Browsing by Author "Bargmann, Bastiaan O. R."

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    Characterization of the F Locus Responsible for Floral Anthocyanin Production in Potato
    Laimbeer, F. Parker E.; Bargmann, Bastiaan O. R.; Holt, Sarah H.; Pratt, Trenton; Peterson, Brenda A.; Doulis, Andreas G.; Buell, C. Robin; Veilleux, Richard E. (Genetics Society of America, 2020-08-27)
    Anthocyanins are pigmented secondary metabolites produced via the flavonoid biosynthetic pathway and play important roles in plant stress responses, pollinator attraction, and consumer preference. Using RNA-sequencing analysis of a cross between diploid potato (Solanum tuberosum L.) lines segregating for flower color, we identified a homolog of the ANTHOCYANIN 2 (AN2) gene family that encodes a MYB transcription factor, herein termed StFlAN2, as the regulator of anthocyanin production in potato corollas. Transgenic introduction of StFlAN2 in white-flowered homozygous doubled-monoploid plants resulted in a recovery of purple flowers. RNA-sequencing revealed the specific anthocyanin biosynthetic genes activated by StFlAN2 as well as expression differences in genes within pathways involved in fruit ripening, senescence, and primary metabolism. Closer examination of the locus using genomic sequence analysis revealed a duplication in the StFlAN2 locus closely associated with gene expression that is likely attributable to nearby genetic elements. Taken together, this research provides insight into the regulation of anthocyanin biosynthesis in potato while also highlighting how the dynamic nature of the StFlAN2 locus may affect expression.
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    Leaf cell-specific and single-cell transcriptional profiling reveals a role for the palisade layer in UV light protection
    Procko, Carl; Lee, Travis; Borsuk, Aleca; Bargmann, Bastiaan O. R.; Dabi, Tsegaye; Nery, Joseph R.; Estelle, Mark; Baird, Lisa; O'Connor, Carolyn; Brodersen, Craig; Ecker, Joseph R.; Chory, Joanne (Oxford University Press, 2022-08-25)
    Leaf cell sorting and scRNA-seq approaches are used to transcriptionally profile the palisade mesophyll layer and provide resources for understanding mesophyll biology. Like other complex multicellular organisms, plants are composed of different cell types with specialized shapes and functions. For example, most laminar leaves consist of multiple photosynthetic cell types. These cell types include the palisade mesophyll, which typically forms one or more cell layers on the adaxial side of the leaf. Despite their importance for photosynthesis, we know little about how palisade cells differ at the molecular level from other photosynthetic cell types. To this end, we have used a combination of cell-specific profiling using fluorescence-activated cell sorting and single-cell RNA-sequencing methods to generate a transcriptional blueprint of the palisade mesophyll in Arabidopsis thaliana leaves. We find that despite their unique morphology, palisade cells are otherwise transcriptionally similar to other photosynthetic cell types. Nevertheless, we show that some genes in the phenylpropanoid biosynthesis pathway have both palisade-enriched expression and are light-regulated. Phenylpropanoid gene activity in the palisade was required for production of the ultraviolet (UV)-B protectant sinapoylmalate, which may protect the palisade and/or other leaf cells against damaging UV light. These findings improve our understanding of how different photosynthetic cell types in the leaf can function uniquely to optimize leaf performance, despite their transcriptional similarities.
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    Protoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.
    Beard, Keely Marie; Boling, Audrey W. H.; Bargmann, Bastiaan O. R. (Elsevier, 2021-06-01)
    Cannabis sativa L. is a valuable, up-and-coming industrial crop with a substantially growing market. However, due to an extended period of legal restriction, research with cannabis has been limited, particularly in laboratory settings. Expanding the application of biotechnological techniques to cannabis can facilitate addressing species-specific impediments to improving crop traits and further fundamental understanding of its intricacies. Here, we describe application of protoplast transformation for the study of transient gene expression in a low-THC cannabis cultivar. To produce explant tissue as a source of protoplasts, a method for hormone-free in vitro micropropagation is established. Protoplasts are isolated from young leaves of the micropropagated stocks and transiently transformed with plasmid DNA carrying a fluorescent marker gene. This is the first report of protoplast transformation in this species. A protoplast isolation yield is achieved of up to 2 × 10 cells per gram of leaf material, vitality staining shows that up to 82 % of isolated protoplasts are viable, and quantification of the cells expressing a fluorescent protein indicates that up to 31 % of the cells can be successfully transformed. Additionally, protoplasts are transformed with an auxin-responsive reporter gene and the reaction to treatment with indole-3-acetic acid is quantified using flow cytometry. This work demonstrates that relatively minor modification of standard techniques can be used to study this important emerging crop. 6
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    Protoplast Regeneration and Its Use in New Plant Breeding Technologies
    Reed, Kelsey M.; Bargmann, Bastiaan O. R. (Frontiers, 2021-09-03)
    The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.
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    Protoplast Regeneration and Its Use in New Plant Breeding Technologies
    Bargmann, Bastiaan O. R.; Reed, Kelsey M. (2021-09-03)
    The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.
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    Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration
    Gonzalez, Juan H.; Taylor, Joseph S.; Reed, Kelsey M.; Wright, R. Clay; Bargmann, Bastiaan O. R. (MDPI, 2021-10-23)
    Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.