Choosing the Best Route: Comparative Optimization of Wheat Transformation Methods for Improving Yield by Targeting TaARE1-D with CRISPR/Cas9

Choosing the Best Route: Comparative Optimization of Wheat Transformation Methods for Improving Yield by Targeting TaARE1-D with CRISPR/Cas9 | 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 Choosing the Best Route: Comparative Optimization of Wheat Transformation Methods for Improving Yield by Targeting TaARE1-D with CRISPR/Cas9 View ORCID Profile Mumin Ibrahim Tek , View ORCID Profile Kubra Budak Tek , Pelin Sarikaya , Abdul Razak Ahmed , View ORCID Profile Hakan Fidan doi: https://doi.org/10.1101/2025.09.11.675438 Mumin Ibrahim Tek 1 Département de Phytologie, Université Laval , Québec City, Québec, Canada 2 Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval , Québec City, Québec, Canada 3 ATG biotek, Izmir , Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mumin Ibrahim Tek Kubra Budak Tek 1 Département de Phytologie, Université Laval , Québec City, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kubra Budak Tek Pelin Sarikaya 4 Plant Protection Department, Akdeniz University , Antalya, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abdul Razak Ahmed 4 Plant Protection Department, Akdeniz University , Antalya, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hakan Fidan 4 Plant Protection Department, Akdeniz University , Antalya, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hakan Fidan For correspondence: hakanfidan{at}akdeniz.edu.tr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Wheat ( Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ∼3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D , a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance. Introduction Wheat ( Triticum aestivum L.) is one of the most important staple crops worldwide, providing a substantial share of the global caloric and protein supply ( 1 ). With the rapidly growing global population and the increasing challenges posed by climate change, there is an urgent need to improve wheat productivity, resilience, and nutritional value. Recent advances in gene-editing technologies, particularly CRISPR/Cas9, have transformed plant breeding by enabling precise and targeted modifications of the genome ( 2 ). This precision facilitates the efficient introduction of desirable traits such as higher yield, enhanced disease resistance, increased tolerance to abiotic stresses, and improved nutritional quality ( 3 , 4 ). To date, many studies have focused on improving wheat yield by either characterizing yield-associated genes or targeting previously identified negative regulators ( 2 ). For example, Grain Weight ( TaGW ) genes, which act as negative regulators of yield, have been frequently targeted, resulting in considerable increases in grain size, grain width, and overall yield ( 5 , 6 ). Another strategy is modifying spike structure-related traits using nucleases such as Cas9. The loss of function of DUO-B1 , which encodes an APETALA2/ethylene response factor (AP2/ERF), was shown to increase the number of spikelets and grains per plant ( 7 ). In addition to morphological traits, targeting physiological regulators has also proven effective. A well-known example is the ARE1 gene, which encodes a regulator of nitrogen uptake in rice. Loss-of-function mutations in ARE1 , known as abnormal cytokinin response1 repressor-1 mutants, improved nitrogen use efficiency ( 8 ). Similarly, targeting the wheat ortholog TaARE1 using CRISPR/Cas9 in the hexaploid AABBDD genome increased nitrogen uptake, leading to longer spikes, delayed senescence, and enhanced grain weight, particularly in AABBdd and aabbDD mutants ( 9 ). Despite the immense potential of gene editing in wheat, its widespread application remains constrained by major bottlenecks in plant transformation and regeneration. Wheat is considered one of the most recalcitrant cereals to genetic transformation, and both the delivery of foreign DNA into cells and the regeneration of whole plants continue to pose significant challenges ( 2 , 10 ). Among these, the regeneration step is the most limiting, as it is highly genotype-dependent, time-consuming, and labor-intensive ( 11 , 12 ). These obstacles hinder the efficient introduction of gene-editing components into elite wheat cultivars. Therefore, overcoming transformation and regeneration barriers is essential for integrating CRISPR/Cas9 and related technologies in breeding programs for wheat ( 7 ). Several methods have been developed for wheat transformation, each with distinct advantages and limitations. The most widely used approach involves the transformation of immature embryos, typically through Agrobacterium -mediated gene transfer or particle bombardment ( 13 – 15 ). However, these methods are often genotype-dependent, labor-intensive, and rely on specialized tissue culture facilities. In planta transformation strategies, which aim to bypass tissue culture by directly targeting germline cells, have also been investigated ( 16 ). However, these approaches have generally been hindered by low transformation efficiencies and poor reproducibility, limiting their suitability for large-scale breeding programs. Therefore, in this study, we optimized existing Agrobacterium -mediated transformation methods to significantly improve efficiency and reduce the time required to obtain transgenic wheat plants by carefully adjusting key parameters. Using CRISPR/Cas9, we targeted TaARE1-D , a well-characterized negative regulator of nitrogen uptake and yield. In addition, we introduced an improved, faster, and more efficient in planta transformation method, providing a promising alternative to conventional approaches. At the same time, we developed accelerated regeneration protocols for both immature and callus explants, achieving efficiencies comparable to traditional but more time-consuming methods. Finally, we comprehensively compared all optimized approaches in terms of transformation efficiency, regeneration rate, gene-editing efficiency, and the overall time from initial transformation to the recovery of edited mature plants with the desired phenotype. MATERIAL & METHODS Plant Material Spring wheat ( Triticum aestivum cv. Kayra), obtained from the Aegean Agricultural Research Institute (AARI, Türkiye), was used as plant material. Plants were cultivated in controlled growth chambers at 24 °C under a 16/8-hour light/dark photoperiod. Water and fertilizer were applied regularly to support growth until spikelet formation and anthesis, at which stage immature embryo explants were collected. Vector Construction We used the modular cloning system for the CRISPR/Cas9 construct ( 17 , 18 ) to target TaARE1 -D in the hexaploid genome of T. aestivum (Supplementary Figure 1). The TaARE1-A (TraesCS7A03G0678600), TaARE1-B (TraesCS7B03G0545300), and TaARE1-D (TraesCS7D03G0650400) sequences were obtained from Phytozome and used to select TaARE1 -D-specific gRNAs (Supplementary Table 1). After Level 1 cloning of the gRNAs, we used the P3 TaU6 guide acceptor (Addgene, 165599) and the P4 TaU6 guide acceptor (Addgene, 165600) in the final assembly with 100 ng of pGoldenGreenGate-M (Addgene, 165422), OsActinP:Hygint:NosT (Addgene, 165423), the pICH41780 linker (Addgene, 48019), and OsUbiP:Cas9:NosT (Addgene, 16542). Each cloning step was performed according to the previously described protocol, after which the plasmids were transferred into E. coli DH5α and subjected to blue-white screening ( 19 ). The final assembly was confirmed by BglII restriction digestion, PCR, and Sanger sequencing (Supplementary Figure 2). Agrobacterium Strain, OD, and Acetosyringone Optimization The CRISPR vector was introduced into Agrobacterium tumefaciens strains AGL1, EHA105, and GV3101 (GoldBio), all carrying the pSOUP helper plasmid, by electroporation ( 20 ). Transformed colonies were selected on LB agar supplemented with kanamycin (50 μg/mL), rifampicin (25 μg/mL), and tetracycline (5 μg/mL) (Supplementary Figure 2). After 2–3 days of incubation, single colonies were transferred into liquid LB medium containing the same antibiotics and incubated at 28 °C. Glycerol stocks (40%) were prepared and stored at –82 °C for long-term use. For transformation experiments, cells were first cultured in antibiotic-free LB medium (5 mL) for 16– 18 hours. On the day of inoculation, cultures were scaled up in 40 mL of fresh LB medium and incubated until an OD 600 of 1.0 was reached. Cells were pelleted by centrifugation (4,000 rpm) and resuspended in inoculation medium (2.2 g/L MS salts, 10 g/L glucose) supplemented with 100, 150, or 200 µM acetosyringone (Phytotech, A104). Suspensions were maintained at 28 °C with shaking (100 rpm) for at least two hours before transformation. Transformation of Callus Derived from Mature Embryos and Regeneration Mature seeds were sterilized in 70% ethanol for 90 seconds, rinsed three times with sterile distilled water, and treated with 1% commercial bleach for 20 minutes. After three additional rinses, seeds were soaked overnight in sterile water to facilitate embryo extraction. Callus induction was performed on MS-based medium modified according to previous protocol ( 21 ). The callus induction (CI) medium consisted of 4.4 g/L MS salts with vitamins (M519, Phytotech), 30 g/L maltose (Sigma, M9171), 1.25 mg/L CuSO 4 (Phytotech, C375), 1 g/L casein hydrolysate (Sigma, 22090), 2.5 mg/L 2,4-D (Phytotech, D295), and 3.5 g/L Phytagel (Sigma, P8169). Excised mature embryos were placed on CI medium and incubated in the dark at 24 °C for 4–6 weeks. The medium was refreshed biweekly. Well-developed calli (6–10 mm diameter) were selected for transformation. For inoculation, 10–15 calli were immersed in 15 mL of Agrobacterium suspension and gently shaken for 10, 15, or 20 minutes. Excess bacterial suspension was removed by blotting on sterile filter paper (Supplementary Figure 3). Then, calli were transferred onto co-culture medium containing 4.4 g/L MS salts with vitamins, 30 g/L maltose, 1 g/L casein hydrolysate, 1.25 mg/L CuSO 4 , 100, 150, or 200 µM acetosyringone (Phytotech, A104), and 3.5 g/L Phytagel, and incubated in the dark at 24 °C for three days. Following co-cultivation, calli were transferred to selection medium (SM) composed of 4.4 g/L MS salts with vitamins, 30 g/L maltose, 1 g/L casein hydrolysate, 1.25 mg/L CuSO 4 , 15 mg/L hygromycin (Phytotech, H397), 200 mg/L Timentin (Phytotech, T869), either 1 mg/L zeatin (Phytotech, Z899) or 0.2 mg/L indole-3-acetic acid (IAA) (Phytotech, I885), and 3.5 g/L Phytagel. Cultures were maintained at 24 °C under a 16/8-hour photoperiod for 2–4 weeks, and calli were monitored daily for shoot formation. Negative transformants were eliminated by hygromycin selection. Regenerated shoots were transferred to rooting medium consisting of 2.2 g/L MS salts with vitamins, 30 g/L maltose, 200 mg/L Timentin, and 3 g/L Phytagel. Healthy plantlets were subsequently acclimatized in pots containing a 3:1 mixture of compost soil and perlite. Pots were covered with transparent plastic bags to maintain high humidity for one week, after which holes (1 cm diameter) were gradually introduced. Bags were removed completely after an additional week. Regenerated plants were subjected to transgene screening, and T1 seeds were harvested from mature plants. Transformation of Immature and Mature Embryos and Regeneration Immature and mature embryos were isolated following seed sterilization as previously described ( 13 , 21 ). Isolated embryos were first placed in liquid inoculation medium containing 0.05% Silwet-L77 (Phytotech, S7777) without Agrobacterium . Samples were centrifuged for 10 minutes at 10,000 rpm, after which the medium was replaced with Agrobacterium suspension. Embryos were incubated for 10, 15, or 20 minutes, with tubes inverted three to four times every five minutes to ensure uniform exposure (Supplementary Figure 3). Excess inoculum was removed by blotting on three layers of sterile filter paper. Embryos were then transferred to co-cultivation medium and incubated in the dark for three days before being placed on CI medium supplemented with 200 mg/L Timentin for seven days. Calli with diameters of 4–6 mm were subsequently transferred to CI medium containing 15 mg/L hygromycin and 200 mg/L Timentin and maintained in the dark at 24 °C for two weeks. Surviving calli were transferred to SM supplemented with either 0.2 mg/L IAA or 1 mg/L zeatin and incubated at 24 °C under a 16/8-hour photoperiod for 2–4 weeks to evaluate shoot regeneration. Healthy shoots that survived selection were subsequently transferred to rooting medium and later acclimatized under greenhouse conditions as described above. In planta Transformation Fifteen to twenty sterilized seeds were placed on pre-wetted paper towels in Petri dishes and incubated in the dark at 24 °C for three days. After germination, an Agrobacterium suspension was delivered using a 1-mL syringe fitted with a 26-gauge needle (0.4 mm × 13 mm). The needle was inserted into the apical meristem to a depth of approximately 0.1 mm, and 10–15 µL of inoculum was applied directly to the wounded tissue. Following injection, seedlings were maintained on wet paper towels in Petri dishes with the lids partially open for two, three, or four days in the dark to ensure high humidity. Explants were then washed with 200 mg/L Timentin for 20 minutes on a shaker (150 rpm, 24 °C) and rinsed thoroughly with sterile distilled water. Treated seedlings were transplanted into pots and fertilized gradually. Two weeks after transformation, newly developed leaves were collected for PCR screening. Plants identified as transgenic were advanced for growth and harvested to obtain T1 seeds. PCR Screening and Gene-Editing Efficiency Genomic DNA was isolated using the CTAB method ( 22 ) from plantlets 2–4 weeks after acclimatization ( in vitro transformation) or sowing ( in planta transformation). Transgene screening was performed by PCR in a 20 µL reaction containing 10 µL 2× EcoTaq Master Mix (Ecotech, ET-5), 1 µL each of HygF/HygR primers ( 13 ). 2 µL DNA template, and 7 µL nuclease-free water. Amplification conditions were: initial denaturation at 94 °C for 10 min, followed by 35 cycles of 95 °C for 35 s, 58 °C for 45 s, and 72 °C for 45 s, with a final extension at 72 °C for 10 min. PCR products were loaded on 1.5% agarose gels, and transgene-positive plants were identified by the presence of the expected band. Negative plants were discarded, while positive plants were maintained under a 16/8-h photoperiod at 24 °C in controlled growth chambers until seed harvesting. Gene-editing efficiency was assessed using the ACT-PCR method (Wang & Wang, 2019). Reaction mixtures and cycling conditions were identical to the screening PCR, except that the annealing temperature was set to 62 °C (Supplementary Figure 1; Supplementary Table 1). Absence of a PCR product was interpreted as a putative mutation caused by disruption of the primer binding site at the Cas9 cleavage site in TaARE1-D . Phenotyping and Data Analysis Edited ( TaARE1-D knockout) and non-edited control plants were undergone phenotyping for agronomic traits including number of grains per spike, spike length, grain length, and thousand-grain weight. Measurements were recorded at maturity using a minimum of three biological replicates per genotype. Statistical analyses were performed in R (v4.5.1). One-way ANOVA was used to compare mean trait values, followed by pairwise t -tests for post hoc comparisons. Data visualization, including phenotypic trait plots and optimization parameter outputs, was generated using the ggplot2, dplyr, and tidyr libraries. RESULTS Optimization of transformation parameters for immature embryo explants To optimize transformation conditions, different Agrobacterium strains were first tested using fixed parameters: OD 600 = 0.8, 150 μM acetosyringone in both inoculum and co-culture media, and an incubation time of 15 minutes. Transformation efficiency was assessed based on the survival of explants following callus formation and selection with 15 mg/L hygromycin ( Figure 1c ). Among the tested strains, AGL1 showed the highest efficiency, with 61.67% of explants surviving after selection, followed by EHA105 at 37.30%, whereas GV3101 showed the lowest survival rate at only 10.42%. Based on these results, AGL1 was chosen for further optimization. Download figure Open in new tab Figure 1. Transformation and regeneration of wheat plants from immature embryo explants. Immature embryos were co-cultured with Agrobacterium tumefaciens strain AGL1 under optimized transformation conditions (a). After one month on callus induction medium (CI), the calli were transferred to selection medium (SM) supplemented with 0.2 mg/L IAA, where shoots and small roots appeared within 3–5 days (b). Surviving shoots were selected on 15 mg/L hygromycin (c) and subsequently transferred to rooting medium (d). Regenerated plantlets were acclimatized in soil under high-humidity conditions (e), and finally well-developed plants were maintained until seed harvest (f). Next, we evaluated bacterial density by varying OD 600 . Transformation with OD 600 = 0.8 yielded the highest survival rate (75.64%), compared with 48.90% at 0.6 and 24.52% at 1.0. Incubation time also influenced efficiency: the lowest transformation rate was observed with 10 minutes (27.47%), whereas 15 minutes increased efficiency to 63.95%. Prolonging incubation to 20 minutes reduced survival to 51.10%. Collectively, these findings indicate that the most efficient conditions for immature embryo transformation were obtained using AGL1 at OD600 = 0.8, with a 15-minute incubation, and 150 μM acetosyringone in both inoculum and co-culture media (Supplementary Table 2). The same strategy was applied to mature embryos. Although callus formation was observed following transformation, no explants survived under selection. Instead, only necrotic calli developed, and shoots lost viability within one to two days in the selection medium. Optimization of transformation parameters for callus Callus formation was successfully induced from mature embryos cultured in the dark on CI medium ( Figure 2 ). Well-developed calli began to appear after 2 weeks, reaching 3–4 mm in diameter, and by four weeks they had expanded to 7–8 mm. These calli were collected and subsequently used for transformation experiments (Supplementary Figure 3). As with immature embryos, different Agrobacterium strains were first tested under fixed parameters: OD 600 = 0.8, 150 µM acetosyringone in inoculum and co-culture media, and a 15-minute incubation. Although overall efficiency was lower than with immature embryo transformation, AGL1 again showed the highest survival rate (51.68%), followed by EHA105 (26.59%) and GV3101 (12.27%). Next, we optimized bacterial density. The highest survival rate was observed at OD 600 = 0.8 (57.51%), while increasing the density to OD 600 = 1.0 reduced survival to 25.71%. No statistically significant difference was observed between OD600 = 0.8 and 0.6, despite a slight difference. The effect of acetosyringone concentration was also examined. While no significant difference was observed between 100 µM and 150 µM, further increasing the concentration substantially reduced survival, with only 12.64% of calli surviving selection. Finally, incubation time was tested. A 10-minute incubation yielded the highest efficiency (73.9%), followed by 15 minutes (63.5%), with no significant difference. However, prolonging the incubation to 20 minutes reduced survival dramatically to 27.68%. In summary, the most efficient conditions for callus transformation were obtained using AGL1 at OD 600 = 0.8, a 10-minute incubation, and 150 µM acetosyringone in the inoculation and co-culture media, with selection on 15 mg/L hygromycin (Supplementary Table 3). Download figure Open in new tab Figure 2. Regeneration of wheat plants from callus derived from mature embryos. Calli were transferred to selection medium (SM) supplemented with 0.2 mg/L IAA (a), where the first shoots appeared within five days (b). After four weeks of selection, surviving plantlets (c) were transferred to rooting medium (d). Well-developed plants were subsequently acclimatized in soil (e) and maintained for seed harvest (f). Regeneration of callus We next tested the effects of IAA (0.2 mg/L) and zeatin (1 mg/L) on the regeneration of wheat calli. After co-cultivation, calli derived from both immature and mature embryos were transferred to selection medium (SM) supplemented with either IAA or zeatin. Shoots regenerated more efficiently on IAA-containing medium than on zeatin-containing medium. To confirm this observation, we evaluated regeneration efficiency in the absence of selection. Explants from immature embryo-derived calli placed on 0.2 mg/L IAA produced shoots within 2–3 days, with a regeneration rate of 52.63%. In contrast, calli placed on 1 mg/L zeatin regenerated at only 26.32% (Supplementary Table 4). Similarly, calli derived from mature embryos regenerated at 47.94% on IAA compared with 24.55% on zeatin. Together, these results demonstrate that 0.2 mg/L IAA is substantially more effective than 1 mg/L zeatin for promoting regeneration in both immature and mature embryo-derived calli. Improving of in planta transformation Similar to the in vitro approach, in planta transformation was optimized ( Figure 3 ) by first testing different Agrobacterium strains under fixed parameters (OD 600 = 0.8, 150 µM acetosyringone in the inoculum, and three days of dark incubation). Based on PCR screening, AGL1 was again the most efficient strain, yielding 30.42% positive plants. We then tested different cell densities (OD 600 = 0.6, 0.8, and 1.0). Increasing bacterial density improved transformation efficiency, with PCR-positive rates rising from 7.01% at OD 600 = 0.6 to 39.12% at OD 600 = 1.0. Next, acetosyringone concentrations were compared (100, 150, and 200 µM). No significant difference was observed between 100 µM (32.54%) and 150 µM (36.51%), but efficiency decreased substantially to 15.28% at 200 µM (Supplementary Table 5). Finally, we evaluated post-transformation dark incubation. Incubation for two days yielded the highest efficiency (39.73%), whereas extending incubation to three or four days reduced efficiency to 24.52% and 27.14%, respectively. Although there was no significant difference between three and four days, extended dark treatment, particularly four days caused etiolation of seedlings. In summary, the most efficient in planta transformation was achieved using AGL1 at OD600 = 1.0, with 150 µM acetosyringone in the inoculum, followed by two days of dark incubation ( Figure 3 ). After treatment, plants were washed with 200 mg/L Timentin for 20 minutes, rinsed three times with distilled water, and transferred to soil for further growth. Interestingly, many in planta -transformed plants exhibited over-tillering, with some producing up to 20–25 tillers (Supplementary Figure 4). Download figure Open in new tab Figure 3. General workflow of in planta transformation. Germinated seedlings were injected with Agrobacterium into the plumule part (a, b). Following transformation, plants were maintained in the dark for at least two days to allow bacterial interaction (c). Subsequently, seedlings were washed with 200 mg/L timentin to eliminate excess Agrobacterium and transferred to soil for further growth. Gene-editing efficiency across transformation methods Using the optimized protocols for each method we evaluated CRISPR/Cas9 gene-editing efficiency. After selection and regeneration, we obtained 43 plants from immature embryo transformation, 40 from callus transformation, and 28 from in planta transformation. Transgene screening was performed with HygF/R primers (Supplementary Figure 5). Of these, 38 out of 42 immature embryo-derived plants and 30 out of 40 callus-derived plants were PCR positive, whereas 7 out of 28 plants from in planta transformation tested positive. We further analyzed randomly selected PCR-positive plants by ACT-PCR to detect mutations induced by CRISPR/Cas9. In total, 22 plants from immature embryos, 15 from callus-derived transformation, and 7 from in planta transformation were screened with target-specific primers. Mutation events at the target region were detected in 16 of 22 immature embryo-derived plants, 9 of 16 callus-derived plants, and 3 of 7 in planta plants (Supplementary Figure 6). Phenotypic analysis of taare1d mutants We compared the taare1d mutants with the unedited control variety ( T. aestivum var. Kayra). Three representative mutant lines; IM33 (immature embryo transformation), MC7 (callus-derived transformation), and IP5 ( in planta transformation) were evaluated for grain yield-related traits, including number of grains per spike, spike length, grain length, and thousand-grain weight. All three mutant lines showed improved yield parameters compared with the control ( Figure 4a ). Kayra produced an average of 57.5 ± 2.89 grains per spike, whereas IM33, MC7, and IP5 produced 65 ± 2.89, 64.5 ± 2.69, and 63.2 ± 1.72 grains, respectively ( Figure 4b ). Spike length also increased, rising from 9.47 cm in Kayra to 10.33 cm in IM33, 10.18 cm in MC7, and 10.20 cm in IP5 ( Figure 4c ). Grain size and weight were likewise improved. The average grain length increased from 6.69 mm in Kayra to 7.91 mm in IM33, 7.46 mm in MC7, and 7.54 mm in IP5, while thousand-grain weight rose from 40.51 g to 43.77 g, 43.57 g, and 43.09 g, respectively ( Figures 4d, 4e ). For grain length specifically, MC7 and IP5 grouped statistically with both NE and IM33, reflecting minor differences between these lines. Download figure Open in new tab Figure 4. Comparison of yield parameters among mutant lines generated by immature embryos, callus-derived, and in planta transformation, alongside the non-edited variety. Representative spikes of mutant and non-edited plants are shown (a). Box plots illustrate differences in the number of grains per spike (b), spike length (c), grain length (d), and thousand-grain weight (e). DISCUSSION Wheat is one of the most important staple crops worldwide, providing nearly 20% of global dietary energy and protein intake ( 23 ). Rising global temperatures have already caused an estimated 5.5% reduction in wheat yields between 1980 and 2010 ( 24 ). Consequently, the development of new wheat varieties with higher yield potential and improved tolerance to abiotic and biotic stress has become a central goal in modern breeding programs. Over the past decade, gene-editing technologies such as CRISPR/Cas9 have emerged as powerful tools to accelerate crop improvement. These approaches enable the precise modification of genomic sequences, allowing rapid introduction of desired traits into elite cultivars ( 2 ). In wheat, the targeted knockout of negative regulators of yield, including TaGW2 and DUO-B1 , has been shown to enhance grain size, spikelet number, and yield ( 6 , 7 ). Despite these advances, the widespread application of gene editing in wheat continues to be constrained by the challenges of stable transformation. The efficiency of established protocols varies widely depending on genotype, delivery strategy, and experimental parameters ( 13 , 25 ). To address these limitations, a comparative optimization of multiple transformation strategies was undertaken in this study, including immature embryo transformation, callus-derived transformation from mature embryos, and injection-based in planta transformation. As a proof of concept, the TaARE1-D gene, previously identified as a negative regulator of nitrogen uptake and yield in rice and wheat ( 8 , 9 ) was targeted for knockout. The transformation parameters were systematically adjusted to optimize efficiency for each method. Across all approaches, AGL1 consistently outperformed EHA105 and GV3101, confirming its suitability for wheat transformation ( Figure 5a ). Previous reports have attributed the high efficiency of AGL1 to the presence of the pSOUP helper plasmid, which is essential for the replication of pGreen-based vectors ( 26 , 27 ). In this study, pSOUP-containing constructs were introduced into all tested strains, suggesting that the superior performance of AGL1 may be linked to intrinsic compatibility with wheat tissues and more effective T-DNA delivery rather than solely the plasmid background. Download figure Open in new tab Figure 5. Bar chart comparing survival and transformation efficiency across different methods. a– d) represent hygromycin selection outcomes for immature embryo transformation, e–h) for callus (from mature embryo) transformation, and i–l) show PCR screening results for in planta transformation. Statistical significance was determined at p = 0.01. After the effect of strain was established, the influence of bacterial density was assessed using AGL1. For in vitro transformation, an OD 600 of 0.8 proved optimal, as both lower (0.6) and higher (1.0) cell densities were associated with reduced survival of explants on hygromycin-containing medium ( Figure 5b, 5f ). Lower densities are likely to reduce the frequency of T-DNA transfer events, while excessive bacterial loads at OD 600 = 1.0 may impair explant viability through tissue damage or bacterial overgrowth ( 28 ). Previous studies have reported successful immature embryo transformation at OD 600 = 0.5 ( 13 ); however, the present findings suggest that efficiency can be improved by increasing the density to 0.8. In callus-derived transformation, no significant difference was observed between 0.6 and 0.8, although a reduction in survival was again detected at OD 600 = 1.0. In contrast, in planta transformation responded differently, as higher bacterial densities correlated with an increased frequency of PCR-positive plants. This divergence may be attributed to the absence of an enriched co-culture medium during in planta inoculation ( Figure 5i ). Without supplemental carbon sources such as glucose, lower bacterial densities may not support sufficient Agrobacterium activity for efficient T-DNA delivery, explaining the superior performance at OD600 = 1.0. The concentration of acetosyringone in the inoculum and co-culture media was subsequently optimized. Among the tested concentrations, 150 μM consistently supported the highest transformation efficiency across all methods. In immature embryo transformation, this concentration produced a statistically significant improvement compared with both lower (100 μM) and higher (200 μM) levels ( Figure 5c ). In callus-derived and in planta transformation, the difference between 100 μM and 150 μM was not statistically significant, although a slight numerical advantage was observed at 150 μM ( Figure 5g ). By contrast, the use of 200 μM acetosyringone negatively affected transformation efficiency, suggesting potential inhibitory effects on either Agrobacterium activity or host tissue responses, as has been noted previously ( 29 , 30 ). The effect of incubation time on transformation efficiency was also evaluated. In immature embryos, a 10-minute incubation with AGL1 was insufficient to promote high transformation efficiency, whereas extended exposure improved outcomes ( Figure 5d ). However, the response was found to be explant dependent. In callus-derived explants, prolonged incubation reduced survival on hygromycin-containing medium, likely due either to inefficient T-DNA transfer at later stages or to detrimental effects of extended bacterial contact on callus tissue integrity ( Figure 5h ). Previous reports have similarly noted that incubation periods of 10–12 minutes are optimal for callus transformation, whereas longer exposures compromise efficiency ( 21 ). In in planta transformation, prolonged dark incubation following bacterial delivery reduced the number of PCR-positive plants ( Figure 5l ). The highest efficiency was obtained when seedlings were maintained for only two days, while extended incubation led not only to reduced transformation success but also to etiolation, particularly after four days in the dark. Shoot regeneration from callus was further evaluated using 0.2 mg/L IAA and 1 mg/L zeatin. Callus from T. aestivum var. Kayra responded rapidly to IAA, with shoots appearing within 4–5 days, accompanied by simultaneous root initiation. A similar response to IAA has been reported in callus derived from mature embryos of the Bobwhite S56 ( 21 ), although such rapid regeneration has not typically been described in callus from immature embryos. In both immature- and mature-derived callus, IAA proved more effective than zeatin, indicating that auxin supplementation promotes more efficient regeneration in this genotype. Although other concentrations of zeatin were not tested, the use of 1 mg/L was clearly less effective for Kayra regeneration. Through optimization of transformation parameters, high efficiencies were achieved with immature embryo- and callus-derived explants, while lower but improved success was obtained with in planta transformation. When efficiencies were normalized using PCR screening to exclude escapes ( Figure 6b ), the values remained comparable to previously reported protocols, confirming that the optimized approaches-maintained transformation quality. A major advance of this study was the modification of the immature embryo protocol. In earlier methods, embryos typically undergo three days of co-cultivation, a seven-day resting period, followed by a prolonged callus induction stage with selection lasting four to six weeks ( 13 , 14 ). In contrast, embryos in this study were maintained for only two weeks on callus induction medium with selection before being transferred directly to regeneration medium containing IAA, under a 16/8-hour photoperiod that allowed regeneration and selection to proceed simultaneously. This adjustment shortened the overall transformation process by approximately one month. Although callus size was smaller and regenerated plantlets appeared less robust, efficiency remained high after normalization, indicating that the shortened induction period did not compromise transformation outcomes. For callus transformation, the using of smaller calluses (6–10 mm) instead of the standard 15–20 mm reduced efficiency slightly but accelerated regeneration, and acclimatized plants showed no developmental abnormalities ( 13 , 21 ). Finally, in in planta transformation, optimization of inoculation parameters increased efficiency remarkably (3.3% to 33%) compared with earlier reports from ( 31 , 32 ), demonstrating its potential as a rapid alternative to tissue culture-dependent approaches. Download figure Open in new tab Figure 6. Effects of hormonal supplementation on regeneration. a) Bar graph comparing the effect of 0.2 mg/l IAA and 1 mg/l zeatin on regeneration from immature and mature embryo–derived calluses. b) Number of plants screened and confirmed as transgenic using the optimized transformation methods. c) Number of plants carrying CRISPR/Cas9-induced mutations CRISPR/Cas9-induced mutations were detected in plants generated by all three transformation methods. Although in planta transformation produced fewer overall transgenics, successful recovery of three TaARE1-D mutants demonstrated that the method can yield edited lines when optimized ( Figure 6c ). The greater number of mutants obtained from immature embryo transformation reflected the higher number of transgenic plants produced, yet the proportion of mutants among tested plants was comparable between immature- and callus-derived approaches. Phenotypic evaluation confirmed that TaARE1-D loss-of-function enhanced yield-related traits, consistent with previous studies in wheat and rice ( 8 , 9 ). Increases in spike length, grain number, and thousand-grain weight were observed in mutants generated by all transformation methods, including in planta . Grain length varied among lines, with IP5 ( in planta ) and MC7 (callus-derived) grouping statistically with both the non-edited control and the immature embryo mutant IM33, likely due to variance within those lines. For other parameters, all mutants exhibited significant improvements compared with the non-edited control. In addition, the stay-green phenotype that is a hallmark of the TaARE1-D disruption was consistently observed, confirming the functional knockout. These findings indicate that by adjusting transformation parameters, multiple methods can be optimized to generate edited wheat plants efficiently. 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Share Choosing the Best Route: Comparative Optimization of Wheat Transformation Methods for Improving Yield by Targeting TaARE1-D with CRISPR/Cas9 Mumin Ibrahim Tek , Kubra Budak Tek , Pelin Sarikaya , Abdul Razak Ahmed , Hakan Fidan bioRxiv 2025.09.11.675438; doi: https://doi.org/10.1101/2025.09.11.675438 Share This Article: Copy Citation Tools Choosing the Best Route: Comparative Optimization of Wheat Transformation Methods for Improving Yield by Targeting TaARE1-D with CRISPR/Cas9 Mumin Ibrahim Tek , Kubra Budak Tek , Pelin Sarikaya , Abdul Razak Ahmed , Hakan Fidan bioRxiv 2025.09.11.675438; doi: https://doi.org/10.1101/2025.09.11.675438 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 (7633) Biochemistry (17680) Bioengineering (13889) Bioinformatics (41927) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13373) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22494) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)