Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present...Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present the development and evaluation of a novel glycosylase-based cytosine base editor(gCBE)for efficient C-to-G editing in rice.Unlike traditional cytosine base editors,which rely on cytosine deamination,gCBE directly excises cytosine to generate an apurinic/apyrimidinic(AP)site,thus circumventing the deamination step and reducing the production of C-to-T byproducts.We constructed several gCBE variants,including N-gCBE,M-gCBE,and C-gCBE,by fusing engineered human UDG2(UNG*)to SpCas9 nickase(nSpCas9,D10A)and tested their editing efficiency and specificity in rice.Our results demonstrate that M-gCBE achieved efficient C-to-G editing(6.3%to 37.5%)similar to OsCGBE(9.4%to 28.1%)at most targets,though with site-dependent variations.Notably,gCBE tools showed a marked reduction in C-to-T byproducts,with average C-to-T mutation rates of 12.5%for N-gCBE and 16.7%for M-gCBE,compared to 53.1%for OsCGBE.Notably,both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T_(0)generation,a key advantage over OsCGBE,which predominantly generated C-to-T mutations.Off-target analysis revealed minimal off-target effects with M-gCBE,highlighting its potential for high-precision genome editing.These findings suggest that gCBE tools,particularly M-gCBE,are highly efficient and precise,providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.展开更多
A numerical and experimental study was conducted to investigate the Laser Ablation(LA)ignition mode in an ethylene-fueled supersonic combustor with a cavity flameholder.Theexperiments were operated under a Mach number...A numerical and experimental study was conducted to investigate the Laser Ablation(LA)ignition mode in an ethylene-fueled supersonic combustor with a cavity flameholder.Theexperiments were operated under a Mach number 2.92 supersonic inflow,with stagnation pressureof 2.4 MPa and stagnation temperature of 1600 K.Reynolds-averaged Navier-Stokes simulationswere conducted to characterize the mixing process and flow field structure.This study identifiedfour distinct LA ignition modes.Under the specified condition,laser ablation in zero and negativedefocusing states manifested two distinct ignition modes termed Laser Ablation Direct Ignition(LADI)mode and Laser Ablation Re-Ignition(LARI)mode,correspondingly.LA ignition in alocal small cavity,created by depressing the flow field regulator,could facilitate the ignition modetransforming from LARI mode to Laser Ablation Transition Ignition(LATI)mode.On the otherhand,the elevation of the flow field regulator effectively inhibited the forward propagation of theinitial flame kernel and reduced the dissipation of LA plasma,further enhancing the LADI mode.Based on these characteristics,the LADI mode was subdivided into strong(LADI-S)and weak(LADI-W)modes.Facilitating the transition of ignition modes through alterations in the local flowfield could contribute to attaining a more effective and stable LA ignition.展开更多
The ignition process and flame propagation with ethylene fuel in cavity-stabilized scramjet by a Multi-Channel Gliding Arc(MCGA)at Mach 2.0 were investigated.Effects of equivalence ratios on the MCGA-assisted ignition...The ignition process and flame propagation with ethylene fuel in cavity-stabilized scramjet by a Multi-Channel Gliding Arc(MCGA)at Mach 2.0 were investigated.Effects of equivalence ratios on the MCGA-assisted ignition process and flame propagation of the scramjet were recorded by two high-speed cameras from different view angles.The discharge characteristics of MCGA are also collected synchronously with the high-speed cameras.The distributions of temperature,velocity,and equivalence ratios in non-reactive flows of the cavity were simulated by Reynolds Averaged Navier-Stokes(RANS)model.The results show that MCGA can achieve reliable ignition with the Global Equivalence Ratios(GER)between 0.06 and 0.17.The ignition process is composed of flame kernel generation,flame development,and stable combustion.The time from flame kernel generation to the establishment of global flame decreases as GER decreases from 0.17 to 0.08.In the streamwise direction,the flame first develops to the Cavity Leading Edge(CLE)because of the influence of the cavity recirculation zone and then uplifts into the cavity shear layer,and finally develops to the Cavity Trailing Edge(CTE).In the spanwise direction,the flame width is less than 50%of the width of the cavity before developing to CLE and begins to develop towards the two sides of the combustor after reaching CLE,which is affected by the angular recirculation zone on both sides of CLE.The ignition processes by MCGA in the scramjet combustor are significantly affected by local distributions of equivalence ratios and velocity in the cavity.展开更多
Mutual effects between a gliding arc(GA)discharge at atmospheric pressure and a premixed CH_(4)/air flame were experimentally investigated.Effects of the flame on the GA were studied using simultaneous measurements of...Mutual effects between a gliding arc(GA)discharge at atmospheric pressure and a premixed CH_(4)/air flame were experimentally investigated.Effects of the flame on the GA were studied using simultaneous measurements of the current,the voltage,and the instantaneous images of the plasma columns.The GA in the flame has a thicker and more diffusive plasma column,and it is more frequently ignited at a smaller breakdown voltage than that in the air.The GA extension velocity and the gliding velocity in the flame are larger than those in the air.The electrode voltage drop of the GA discharge in the flame is about 160 V,whereas that in the air is about 220 V.Compared with the GA in the air,the different features of the GA in the flame can be explained by high-temperature,weakly ionized,and species-abundant environment that are generated by the premixed CH_(4)/air flame.Effects of the gliding arc discharge on the premixed flames were demonstrated using planar laser-induced fluorescence of hydroxyl radicals(OH)and formaldehyde(CH_(2)O).OH and CH_(2)O can be formed in the CH_(4)/air mixture in the presence of the GA due to kinetic effects,and the increase of OH and CH_(2)O shows the great potential of the GA for combustion enhancement.展开更多
Of the more than 370000 species of higher plants in nature,fewer than 0.1%can be geneticallymodified due to limitations of the current gene delivery systems.Even for those that can be genetically modified,the modifica...Of the more than 370000 species of higher plants in nature,fewer than 0.1%can be geneticallymodified due to limitations of the current gene delivery systems.Even for those that can be genetically modified,the modification involves a tedious and costly tissue culture process.Here,we describe an extremely simple cut-dip-budding(CDB)delivery system,which uses Agrobacterium rhizogene to inoculate explants,generating transformed roots that produce transformed buds due to root suckering.We have successfully used CDB to achieve the heritable transformation of plant species inmultiple plant families,including two herbaceous plants(Taraxacum kok-saghyz and Coronilla varia),a tuberous root plant(sweet potato),and three woody plant species(Ailanthus altissima,Aralia elata,and Clerodendrum chinense).These plants have previously been difficult or impossible to transform,but the CDB method enabled efficient transformation or gene editing in them using a very simple explant dipping protocol,under non-sterile conditions and without the need for tissue culture.Our work suggests that large numbers of plants could be amenable to genetic modifications using the CDB method.展开更多
With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a ge...With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate(WDV-surrogate), to facilitate high-throughput genome editing.WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene(HygM) for surrogate selection;the other is a WDV-based construct expressing two single guide RNAs(sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T_0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency(66.8% vs. 90.1%), plantlet regeneration efficiency(2.31-fold increase), and numbers of homozygous-edited plants(36.3%vs. 70.7%). Large-scale editing using pooled sg RNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDVGate on sequence knock-in for protein tagging.By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.展开更多
Deaminase-based cytosine base editors(CBEs)and adenine base editors(ABEs)induce C-to-T and A-to-G transitions,respectively,enabling single-nucleotide variants(SNVs)in plants for research and crop enhancement(Li et al....Deaminase-based cytosine base editors(CBEs)and adenine base editors(ABEs)induce C-to-T and A-to-G transitions,respectively,enabling single-nucleotide variants(SNVs)in plants for research and crop enhancement(Li et al.,2023a).The C-to-G base editors(CGBEs)and A-to-Y base editors(AYBEs),developed by incorporating glycosylases with CBEs and ABEs,expand the repertoire of base editing products,allowing C-to-T/G and A-to-T/G transversions in plants(Li et al.,2023b,2023c;Sretenovic et a.,2021).展开更多
Geneupregulation through genome editing is important for plant research and breeding.Targeted insertion of short transcriptional enhancers(STEs)into gene promoters may offer a universal solution akin to transgene-medi...Geneupregulation through genome editing is important for plant research and breeding.Targeted insertion of short transcriptional enhancers(STEs)into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis.Here,we introduce an“in locus activation”technique in rice that leverages well-characterized STEs for refined,heritable,and multiplexed gene upregulation.To address the scarcity of potent enhancers,we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts.The in locus integration of these STEs into eight rice genes resulted in substantial tran-scriptional upregulation in the edited plants,with up to 869.1-fold increases in their transcript levels.Em-ploying a variety of STEs,we achieved delicate control of gene expression,enabling the fine-tuning of key phenotypic traits such as plant height.Our approach also enabled efficient multiplexed gene upregu-lation,with up to four genes activated simultaneously,significantly enhancing the nicotinamide mononucleotide metabolic pathway.Importantly,heritability studies from the To to T3 generations confirmed the stable and heritable nature of STE-driven gene activation.Collectively,our work demon-strates that coupled with STE mining,leveraging genome editing for in locus activation and gene upregu-lation holds great promise to be widely adopted in fundamental plant research and crop breeding.展开更多
Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tag...Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remains unexplored in plants. Here, we introduce the in locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, a result attributable to low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence up to 6.9 fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in situ visualization using a cross-breeding strategy, and effective immunoprecipitation for analysis of protein interactions. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.展开更多
基金supported by the National Natural Science Foundation of China(82404798)the Natural Science Foundation of Sichuan Province(2024NSFSC1831)+1 种基金the National Key Laboratory for Tropical Crop Breeding(NKLTCB-RC202403,NKLTCBZRJJ4)the Hainan Seed Industrial Laboratory(B22C1000P).
文摘Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present the development and evaluation of a novel glycosylase-based cytosine base editor(gCBE)for efficient C-to-G editing in rice.Unlike traditional cytosine base editors,which rely on cytosine deamination,gCBE directly excises cytosine to generate an apurinic/apyrimidinic(AP)site,thus circumventing the deamination step and reducing the production of C-to-T byproducts.We constructed several gCBE variants,including N-gCBE,M-gCBE,and C-gCBE,by fusing engineered human UDG2(UNG*)to SpCas9 nickase(nSpCas9,D10A)and tested their editing efficiency and specificity in rice.Our results demonstrate that M-gCBE achieved efficient C-to-G editing(6.3%to 37.5%)similar to OsCGBE(9.4%to 28.1%)at most targets,though with site-dependent variations.Notably,gCBE tools showed a marked reduction in C-to-T byproducts,with average C-to-T mutation rates of 12.5%for N-gCBE and 16.7%for M-gCBE,compared to 53.1%for OsCGBE.Notably,both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T_(0)generation,a key advantage over OsCGBE,which predominantly generated C-to-T mutations.Off-target analysis revealed minimal off-target effects with M-gCBE,highlighting its potential for high-precision genome editing.These findings suggest that gCBE tools,particularly M-gCBE,are highly efficient and precise,providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.
基金supported by the National Natural Science Foundation of China(Nos.12272408 and 11925207)the Natural Science Foundation for Distinguished Young Scholars of Hunan Province,China(No.2024J12057)。
文摘A numerical and experimental study was conducted to investigate the Laser Ablation(LA)ignition mode in an ethylene-fueled supersonic combustor with a cavity flameholder.Theexperiments were operated under a Mach number 2.92 supersonic inflow,with stagnation pressureof 2.4 MPa and stagnation temperature of 1600 K.Reynolds-averaged Navier-Stokes simulationswere conducted to characterize the mixing process and flow field structure.This study identifiedfour distinct LA ignition modes.Under the specified condition,laser ablation in zero and negativedefocusing states manifested two distinct ignition modes termed Laser Ablation Direct Ignition(LADI)mode and Laser Ablation Re-Ignition(LARI)mode,correspondingly.LA ignition in alocal small cavity,created by depressing the flow field regulator,could facilitate the ignition modetransforming from LARI mode to Laser Ablation Transition Ignition(LATI)mode.On the otherhand,the elevation of the flow field regulator effectively inhibited the forward propagation of theinitial flame kernel and reduced the dissipation of LA plasma,further enhancing the LADI mode.Based on these characteristics,the LADI mode was subdivided into strong(LADI-S)and weak(LADI-W)modes.Facilitating the transition of ignition modes through alterations in the local flowfield could contribute to attaining a more effective and stable LA ignition.
基金supported by the National Natural Science Foundation of China(Nos.12172379,11925207,and 91741205)the Foundation for Outstanding Young Scholars of National University of Defense Technology,China.
文摘The ignition process and flame propagation with ethylene fuel in cavity-stabilized scramjet by a Multi-Channel Gliding Arc(MCGA)at Mach 2.0 were investigated.Effects of equivalence ratios on the MCGA-assisted ignition process and flame propagation of the scramjet were recorded by two high-speed cameras from different view angles.The discharge characteristics of MCGA are also collected synchronously with the high-speed cameras.The distributions of temperature,velocity,and equivalence ratios in non-reactive flows of the cavity were simulated by Reynolds Averaged Navier-Stokes(RANS)model.The results show that MCGA can achieve reliable ignition with the Global Equivalence Ratios(GER)between 0.06 and 0.17.The ignition process is composed of flame kernel generation,flame development,and stable combustion.The time from flame kernel generation to the establishment of global flame decreases as GER decreases from 0.17 to 0.08.In the streamwise direction,the flame first develops to the Cavity Leading Edge(CLE)because of the influence of the cavity recirculation zone and then uplifts into the cavity shear layer,and finally develops to the Cavity Trailing Edge(CTE).In the spanwise direction,the flame width is less than 50%of the width of the cavity before developing to CLE and begins to develop towards the two sides of the combustor after reaching CLE,which is affected by the angular recirculation zone on both sides of CLE.The ignition processes by MCGA in the scramjet combustor are significantly affected by local distributions of equivalence ratios and velocity in the cavity.
基金financially supported by National Natural Science Foundation of China(Nos.12172379,12322211,and 11925207)。
文摘Mutual effects between a gliding arc(GA)discharge at atmospheric pressure and a premixed CH_(4)/air flame were experimentally investigated.Effects of the flame on the GA were studied using simultaneous measurements of the current,the voltage,and the instantaneous images of the plasma columns.The GA in the flame has a thicker and more diffusive plasma column,and it is more frequently ignited at a smaller breakdown voltage than that in the air.The GA extension velocity and the gliding velocity in the flame are larger than those in the air.The electrode voltage drop of the GA discharge in the flame is about 160 V,whereas that in the air is about 220 V.Compared with the GA in the air,the different features of the GA in the flame can be explained by high-temperature,weakly ionized,and species-abundant environment that are generated by the premixed CH_(4)/air flame.Effects of the gliding arc discharge on the premixed flames were demonstrated using planar laser-induced fluorescence of hydroxyl radicals(OH)and formaldehyde(CH_(2)O).OH and CH_(2)O can be formed in the CH_(4)/air mixture in the presence of the GA due to kinetic effects,and the increase of OH and CH_(2)O shows the great potential of the GA for combustion enhancement.
基金supported by Shandong Shunfeng BiotechnologyCo.Ltd.,Jinan,China.
文摘Of the more than 370000 species of higher plants in nature,fewer than 0.1%can be geneticallymodified due to limitations of the current gene delivery systems.Even for those that can be genetically modified,the modification involves a tedious and costly tissue culture process.Here,we describe an extremely simple cut-dip-budding(CDB)delivery system,which uses Agrobacterium rhizogene to inoculate explants,generating transformed roots that produce transformed buds due to root suckering.We have successfully used CDB to achieve the heritable transformation of plant species inmultiple plant families,including two herbaceous plants(Taraxacum kok-saghyz and Coronilla varia),a tuberous root plant(sweet potato),and three woody plant species(Ailanthus altissima,Aralia elata,and Clerodendrum chinense).These plants have previously been difficult or impossible to transform,but the CDB method enabled efficient transformation or gene editing in them using a very simple explant dipping protocol,under non-sterile conditions and without the need for tissue culture.Our work suggests that large numbers of plants could be amenable to genetic modifications using the CDB method.
基金supported by the National Key R&D Program of China (2021YFD1201300 to Y.L., 2021YFA1300404 to J.-K.Z.)the National Natural Science Foundation of China (32070396 to Y.L., 32188102 to J.-K.Z.)the China Postdoctoral Science Foundation (BX20220098 to Y.T.)。
文摘With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate(WDV-surrogate), to facilitate high-throughput genome editing.WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene(HygM) for surrogate selection;the other is a WDV-based construct expressing two single guide RNAs(sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T_0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency(66.8% vs. 90.1%), plantlet regeneration efficiency(2.31-fold increase), and numbers of homozygous-edited plants(36.3%vs. 70.7%). Large-scale editing using pooled sg RNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDVGate on sequence knock-in for protein tagging.By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.
基金supported by the National Key R&D Program of China(2021YFA1300404 to J.-K.Z.)the National Natural Science Foundation of China(32188102 to J.-K.Z.)+2 种基金the China Postdoctoral Science Foundation(BX20220098 and 2022M720973 to Y.T.)the Hainan Seed Industry Laboratory(B22C1000P to Y.T.)Nanfan special project,CAAS(ZDXM2314 to M.W.)。
文摘Deaminase-based cytosine base editors(CBEs)and adenine base editors(ABEs)induce C-to-T and A-to-G transitions,respectively,enabling single-nucleotide variants(SNVs)in plants for research and crop enhancement(Li et al.,2023a).The C-to-G base editors(CGBEs)and A-to-Y base editors(AYBEs),developed by incorporating glycosylases with CBEs and ABEs,expand the repertoire of base editing products,allowing C-to-T/G and A-to-T/G transversions in plants(Li et al.,2023b,2023c;Sretenovic et a.,2021).
基金supported by the National Key R&D Program of China(2021YFD1201300 to Y.L.and 2021YFA1300404 to J.-K.Z.)the Shanghai Agricultural Science and Technology Innovation Program of China(K2023001 to Y.L.)the National Natural Science Foundation of China(32070396 to Y.L.and 32188102 to J.-K.Z.).
文摘Geneupregulation through genome editing is important for plant research and breeding.Targeted insertion of short transcriptional enhancers(STEs)into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis.Here,we introduce an“in locus activation”technique in rice that leverages well-characterized STEs for refined,heritable,and multiplexed gene upregulation.To address the scarcity of potent enhancers,we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts.The in locus integration of these STEs into eight rice genes resulted in substantial tran-scriptional upregulation in the edited plants,with up to 869.1-fold increases in their transcript levels.Em-ploying a variety of STEs,we achieved delicate control of gene expression,enabling the fine-tuning of key phenotypic traits such as plant height.Our approach also enabled efficient multiplexed gene upregu-lation,with up to four genes activated simultaneously,significantly enhancing the nicotinamide mononucleotide metabolic pathway.Importantly,heritability studies from the To to T3 generations confirmed the stable and heritable nature of STE-driven gene activation.Collectively,our work demon-strates that coupled with STE mining,leveraging genome editing for in locus activation and gene upregu-lation holds great promise to be widely adopted in fundamental plant research and crop breeding.
基金National Key R&D Program of China(no.2021YFD1201300 to Y.L.and 2021YFA1300404 to J.-K.Z.)National Natural Science Foundation of China(32070396 to Y.L.)China Postdoctoral Science Foundation(BX20220098 and 2022M720973 to Y.T.).
文摘Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remains unexplored in plants. Here, we introduce the in locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, a result attributable to low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence up to 6.9 fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in situ visualization using a cross-breeding strategy, and effective immunoprecipitation for analysis of protein interactions. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.
