Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana

Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana View ORCID Profile Anthony Garcia , Jo Trang Bùi , View ORCID Profile Todd P. Michael , View ORCID Profile Stefanie M. Ickert-Bond , View ORCID Profile Verónica S. Di Stilio doi: https://doi.org/10.1101/2025.02.07.637168 Anthony Garcia 1 University of Washington, Department of Biology , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anthony Garcia Jo Trang Bùi 1 University of Washington, Department of Biology , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Todd P. Michael 2 Salk Institute , La Jolla, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Todd P. Michael Stefanie M. Ickert-Bond 3 University of Alaska , Fairbanks, AK, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefanie M. Ickert-Bond Verónica S. Di Stilio 1 University of Washington, Department of Biology , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Verónica S. Di Stilio For correspondence: distilio{at}uw.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Premise As the sister clade to angiosperms, gymnosperms are key to enabling the reconstruction of ancestral gene regulatory networks for seed plants. However, tools to rapidly and efficiently investigate gene function in gymnosperms remain limited due to the challenges of long life cycles and large genome sizes. Species within the xerophytic genus Ephedra (Gnetales) have comparatively smaller genomes and shrubby growth habits with shorter life spans, making them better suited for greenhouse cultivation and laboratory experiments. Methods and Results Here, we implement Virus-Induced Gene Silencing (VIGS) to manipulate gene expression in Ephedra tweedieana. Agrobacterium -mediated vacuum infiltration of Tobacco Rattle Virus (TRV2 and TRV1) in seedlings resulted in highly efficient silencing of the E. tweedieana PHYTOENE DESATURASE ortholog EtwPDS . The expected photobleaching phenotype was observed as early as two weeks. It lasted at least three months, in stems, shoot tips, leaves, axillary meristems, and lateral branches of treated plants. Conclusions This first report of transient transformation and targeted gene silencing in a gymnosperm will further enable functional studies of the genetic mechanisms underpinning adaptations in this important and underrepresented lineage of seed plants. INTRODUCTION Gymnosperms abound in present-day ecosystems, including some of the most important timber species (e.g., pine, spruce, and fir), and the largest (giant redwoods) and oldest (bristlecone pine) species on earth. Ephedra L. comprises approximately 54 species distributed worldwide, New World species occur in the North American deserts of the southwest and arid and semi-arid regions of South America ( Ickert-Bond and Renner, 2016 ). Many aspects of diversification within Ephedra remain unresolved as a result of widespread polyploidy and the paucity of comparative data. Along with two other disparate genera, Gnetum L. and Welwitschia Hook.f., they form the gymnosperm order Gnetales nested within the conifers, which together with cycads, ginkgoes, and flowering plants (angiosperms) comprises the seed plants. Gnetales have interesting convergences with the flowering plants (angiosperms), including double fertilization and flower-like reproductive structures ( Bowe et al., 2000 ), and the occasional occurrence of structurally bisexual cones ( Ickert-Bond and Renner, 2016 ). Climate is shifting towards warmer temperatures and more weather extremes, threatening agriculture worldwide ( Heino et al., 2023 ). Ephedra ’s success has been largely attributed to its ability to remain metabolically active year-round, with photosynthetic stems, highly reduced ephemeral leaves, sunken stomata, and large taproots, becoming the dominant element of the flora in extreme environments (high altitude and arid ecosystems; Ickert-Bond and Renner, 2016 ). Research on the genetic and developmental basis of adaptations to extreme environments and of seed plant character evolution has been hindered by a lack of gymnosperm model systems since most are trees with decades-long generation times. Having relatively small genomes, 7-8 giga base pairs (Gbp), and shorter generation times (as short as 2 years), Ephedra is amenable to investigations on the evolution of gymnosperm and seed plant innovations. Uncovering the genes underlying key innovations in this lineage requires a genetic toolkit including transformation protocols for functional studies. Virus-Induced Gene Silencing (VIGS) has proven successful as a transient transformation technique for evo-devo studies across a variety of plants ( Di Stilio, 2011 ), but still remains limited to angiosperms ( Lange et al., 2013 ; Dommes et al., 2019 ; Rössner et al., 2022 ). In bryophytes, stable transformation techniques are facilitated by their haploid, gametophyte-dominant life cycles ( Yadav et al., 2023 ). In the model fern Ceratopteris richardii Brongn., particle bombardment ( Rutherford et al., 2004 ; Plackett et al., 2014 ) and Agrobacterium -mediated transformation of spores ( Muthukumar et al., 2013 ) and gametophytes ( Jiang et al., 2024 ) have facilitated functional gene studies via stable transformation. In gymnosperms, transformation has been achieved in conifers and Ginkgo biloba L. (ginkgo). Stable and transient transformation in conifers relies on particle bombardment or Agrobacterium -mediated transformation and regeneration ( Morris et al., 1989 ; Moyle et al., 2002 ; Wagner et al., 2005 ; Tahir et al., 2011 ; Perez-Matas et al., 2023 ; Zhao et al., 2024 ). In ginkgo, Agrobacterium- mediated stable transformation ( Dupré et al., 2000 ; Ayadi and Trémouillaux-Guiller, 2003 ) and protoplast transient transformation ( Han et al., 2023 ) have also been described. However, the challenges of long life cycles and complicated protocols have limited the transformation of those species to forestry and industry applications. VIGS, on the other hand, has not been previously described in a gymnosperm and would allow for faster and more cost-effective functional analyses. Here, we describe a method for efficient virus-mediated transient transformation in Ephedra tweedieana Fisch. & C.A.Mey., a key tool in developing a gymnosperm model organism. METHODS Plant cultivation Ephedra tweedieana seeds (from a population near Montevideo, Uruguay) were germinated on 1% Agar plates in growth chambers under 50% humidity, 120 µm light (Red: Far-Red ratio =1.0), and 16 hr. light/8 hr. dark cycle. Germination success was 93% (51/55 seeds). After the emergence of a taproot, seedlings were transplanted to soil (Sunshine Mix #4, Sun Gro Horticulture, Agawam, MA) and kept in growth chambers under the above conditions, with 85% survival rate (47 seedlings). Identification of the Ephedra tweedieana PHYTOENE DESATURASE ortholog EtwPDS To identify the PHYTOENE DESATURASE ortholog, we queried the E. tweedieana proteome with the Arabidopsis PHYTOENE DESATURASE (AT4G14210) PDS3 coding sequence. The proteome was first searched using a gene family analysis (Orthofinder) based on closely related gymnosperm and model plant proteomes (resources.michael.salk.edu/resources/ephedra_genomes/). Then, the E. tweedieana proteome was searched using BLASTP to ensure that all potential orthologs were identified. Two potential orthologs were identified on Chromosome 7 (Chr07) and Chr03. Multiple sequencing alignment (MSA) revealed that the ortholog on Chr07 was truncated. Therefore, we utilized the ortholog on Chr03: EtweTM011.hap2.chr3.a04.g228080.t1 to build our construct (Fig. S1) and named it Etw PDS . Generation of Tobacco Rattle Virus (TRV) constructs A 425 base-pair fragment of the EtwPDS gene was amplified from cDNA with locus-specific primers (Table S1), digested with KpnI-HF and XhoI (New England Biolabs, Ipswich, MA, USA) and ligated into pYL156 (TRV2, Addgene plasmid #148969, Liu et al., 2002 ) using T4 DNA Ligase (New England Biolabs, Ipswich, MA, USA). Three clones were verified by Sanger sequencing (Genewiz, South Plainfield, NJ, USA). Agrobacterium tumefaciens GV3101 was transformed by electroporation with TRV2- EtwPDS , empty TRV2 (EV) or TRV1 (pYL192, Addgene plasmid # 148968, Liu et al., 2002 ), and the resulting colonies were confirmed by PCR. Agroinfiltration of Ephedra tweedieana seedlings Virus-induced gene silencing of EtwPDS was performed on one to two-week-old seedlings as previously described for Thalictrum dioicum L. ( Di Stilio et al., 2010 ) with the following modifications: 10 mL of 0.5 M MES (2-(4-Morpholino)-Ethane Sulfonic Acid) was used instead of 5 mL 1.0 M MES and two vacuum infiltration regimes of 2 and 5 min instead of a single infiltration time of 2 minutes. In addition to VIGS-treated plants (n=21), we included two negative controls: TRV1 + TRV2 empty vector (EV, n=10) to control for virus infection effects, and infiltration media (IM, n=3) to control for the vacuum infiltration treatment. Treated seedlings were returned to pots containing Sunshine#4 soil, covered with a plastic dome, and allowed to recover and grow under the conditions specified above. Validation of TRV infection and gene silencing Three weeks following VIGS treatment, approximately 10 mg of cotyledon or stem tissue was collected and flash frozen in liquid nitrogen, and RNA was extracted from using Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA). First-strand synthesis was carried out with iScript™ cDNA Synthesis Kit (Bio-Rad, Laboratories, Hercules, CA, USA) from 1 µg of RNA and used in reverse transcription PCR (RT-PCR) for TRV RNA detection and in real-time quantitative PCR (qPCR) for the validation of gene expression. RT-PCR was conducted to detect TRV1, TRV2, and the EF1b reference gene with locus-specific primers (Table S1). Samples were amplified in a thermocycler (DNA Engine DYAD Peltier Thermal Cycler, MJ Research, St. Bruno, QC, Canada) using the following conditions: initial denaturation 2 min at 95°C, 30 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 51°C, and extension for 1 min at 72°C, followed by a final extension at 72°C for 5 min. Samples were loaded on a 1% TAE gel, run at 100V for 60 minutes, and visualized using the Bio-Rad Gel Doc EZ Imager (Bio-Rad Laboratories, Hercules, CA, USA). qPCR was conducted using the iTaq Universal SYBR Green Supermix and gene-specific primers to detect target EtwPDS and housekeeping EtwEF1b expression levels (Supplemental Table 1). Samples were run using a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) set to the following conditions: initial denaturation for 5 min at 94°C, 40 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 57°C, extension for 30 sec at 72°C, and plate read, with cycling followed by melt curve analysis increasing in 0.5°C every 5 sec from 65°C to 95°C. EtwPDS expression was normalized to EtwEF1b using the delta delta CT method ( Livak and Schmittgen, 2001 ), and a one-way ANOVA was performed to compare expression levels of treatments and controls. Imaging Plants were photographed using a Nikon D3400 hand-held digital camera or a dissecting microscope (Nikon SMZ800, Nikon Instruments Inc., Melville, NY, USA) equipped with a QImaging MicroPublisher 3.3 RTV digital camera (Surrey, BC, Canada). Images were edited and compiled using Affinity Publisher v1.10.6 and Inkscape v1.4. RESULTS Vacuum Agrobacterium infiltration triggers TRV infection and targeted gene silencing To enable the investigation of gene function in the gymnosperm E. tweedieana , we aimed to obtain proof-of-principle evidence of transient transformation by Virus-Induced Gene Silencing (VIGS). To that end, we tested whether Agrobacterium -mediated infiltration of Tobacco Rattle Virus (TRV) can trigger targeted gene silencing of the commonly used marker PHYTOENE DESATURASE resulting in photobleaching of green photosynthetic tissues ( Figure 1 ). Vacuum infiltration of seedlings with Agrobacterium tumefaciens carrying E. tweedieana PHYTOENE DESATURASE ( TRV2- EtwPDS) resulted in high rates of viral infection, between 30% and 80% depending on the specific treatment ( Table 1 ). Download figure Open in new tab Figure 1: VIGS workflow in the gymnosperm Ephedra tweedieana . Agrobacterium- mediated infiltration of TRV in E. tweedieana seedlings triggers virus-induced gene silencing of the PHYTOENE DESATURASE ortholog Etw PDS . Seeds germinate on agar after one week and are transplanted to soil. Two-week old seedlings are infiltrated under vacuum for two minutes with equal parts of Agrobacteria cultures carrying TRV1 and TRV2 vectors, the latter with a fragment of the target gene. Photobleaching of green tissues, a sign of Etw PDS silencing, was observed ten days after treatment. View this table: View inline View popup Download powerpoint Table 1: Summary statistics for VIGS of the PHYTOENE DESATURASE ortholog in Ephedra tweedieana . Molecular validation via the detection of Tobacco Rattle Virus (TRV) RNA and photobleaching phenotype in different tissues of controls (infiltration media and empty vector, EV) and of plants treated under two or five-min vacuum infiltration. Infection by TRV, validated by amplification of viral RNA, was only observed in plants treated with TRV1 and TRV2, not in infiltration media (IM) control plants ( Fig. 2g ). Plants treated with an empty vector, TRV2-EV, were no different than IM control plants in leaf morphology, shoot morphology, or growth habit, suggesting Agrobacterium infection with TRV alone does not alter E. tweedieana growth and development (Supplemental Figure 1). Vacuum infiltration for two minutes resulted in a higher infection rate (64%) than five minutes (40%, Table 1 ), hence longer infiltration times are not warranted, nor beneficial for increasing infiltration rate. Download figure Open in new tab Figure 2: Photobleaching in Ephedra tweedieana by Virus-induced Gene Silencing. Full plant images show a range of bleaching severity, and the corresponding narrow panel displays magnified detail of the region marked in a white box. a) Empty-vector control plant. b-f) Representative TRV 2 - EtwPDS treated plants displaying a range of phenotypes. Scale bars 1 cm, or 1 mm (insets). Arrowheads point to leaves, and asterisk indicates lateral branch. g) RT-PCR validation of TRV1 RNA and TRV2 RNA presence, including the reference gene EF1b as a loading control. h) qPCR validation of PDS silencing in bleached tissues of virus-validated plants, relative to EF1b. Different letters indicate statistical significance ( P <0.05) in a one-way ANOVA ( F 2,15 = 18.42, P = 9.13e-05) followed by a Tukey HSD post-hoc test ( P EV vs IM = 0.126, P EV vs pds = 0.00006, P IM vs pds = 0.06). Plants treated with TRV2 -EtwPDS experienced down-regulation of EtwPDS , to an approximately 10-fold decrease in EtwPDS expression compared to TRV2-EV controls ( Figure 2h ). One 5-minute treated plant showed similar levels of EtwPDS expression to TRV-EV plants (Supplemental Figure 2). Taken together, our results indicate that Agrobacterium- mediated infiltration of TRV causes TRV infection that triggers gene silencing in E. tweedieana . Ephedra tweedieana PDS silencing causes photobleaching in a range of tissues PDS silencing typically results in photobleaching of photosynthetic tissue by disrupting carotenoid biosynthesis ( Wang et al., 2009 ) and is commonly used as a visual marker in targeted gene silencing experiments. Photobleaching of E. tweedieana tissues was first observed 10 d after infiltration in TRV- EtwPDS silenced plants ( Table 1 , Supplemental Figure 3). Three weeks following treatment, a range of photobleaching was observed compared to controls, with some plants overcoming silencing ( Figure 1a-c ) while others displayed stunted growth and severe silencing ( Figure 2f ). To determine the range of tissues susceptible to VIGS in E. tweedieana , we examined specific tissues affected by photobleaching by light microscopy ( Table 1 ). All plants undergoing silencing of EtwPDS exhibited photobleaching of stem internodes ( Figure 2f ), and most also showed leaf photobleaching. Additionally, two plants showed photobleaching of lateral shoots ( Figure 2e ) and apical leaves ( Figure 2f ). This suggests that, while stem internodes were most susceptible to VIGS, gene silencing can also encompass lateral and apical meristematic tissue, extending the phenotypic effects to tissues that develop after treatment. DISCUSSION To the best of our knowledge, this is the first report of VIGS in a non-flowering plant. The gene silencing efficiency of 64 % we observed in E. tweedieana is comparable to or higher than in other species where VIGS has been implemented. This includes several species in the Ranunculales: Eschscholzia californica Cham. (up to 94%, Wege et al., 2007 ), Thalictrum dioicum (42%, Di Stilio et al., 2010 ), Papaver somniferum L. (23%, Hileman et al., 2005 ), and Aquilegia coerulea E.James (up to 12%, Gould and Kramer, 2007 ; Sharma and Kramer, 2013 ) and across eudicots and monocots ( Di Stilio, 2011 ). As the first described example of VIGS in a gymnosperm species, the rapid and high efficiency of silencing by VIGS in Ephedra will facilitate testing of candidate genes to investigate the evolution of gene function, bridging the gap across seed plants and land plants more broadly. This tool also opens the door for studying the wealth of gymnosperm diversity across clades such as cycads and Gnetales. Within Ephedra , a number of avenues exist for studying stress response and convergent evolution of fleshy reproductive structures analogous to angiosperm fruits ( Di Stilio and Ickert-Bond, 2021 ). Functional validation of candidate genes in Ephedra would provide insight into the conservation, co-option, and evolution of genetic networks. Genetic transformation also complements transcriptomic analyses ( Zumajo-Cardona and Ambrose, 2022 ) by providing an avenue to test the function of candidate genes emerging from gene expression analysis. While short compared to other gymnosperms, the 2–4-year generation time of Ephedra species may pose challenges for applying VIGS in reproductive tissues. Within the scope of our study, photobleaching continued beyond three months, but the full duration of gene silencing remains to be tested. However, there are proven alternatives to address the eventual loss of gene silencing. Wounding and injecting previously infected adult plants can help reestablish silencing, as found in other perennials such as Aquilegia coerulea ( Gould and Kramer, 2007 ) and Thalictrum dioicum ( LaRue et al., 2013 ), and tubers can be treated in Spring ephemerals, accelerating the gap between treatment and flowering ( Di Stilio et al., 2010 ). Additionally, since various Ephedra species are amenable to clonal propagation ( O’Dowd and Richardson, 1993 ), infiltration of reproductive explants prior to rooting would circumvent time to reproduction and provide an opportunity to silence candidate genes in reproductively mature plants. In conclusion, the implementation of VIGS in E. tweedieana represents an exciting opportunity to expand functional genetic studies to gymnosperms and, in doing so, bridge the gap between angiosperms and the rest of the seed plants. Acknowledgements Thanks to Dr. Dinesh-Kumar for encouraging this study and sharing plasmids pYL156 and pYL192. Mauricio Bonifacino provided the E. tweedieana seeds. This work was funded by the Royalty Research Fund, University of Washington (to VSD), AGG was funded by NSF-GRFP, and JTB by UW Biology Department Top Scholar Award. Footnotes Author name misspelling (Stefanie, not Stephanie) Funding added for one author A few other minor typos in the body of the manuscript REFERENCES ↵ Ayadi , R. , and J. Trémouillaux-Guiller . 2003 . Root formation from transgenic calli of Ginkgo biloba . Tree Physiology 23 : 713 – 718 . OpenUrl CrossRef PubMed ↵ Bowe , L. M. , G. Coat , and C. W. dePamphilis . 2000 . Phylogeny of seed plants based on all three genomic compartments: Extant gymnosperms are monophyletic and Gnetales’ closest relatives are conifers . Proceedings of the National Academy of Sciences of the United States of America 97 : 4092 – 4097 . OpenUrl Abstract / FREE Full Text ↵ Di Stilio , V. S. 2011 . Empowering plant evo-devo: Virus induced gene silencing validates new and emerging model systems . Bioessays 33 : 711 – 718 . OpenUrl CrossRef PubMed ↵ Di Stilio , V. S. , and S. M. Ickert-Bond . 2021 . Ephedra as a gymnosperm evo-devo model lineage . Evolution & Development 23 : 256 – 266 . OpenUrl CrossRef PubMed ↵ Di Stilio , V. S. , R. A. Kumar , A. M. Oddone , T. R. Tolkin , P. Salles , and K. McCarty . 2010 . Virus-Induced Gene Silencing as a Tool for Comparative Functional Studies in Thalictrum E. Newbigin [ed.] ,. PLoS ONE 5 : e12064 . OpenUrl CrossRef PubMed ↵ Dommes , A. B. , T. Gross , D. B. Herbert , K. I. Kivivirta , and A. Becker . 2019 . Virus-induced gene silencing: empowering genetics in non-model organisms . Journal of Experimental Botany 70 : 757 – 770 . OpenUrl CrossRef PubMed ↵ Dupré , P. , J. Lacoux , G. Neutelings , D. Mattar-Laurain , M.-A. Fliniaux , A. David , and A. Jacquin-Dubreuil . 2000 . Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens . Physiologia Plantarum 108 : 413 – 419 . OpenUrl CrossRef ↵ Gould , B. , and E. M. Kramer . 2007 . Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae) . Plant Methods 3 : 6 . OpenUrl CrossRef PubMed ↵ Han , X. , H. Rong , Y. Feng , Y. Xin , X. Luan , Q. Zhou , M. Xu , and L. Xu . 2023 . Protoplast isolation and transient transformation system for Ginkgo biloba L . Frontiers in Plant Science 14 . ↵ Heino , M. , P. Kinnunen , W. Anderson , D. K. Ray , M. J. Puma , O. Varis , S. Siebert , and M. Kummu . 2023 . Increased probability of hot and dry weather extremes during the growing season threatens global crop yields . Scientific Reports 13 : 3583 . OpenUrl CrossRef PubMed ↵ Hileman , L. C. , S. Drea , G. de Martino , A. Litt , and V. F. Irish . 2005 . Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy) . The Plant Journal 44 : 334 – 341 . OpenUrl CrossRef PubMed Web of Science ↵ Ickert-Bond , S. M. , and S. S. Renner . 2016 . The Gnetales: Recent insights on their morphology, reproductive biology, chromosome numbers, biogeography, and divergence times . Journal of Systematics and Evolution 54 : 1 – 16 . OpenUrl CrossRef ↵ Jiang , W. , F. Deng , M. Babla , C. Chen , D. Yang , T. Tong , Y. Qin , et al. 2024 . Efficient gene editing of a model fern species through gametophyte-based transformation . Plant Physiology 196 : 2346 – 2361 . OpenUrl CrossRef PubMed ↵ A. Becker Lange , M. , A. L. Yellina , S. Orashakova , and A. Becker . 2013 . Virus-Induced Gene Silencing (VIGS) in Plants: An Overview of Target Species and the Virus-Derived Vector Systems . In A. Becker [ed.], Virus-Induced Gene Silencing: Methods and Protocols , 1 – 14 . Humana Press , Totowa, NJ . ↵ LaRue , N. C. , A. M. Sullivan , and V. S. Di Stilio . 2013 . Functional recapitulation of transitions in sexual systems by homeosis during the evolution of dioecy in Thalictrum . Frontiers in Plant Science 4 . ↵ Liu , Y. , M. Schiff , R. Marathe , and S. P. Dinesh-Kumar . 2002 . Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus . The Plant Journal 30 : 415 – 429 . OpenUrl CrossRef PubMed Web of Science ↵ Livak , K. J. , and T. D. Schmittgen . 2001 . Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method . Methods 25 : 402 – 408 . OpenUrl CrossRef PubMed Web of Science ↵ Morris , J. W. , L. A. Castle , and R. O. Morris . 1989 . Efficacy of different Agrobacterium tumefaciens strains in transformation of pinaceous gymnosperms . Physiological and Molecular Plant Pathology 34 : 451 – 461 . OpenUrl CrossRef ↵ Moyle , R. , J. Moody , L. Phillips , C. Walter , and A. Wagner . 2002 . Isolation and characterization of a Pinus radiata lignin biosynthesis-related O-methyltransferase promoter . Plant Cell Reports 20 : 1052 – 1060 . OpenUrl CrossRef ↵ Muthukumar , B. , B. L. Joyce , M. P. Elless , and C. N. Stewart Jr . . 2013 . Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides . Plant Physiology 163 : 648 – 658 . OpenUrl Abstract / FREE Full Text ↵ O’Dowd , N. A. , and D. H. S. Richardson . 1993 . In vitro micropropagation of Ephedra . Journal of Horticultural Science 68 : 1013 – 1020 . OpenUrl CrossRef ↵ Perez-Matas , E. , D. Hidalgo-Martinez , A. Escrich , M. A. Alcalde , E. Moyano , M. Bonfill , and J. Palazon . 2023 . Genetic approaches in improving biotechnological production of taxanes: An update . Frontiers in Plant Science 14 . ↵ Plackett , A. R. G. , L. Huang , H. L. Sanders , and J. A. Langdale . 2014 . High-Efficiency Stable Transformation of the Model Fern Species Ceratopteris richardii via Microparticle Bombardment . Plant Physiology 165 : 3 – 14 . OpenUrl Abstract / FREE Full Text ↵ Rössner , C. , D. Lotz , and A. Becker . 2022 . VIGS Goes Viral: How VIGS Transforms Our Understanding of Plant Science . Annual Review of Plant Biology 73 : 703 – 728 . OpenUrl CrossRef PubMed ↵ Rutherford , G. , M. Tanurdzic , M. Hasebe , and J. A. Banks . 2004 . A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes . BMC Plant Biology 4 : 6 . OpenUrl CrossRef PubMed ↵ A. Becker Sharma , B. , and E. M. Kramer . 2013 . Virus-Induced Gene Silencing in the Rapid Cycling Columbine Aquilegia coerulea “Origami” . In A. Becker [ed.], Virus-Induced Gene Silencing: Methods and Protocols , 71 – 81 . Humana Press , Totowa, NJ . ↵ T. A. Thorpe , and E. C. Yeung Tahir , M. , E. A. Waraich , and C. Stasolla . 2011 . Genetic Transformation Protocols Using Zygotic Embryos as Explants: An Overview . In T. A. Thorpe , and E. C. Yeung [eds.], Plant Embryo Culture: Methods and Protocols , 309 – 326 . Humana Press , Totowa, NJ . ↵ Wagner , A. , L. Phillips , R. D. Narayan , J. M. Moody , and B. Geddes . 2005 . Gene silencing studies in the gymnosperm species Pinus radiata . Plant Cell Reports 24 : 95 – 102 . OpenUrl CrossRef PubMed Web of Science ↵ Wang , M. , G. Wang , J. Ji , and J. Wang . 2009 . The effect of pds gene silencing on chloroplast pigment composition, thylakoid membrane structure and photosynthesis efficiency in tobacco plants . Plant Science 177 : 222 – 226 . OpenUrl CrossRef Web of Science ↵ Wege , S. , A. Scholz , S. Gleissberg , and A. Becker . 2007 . Highly efficient virus-induced gene silencing (VIGS) in California poppy (Eschscholzia californica): an evaluation of VIGS as a strategy to obtain functional data from non-model plants . Annals of Botany 100 : 641 – 649 . OpenUrl CrossRef PubMed ↵ Yadav , S. , S. Basu , A. Srivastava , S. Biswas , R. Mondal , V. K. Jha , S. K. Singh , and Y. Mishra . 2023 . Bryophytes as Modern Model Plants: An Overview of Their Development, Contributions, and Future Prospects . Journal of Plant Growth Regulation 42 : 6933 – 6950 . OpenUrl CrossRef ↵ Zhao , H. , J. Zhang , J. Zhao , S. Niu , H. Zhao , J. Zhang , J. Zhao , and S. Niu . 2024 . Genetic transformation in conifers: current status and future prospects . Forestry Research 4 . ↵ Zumajo-Cardona , C. , and B. A. Ambrose . 2022 . Fleshy or dry: transcriptome analyses reveal the genetic mechanisms underlying bract development in Ephedra . EvoDevo 13 : 10 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 12, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana Anthony Garcia , Jo Trang Bùi , Todd P. Michael , Stefanie M. Ickert-Bond , Verónica S. Di Stilio bioRxiv 2025.02.07.637168; doi: https://doi.org/10.1101/2025.02.07.637168 Share This Article: Copy Citation Tools Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana Anthony Garcia , Jo Trang Bùi , Todd P. Michael , Stefanie M. Ickert-Bond , Verónica S. Di Stilio bioRxiv 2025.02.07.637168; doi: https://doi.org/10.1101/2025.02.07.637168 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13871) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18561) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22475) Immunology (17713) Microbiology (40328) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)