Reporter genes confer new-to-nature ornamental traits in plants

Yuan, Guoliang; Lu, Haiwei; Weston, David J.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.; Yang, Xiaohan

doi:10.1093/hr/uhac077

Cited by 9 publications

(5 citation statements)

References 11 publications

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“…To date, the transformation methods for P. tremula × P. alba 717-1B4 are well-developed and optimized [38][39][40]. In general, it is simple to induce transgenic shoots selected from shoot elongation medium to rooting in root medium in P. tremula × P. alba 717-1B4 transformation [34,35]. In contrast, we found that the rooting rate of P. deltoides WV94 transformation is generally less than 10% under the same condition.…”

Section: Discussionmentioning

confidence: 83%

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

Yuan

Liu

Yao

et al. 2023

Plants

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Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone ‘52-225’ (P. trichocarpa × P. deltoides clone ‘52-225’). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.

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Section: Discussionmentioning

confidence: 83%

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

Yuan

Liu

Yao

et al. 2023

Plants

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“…In in vitro gene transformation studies, RUBY is a valuable reporter gene as it did not affect callus induction, plant regeneration, development or fertility of Plukenetia volubilis, Nicotiana benthamiana, and Arabidopsis (Yu et al, 2023). In addition to "in root" gene functional studies, exciting opportunities for creating new horticultural value is achievable in breeding of ornamental plants through RUBY gene expression and heterologous betalain production (Yuan et al, 2022). Single insertion of transgene is the most optimal mode of integration in a transformation assay.…”

Section: Discussionmentioning

confidence: 99%

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY

et al. 2023

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In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD600), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

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“…A new reporter RUBY that converts tyrosine to vividly red betalain can be detected without the need of special equipment or chemical treatment [ 99 ]. Another potential reporter for biosensor is eYGFPuv, which has been successfully used as a reporter for plant transformation [ 100 , 101 ]. Both RUBY and eYGFPuv could be used as suitable reporters for building transcriptional- or translational-based biosensors.…”

Section: Next-generation Identification and Design Of Biological Comp...mentioning

confidence: 99%

Biological and Molecular Components for Genetically Engineering Biosensors in Plants

et al. 2022

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Plants adapt to their changing environments by sensing and responding to physical, biological, and chemical stimuli. Due to their sessile lifestyles, plants experience a vast array of external stimuli and selectively perceive and respond to specific signals. By repurposing the logic circuitry and biological and molecular components used by plants in nature, genetically encoded plant-based biosensors (GEPBs) have been developed by directing signal recognition mechanisms into carefully assembled outcomes that are easily detected. GEPBs allow for in vivo monitoring of biological processes in plants to facilitate basic studies of plant growth and development. GEPBs are also useful for environmental monitoring, plant abiotic and biotic stress management, and accelerating design-build-test-learn cycles of plant bioengineering. With the advent of synthetic biology, biological and molecular components derived from alternate natural organisms (e.g., microbes) and/or de novo parts have been used to build GEPBs. In this review, we summarize the framework for engineering different types of GEPBs. We then highlight representative validated biological components for building plant-based biosensors, along with various applications of plant-based biosensors in basic and applied plant science research. Finally, we discuss challenges and strategies for the identification and design of biological components for plant-based biosensors.

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Reporter genes confer new-to-nature ornamental traits in plants

Cited by 9 publications

References 11 publications

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY

Biological and Molecular Components for Genetically Engineering Biosensors in Plants

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