of paralogue evolution affecting fruit size. The loss of a redundant paralogue of the classical fruit size regulator CLAVATA3 (CLV3)10,11 was compensated by a lineage-specific tandem duplication. Subsequent pseudogenization of the derived copy, followed by a large cultivar-specific deletion, created a single fused CLV3 allele that modulates fruit organ number alongside an enzymatic gene controlling the same trait. Our findings demonstrate that paralogue diversifications over short timescales are underexplored contingencies in trait evolvability. Exposing and navigating these contingencies is crucial for translating genotype-to-phenotype relationships across species.

Plants regenerated from seedling explants (hypocotyls and cotyledons) of the Solanaceae family members Physalis grisea (groundcherry), Solanum lycopersicum (tomato), and Solanum prinophyllum (forest nightshade) were used to determine the in vitro culture parameters that contribute to the incidence in polyploidization of tissue culture-derived plants (regenerants) from these species. We examined the possible effects of zeatin concentration in the plant regeneration medium, explant source, and species. Plants were grown to maturity under greenhouse conditions, pollen was collected and germinated. Flow cytometry analysis verified the utility of the pollen germination method for determining differences in ploidy, which was based on the number of pollen tubes produced with one tube representing diploid and two indicating polyploid. As for zeatin concentration, we assessed the effect of our standard method of initiation on medium containing 2 mg/l followed by 1 mg/l 2 weeks after culture initiation in comparison with 0.25, 0.5, and 1 mg/l throughout the culture lifetime. There were no major correlations for zeatin concentration on ploidy status across the species except for plants regenerated from S. lycopersicum hypocotyl explants where the percentage of polyploid regenerants increased with increasing concentrations. As for species and explant effects, P. grisea plants regenerated from hypocotyl explants had the highest percentage of polyploid plants at 81% compared to 43% and 35% for S. lycopersicum and S. prinophyllum , respectively. From cotyledons, 8% of S. lycopersicum and 20% of S. prinophyllum were polyploid. A comparison with P. grisea could not be made because cotyledon explants do not regenerate on zeatin-containing medium. The results indicated the incidence of polyploidization cannot be generalized for zeatin concentration, however, an influence of explant type and species was observed. Effects of increased ploidy on plant morphology were primarily larger flower and seed size; however, no significant differences were observed in plant or fruit size. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, https://ror.org/021nxhr62, National Science Foundation Plant Genome Research Program grant IOS-2216612

Plant transformation is an important part of plant research and crop improvement. Transformation methods remain complex, labor intensive, and inefficient. PlantGENE is a community of scientists from academia, industry, non-profit research institutes, and government organizations working to improve plant transformation. PlantGENE hosts virtual training, interactive webinars, and a website with career opportunities, directories, and more. The plant science community has shown great interest and support for PlantGENE.

Pan-genomics and genome editing technologies are revolutionizing the breeding of globally cultivated crops. A transformative opportunity lies in the reciprocal exchange of genotype-to-phenotype knowledge of agricultural traits between these major crops and hundreds of locally cultivated indigenous crops, thereby enhancing the diversity and resilience of our food system. However, species-specific genetic variants and their interactions with desired natural or engineered mutations pose barriers to achieving predictable phenotypic effects, even between closely related crops or genotypes. Here, by establishing a pan-genome of the crop-rich genus Solanum and integrating functional genomics and genetics, we show that gene duplication and subsequent paralog diversification are a major obstacle to genotype-phenotype predictability. Despite broad conservation of gene macrosynteny among chromosome-scale references for 22 species, including 13 indigenous crops, hundreds of global and lineage-specific gene duplications exhibited dynamic evolutionary trajectories in paralog sequence, expression, and function, including among members of key domestication gene families. Extending our pan-genome with 10 cultivars of African eggplant and leveraging quantitative genetics and genome editing, we uncovered an intricate history of paralog emergence and evolution within this indigenous crop. The loss of an ancient redundant paralog of the classical regulator of stem cell proliferation and fruit organ number, CLAVATA3 ( CLV3 ), was compensated by a lineage-specific tandem duplication. Subsequent pseudogenization of the derived copy followed by a cultivar-specific structural variant resulted in a single fused functional copy of CLV3 that modifies locule number alongside a newly identified gene controlling the same trait. Our findings demonstrate that paralog diversifications over short evolutionary periods are critical yet underexplored contingencies in trait evolvability and independent crop domestication histories. Unraveling these contingencies is crucial for translating genotype-to-phenotype relationships across related species. ### Competing Interest Statement WRM is a founder and shareholder in Orion Genomics, a plant genomics company. Z.B.L. is a consultant for and a member of the Scientific Strategy Board of Inari Agriculture.

Hornworts are a deeply diverged lineage of bryophytes that are sister to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO 2 -concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient-expression and stable transgenic lines in the model hornwort, Anthoceros agrestis . An average of 569 (± 268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48 hours. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species. Highlight We developed a new transformation method for hornworts, a lineage of understudied bryophytes, and demonstrated its effectiveness in studying subcellular localization of proteins involved in pyrenoid-based CO 2 -concentrating mechanism.

Studying morphological novelties offers special insights into developmental biology and evolution. The inflated calyx syndrome (ICS) is a largely unrecognized but fascinating feature of flower development, where sepals form balloon-like husks that encapsulate fruits. Despite its independent emergence in many lineages of flowering plants, the genetic and molecular mechanisms of ICS remain unknown. Early studies in the Solanaceae genus Physalis put forth key roles of MADS-box genes in ICS. However, recent work suggests these classical floral identity transcription factors were false leads. With newfound capabilities that allow rapid development of genetic systems through genomics and genome editing, Physalis has re-emerged as the most tractable model species for dissecting ICS. This review revisits current understanding of ICS and highlights how recent advancements enable a reset in the search for genetic and molecular mechanisms using unbiased, systematic approaches.

Societal Impact Statement Groundcherry (Physalis grisea) is a plant species grown for its flavorful fruit. The fruit drops from the plant, hence the common name groundcherry. This makes harvest cumbersome and puts the fruit at risk for carrying soil‐borne pathogens, therefore making them unsellable. Furthermore, insects often damage the plants, reducing yield. Advances in gene editing offer promise for addressing these issues and aiding home gardeners and farmers. Improvement will expand access to this nutritious fruit, rich in potassium, vitamin C, and antioxidants. Additionally, studies of its biology could serve as a model for improving other fruiting plants, particularly underutilized species. Summary P. grisea is an underutilized, semidomesticated fruit crop with rising agronomic value. Several resources have been developed for its use in fundamental biological research, including a plant transformation system and a high‐quality reference genome. Already, P. grisea has been used as a model to investigate biological phenomena including inflated calyx syndrome and gene compensation. P. grisea has also been used to demonstrate the potential of fast‐tracking domestication trait improvement through approaches such as CRISPR/Cas9 gene editing. This work has led to the Physalis Improvement Project, which relies on reverse genetics to understand the mechanisms that underlie fruit abscission and plant–herbivore interactions to guide approaches for improvement of undesirable characteristics. CRISPR/Cas9 gene editing has been used to target P. grisea genes that are suspected to act in fruit abscission, particularly orthologs of those that are reported in tomato abscission zone development. A similar approach is being taken to target P. grisea genes involved in the withanolide biosynthetic pathway to determine the impact of withanolides on plant–herbivore interactions. Results from these research projects will lead to a greater understanding of important biological processes and will also generate knowledge needed to develop cultivars with reduced fruit drop and increased resistance to insect herbivory.

An enduring question in evolutionary biology concerns the degree to which episodes of convergent trait evolution depend on the same genetic programs, particularly over long timescales. In this work, we genetically dissected repeated origins and losses of prickles—sharp epidermal projections—that convergently evolved in numerous plant lineages. Mutations in a cytokinin hormone biosynthetic gene caused at least 16 independent losses of prickles in eggplants and wild relatives in the genus Solanum . Homologs underlie prickle formation across angiosperms that collectively diverged more than 150 million years ago, including rice and roses. By developing new Solanum genetic systems, we leveraged this discovery to eliminate prickles in a wild species and an indigenously foraged berry. Our findings implicate a shared hormone activation genetic program underlying evolutionarily widespread and recurrent instances of plant morphological innovation.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato. Whole-genome sequencing and transcriptome sequencing reveal high editing specificity of an efficient adenine base editor in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.