Roger Thilmony

Microvine 04C023V0004 (V4) is a model Vitis vinifera genotype that carries a heterozygous g ibberellin insensitive 1 ( Vvigail ) mutation making the plants compact in stature and constantly fruiting, yet its utility is hindered by long regeneration times and modest transformation efficiency levels. To improve microvine V4 transformation, we developed a method utilizing embryogenic cell suspension (ECS) cultures and a novel protocol for Agrobacterium- mediated transformation. Friable globular (translucent or cream-colored) somatic embryos dissected from microvine embryogenic calli were used to initiate suspension cultures. ECS cultures require a modest amount of time and effort to maintain and produce abundant, rapidly growing explant material within several months. For transformation, the ECS cells were heat shocked at 45 °C and co-cultivated with Agrobacterium tumefaciens carrying a binary vector with the microvine Ubiquitin 7 ( VviUbi7 ) promoter controlling mCherry expression allowing the use of red fluorescence as a visible marker. Development of transgenic microvine plants was improved with the addition of gibberellic acid to the shooting medium. Eighteen independent transgenic plants were characterized using droplet digital PCR (ddPCR) demonstrating that nine (50%) had one or two copies of the introduced selection marker transgene. This method produced approximately 30 transgenic plants per 100 mg fresh weight of ECS culture five months after co-cultivation. Use of microvine V4 ECS cultures and a modified transformation protocol can efficiently generate transgenic plants advancing grapevine biotechnology research.

The GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technology) system enables efficient gene stacking within an Agrobacterium T-DNA. Using unidirectional site-specific recombinases and alternating selection markers, it allows precise, sequential assembly of multiple genes directly within an Agrobacterium virulence plasmid. Here, we modified Agrobacterium tumefaciens strain EHA105 to create JGT105 as a GAANTRY recipient and constructed a 15.8 kb T-DNA containing five cargo sequences. We compared the performance of the JGT105 5-stack strain against a conventional binary vector carrying the same cargo sequences in Arabidopsis and potato transformation. The transformation efficiencies were comparable for the GAANTRY strain and the binary vector (potato: 83% vs. 82%; Arabidopsis: 1.73% vs. 1.95%). Single T-DNA insertion frequencies were also similar between the two systems (17.6% for GAANTRY vs. 24.5% for the binary vector construct in potato; 10.3% vs. 18.2% in Arabidopsis, respectively). Notably, the GAANTRY construct had significantly reduced vector backbone transfer in potato (10.0% vs. 26.5%) for the binary vector, whereas rates were higher in Arabidopsis (37.5% vs. 48.9%). These results show that the JGT105 GAANTRY strain is an effective T-DNA delivery system, matching binary vector transformation efficiency while offering lower backbone integration frequency, facilitating the generation of high-quality, multi-gene transgenic plants.

Wildfires pose a growing threat to viticulture, with smoke exposure causing smoke taint—off-flavors described as ‘smoky’ or ‘ashy’ that can make grape unmarketable. This taint results from smoke-derived volatile phenols (VPs) absorbed by grape berries and converted into stable glycoconjugates via glycosyltransferases (GTs). We present the dwarf grapevine cultivar Pixie as a model system for smoke taint research. Using HiFi long-read sequencing, we produced a haplotype-resolved, telomere-to-telomere (T2T) diploid genome and a tissue- and stage-resolved expression atlas across 30 replicated samples. We constructed a second T2T assembly for Pinot Noir to assess GT expression during berry ripening in the field. Controlled smoke exposure experiments, combined with transcriptomics and VP quantification, revealed 12 GT1 genes highly up-regulated by smoke. We designated these as Vitis vinifera smoke-inducible UGT (VviSIUGT) genes. Their expression peaked 1 d post-exposure and declined by day 3, mirroring glycoside accumulation dynamics. Gene co-expression network analyses highlighted stress-responsive modules enriched in these VviSIUGT genes, while transcription factor-binding site analyses pinpointed stress-related regulatory elements in their promoters. These findings uncover early transcriptional responses to smoke in grape berries and identify candidate genes that can serve as molecular markers or engineering targets for developing grapevine cultivars with reduced susceptibility to smoke taint.

With the increasing frequency of wildfires, vineyards are more often exposed to smoke, resulting in a higher risk of smoke taint in wine. This taint imparts undesirable “smoky” and “ashy” aromas and causes significant economic losses. Smoke-derived volatile phenols (VPs) in grape berries, metabolized into stable, non-volatile glycoconjugates via glycosyltransferases (GTs), underlie smoke taint formation. Here, we present the dwarf grapevine cultivar Pixie as a model system for smoke taint research. We generated a phased, telomere-to-telomere diploid genome using HiFi long-read sequencing, along with an expression atlas spanning 30 replicated samples across various tissues and developmental stages. We also constructed a second telomere-to-telomere assembly for Pinot Noir to assess GT expression during berry ripening in the field. Controlled smoke-exposure experiments profiled GT expression and enabled transcriptome-wide analyses at multiple time points. We measured VPs in both smoke-exposed and control samples, revealing 12 GT1 genes significantly upregulated by smoke, named Vitis vinifera smoke-inducible UGTs ( VviSIUGTs ). Their expression peaked one day post-exposure and declined by day three, mirroring glycoside accumulation dynamics. Gene co-expression network analyses highlighted stress-responsive modules enriched in these VviSIUGTs , while transcription factor binding site analyses pinpointed stress-related regulatory elements in their promoters. These findings provide new insights into the molecular basis of smoke taint and identify potential targets for breeding and biotechnological interventions aimed at developing grapevine cultivars with reduced susceptibility to smoke damage.

Perennial grasses are important forage crops and emerging biomass crops and have the potential to be more sustainable grain crops. However, most perennial grass crops are difficult experimental subjects due to their large size, difficult genetics, and/or their recalcitrance to transformation. Thus, a tractable model perennial grass could be used to rapidly make discoveries that can be translated to perennial grass crops. Brachypodium sylvaticum has the potential to serve as such a model because of its small size, rapid generation time, simple genetics, and transformability. Here, we provide a high-quality genome assembly and annotation for B. sylvaticum, an essential resource for a modern model system. In addition, we conducted transcriptomic studies under 4 abiotic stresses (water, heat, salt, and freezing). Our results indicate that crowns are more responsive to freezing than leaves which may help them overwinter. We observed extensive transcriptional responses with varying temporal dynamics to all abiotic stresses, including classic heat-responsive genes. These results can be used to form testable hypotheses about how perennial grasses respond to these stresses. Taken together, these results will allow B. sylvaticum to serve as a truly tractable perennial model system.

Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3’(2’), 5’-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M₁ progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

The highly conserved Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3′ (2′), 5′-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted. Precise gene editing techniques were used to generate Sal1 mutants in hexaploid bread wheat. The CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) Cas9 system with three guide RNAs (gRNAs) was used to inactivate six Sal1 homologous genes within the Bobwhite wheat genome. The resulting mutant wheat plants with all their Sal1 genes disabled had slimmer stems, had a modest reduction in biomass and senesced more slowly in water limiting conditions, but did not exhibit improved yield under drought conditions. Our results show that multiplexed gRNAs enabled effective targeted gene editing of the Sal1 gene family in hexaploid wheat. These Sal1 mutant wheat plants will be a resource for further research studying the function of this gene family in wheat.

Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.

Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.

Background: Promoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development. Results: In this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity. Conclusions: Among the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.