T-DNA Tagging Research Articles

Rhamnogalacturonan lyase 1 (RGL1), as a suppressor of E3 ubiquitin ligase Arabidopsis thaliana ring zinc finger 1 (AtRZF1), is involved in dehydration response to mediate proline synthesis and pectin rhamnogalacturonan-I composition.

Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.

BES1/BZR1 Homolog 3 cooperates with E3 ligase AtRZF1 to regulate osmotic stress and brassinosteroid responses in Arabidopsis

Proline (Pro) metabolism plays important roles in protein synthesis, redox balance, and abiotic stress response. However, whether they share a cross-talk with brassinosteroid (BR) signaling regulation remains unclear. Here, we introduced an Arabidopsis intergenic enhancer double mutant namely proline content alterative 41 (pca41) which T-DNA tagged in atrzf1 mutant background. The pca41 showed that T-DNA inserted at the site of gene encoded for BEH3. The pca41 possessed an insensitive phenotype that was stronger than atrzf1 observed under osmotic stress including high Pro accumulation and decreased reactive oxygen species levels. Analysis of physiological, genetic, and molecular networks revealed that negative regulation of BEH3 in abiotic stress was linked to BR-signaling pathway. Obtained data also suggested that AtRZF1, an E3 ubiquitin ligase, might control dehydration, abscisic acid, and BR responses in a BEH3-dependent manner. Under darkness, the pca41 displays the long hypocotyl phenotype, which is similar to atrzf1 and beh3, suggesting that BEH3 acts in the same pathway as AtRZF1. Furthermore, overexpression of BEH3 resulted in dehydration-induced sensitive phenotype, and this sensitive phenotype is reversed by exogenous BR application. Taken together, our results suggest that AtRZF1 and BEH3 might play an important role in osmotic stress response via ubiquitination and BR-signaling cross-talk.

T-DNA activation tagging in rice results in a variable response to Meloidogyne graminicola infection

Plant-parasitic nematodes (PPNs) are known to be devastating pests of agricultural crops; the rice root knot nematode Meloidogyne graminicola being the most damaging in rice. Variability in response to any trait is important not only to understand plant-stress interactions, but also its utility in stress mitigation. In this direction, the focus of the study was to exploit activation tagging, a well-established functional genomics approach in rice as a plausible strategy to assess variability in the phenotypic and molecular response to M. graminicola infection. T-DNA insertional mutants were developed in rice (acc. JBT 36/14) using an apical meristem-targeted in planta transformation strategy. A panel of 30 mutants that demonstrated molecular evidences for T-DNA integration and portraying a robust phenotype were chosen for the study. Vigorous phenotyping of these mutants against M. graminicola second stage juveniles both under in vitro pluronic gel medium and soil conditions depicted variable response based on four disease scoring parameters and the derived nematode reproductive factor. Among 30 mutants that were evaluated, five showed a resistant phenotype while 25 depicted a susceptible reaction. The resistant plants clearly demonstrated a reduction in all the disease parameters both under in vitro as well as greenhouse conditions. Quantification of transcripts of 25 genes associated with host defence responses belonging to salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways also indicated corroboration with the phenotype exhibited by the mutants. Distinct pattern of gene expression profiles was observed not only between the susceptible and resistant mutants, but also amongst the resistant ones, demonstrating the possibility of lesser known defense pathways being operated in these plants, apparently depending upon the T-DNA integration loci. The variability in the response to nematode challenge demonstrated in this study can be a step towards deciphering of compatible/incompatible relationships that can form a knowledge base towards not only understanding plant-nematode interactions but also extrapolation of their utility in rice improvement programmes.

Activity characterization of the plantain promoter from the heavy metal-associated isoprenylated plant gene (MabHIPP) using the luciferase reporter gene

Banana and plantain crops may be subjected to different abiotic stress factors such as temperature, salinity and presence of heavy metals. Genetic engineering has been a valuable tool to accelerate modifications in plants by expressing genes to improve tolerance to different abiotic stress. Promoters have a fundamental role to drive the specific expression of useful genes in genetically modified plants. For promoter activity characterization, fusion with reporter genes are often used. The American firefly luciferase reporter gene system (LUC) could be used to characterize the promoter activity in vivo and with high sensitivity when compared to other reporter genes including β-glucuronidase (GUS) and Green fluorescent protein (GFP). In this study, activity of the heavy metal-associated isoprenylated plant protein (HIPP) promoter, originally discovered in a T-DNA tagging approached in plantain, was tested in the banana ‘Williams’ (genotype AAA) by Agrobacterium-mediated transformation of in vitro plants. A 625 bp promoter region was fused to the luc2 reporter gene in a pCAMBIA 1301-GUS less vector backbone. In silico promoter analysis revealed candidate cis-acting elements and a TATA box. Luciferase activity was up-regulated after 6 h of 60 μM of cadmium application in banana in vitro plants, indicating an increase in promoter activity. The luciferase reporter gene proved to be a useful tool to evaluate differential promoter activity after metal application in plants in vivo.

Loss of Arabidopsis Halotolerance 2-like (AHL), a 3'-phosphoadenosine-5'-phosphate phosphatase, suppresses insensitive response of Arabidopsis thaliana ring zinc finger 1 (atrzf1) mutant to abiotic stress.

Destruction of PAP phosphatase AHL suppresses atrzf1 phenotype in abiotic stress responses. AHL plays an intermediate role in the regulation of proline accumulation by PAP nucleotidase. Proline (Pro) metabolism is important for environmental responses, plant development, and growth. However, the role of Pro in abiotic stress process is unclear. Using atrzf1 (Arabidopsis thaliana ring zinc finger 1) mutant as a parental line for T-DNA tagging mutagenesis, we identified a suppressor mutant designated as proline content alterative 17 (pca17) that suppressed insensitivity of atrzf1 to abiotic stresses during early seedling growth. Pro content of pca17 was lower than that in both wild type (WT) and atrzf1 while complementary lines were less sensitive to abscisic acid (ABA) and abiotic stresses compared to WT. Thermal Asymmetric Interlaced (TAIL)-PCR of pca17 showed that T-DNA was inserted at site of At5g54390 (AHL for Arabidopsis Halotolerance 2-like) encoding 3'-phosphoadenosine-5'-phosphate (PAP) phosphatase. Under drought stress condition, products of sulfate metabolism such as PAP and adenosine monophosphate were significantly lower in pca17 than those in WT and atrzf1. Furthermore, pca17 showed significantly higher levels of several important drought parameters including malondialdehyde, ion leakage, and water loss than WT and atrzf1. Fluorescence signal of green fluorescent protein (GFP)-tagged AHL was quite strong in nuclei of the root and guard cells of transgenic seedlings. Additionally, AHL promoter-β-glucuronidase (GUS) construct revealed substantial gene expression in vasculature tissues and pollen. Collectively, these findings demonstrate that pca17 acts as a dominant suppressor mutant of atrzf1 in abiotic stress response by modulating proline and sulfate metabolism.

Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome

CRISPR/Cas9-induced genome editing is a powerful tool for studying gene function in a variety of organisms, including plants. Using multi-sgRNAs to target one or more genes is helpful to improve the efficacy of gene editing and facilitate multi-gene editing. Here, we describe a CRISPR/Cas9 system which can be conveniently developed as a CRISPR kit. SgRNA expression cassettes can be rapidly generated by one-step PCR using our CRISPR kit. In our kit, there are two binary vectors pHNCas9 and pHNCas9HT. The binary vector pHNCas9 was constructed to allow to assemble up to eight sgRNA expression cassettes by one-step Golden Gate cloning. Another binary vector pHNCas9HT can be used to generate a large number of single target constructs by directly transforming ligation reactions products into A. tumefaciens without several procedures, such as PCR and plasmid extraction. The two binary vectors are designed according to the principles of standard BioBrick assembly to be used as an open-source tool. For example, we used BioBrick as a visual T-DNA tag. We also developed a primer design aid to complement the system. With this primer design aid, researchers can rapidly obtain primers and GC content, and sgRNA sequence of target site. Our CRISPR/Cas9 system can perform single- and multi-site editing and multiple gene editing to produce various types of mutations in tomato. This rapid and user-friendly CRISPR/Cas9 system for tomato can be potentially used for mutagenesis of important crop species for genetic improvement and is suitable for research into the function of genes.

OsWRKY67 Plays a Positive Role in Basal and XA21-Mediated Resistance in Rice.

WRKY proteins play important roles in transcriptional reprogramming in plants in response to various stresses including pathogen attack. In this study, we functionally characterized a rice WRKY gene, OsWRKY67, whose expression is upregulated against pathogen challenges. Activation of OsWRKY67 by T-DNA tagging significantly improved the resistance against two rice pathogens, Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo). Reactive oxygen species (ROS) rapidly accumulated in OsWRKY67 activation mutant lines in response to elicitor treatment, compared with the controls. Overexpression of OsWRKY67 in rice confirmed enhanced disease resistance, but led to a restriction of plant growth in transgenic lines with high levels of OsWRKY67 protein. OsWRKY67 RNAi lines significantly reduced resistance to M. oryzae and Xoo isolates tested, and abolished XA21-mediated resistance, implying the possibility of broad-spectrum resistance from OsWRKY67. Transcriptional activity and subcellular localization assays indicated that OsWRKY67 is present in the nucleus where it functions as a transcriptional activator. Quantitative PCR revealed that the pathogenesis-related genes, PR1a, PR1b, PR4, PR10a, and PR10b, are upregulated in OsWRKY67 overexpression lines. Therefore, these results suggest that OsWRKY67 positively regulates basal and XA21-mediated resistance, and is a promising candidate for genetic improvement of disease resistance in rice.

Model Legumes: Functional Genomics Tools in Medicago truncatula.

Many researchers have sought along the last two decades a legume species that could serve as a model system for genetic studies to resolve specific developmental or metabolic processes that cannot be studied in other model plants. Nitrogen fixation, nodulation, compound leaf, inflorescence and plant architecture, floral development, pod formation, secondary metabolite biosynthesis, and other developmental and metabolic aspects are legume-specific or show important differences with those described in Arabidopsis thaliana, the most studied model plant. Mainly Medicago truncatula and Lotus japonicus were proposed in the 1990s as model systems due to their key attributes, diploid genome, autogamous nature, short generation times, small genome sizes, and both species can be readily transformed. After more than decade-long, the genome sequences of both species are essentially complete, and a series of functional genomics tools have been successfully developed and applied. Mutagens that cause insertions or deletions are being used in these model systems because these kinds of DNA rearrangements are expected to assist in the isolation of the corresponding genes by Target-Induced Local Lesions IN Genomes (TILLING) approaches. Different M. truncatula mutants have been obtained following γ-irradiation or fast neutron bombardment (FNB), ethyl-nitrosourea (ENU) or ethyl-methanesulfonate (EMS) treatments, T-DNA and activation tagging, use of the tobacco retrotransposon Tnt1 to produce insertional mutants, gene silencing by RNAi, and transient post-transcriptional gene silencing by virus-induced gene silencing (VIGS). Emerging technologies of targeted mutagenesis and gene editing, such as the CRISPR-Cas9 system, could open a new era in this field. Functional genomics tools and phenotypic analyses of several mutants generated in M. truncatula have been essential to better understand differential aspects of legumes development and metabolism.

Loss of Ribosomal Protein L24A (RPL24A) suppresses proline accumulation of Arabidopsis thaliana ring zinc finger 1 (atrzf1) mutant in response to osmotic stress

Proline (Pro) metabolism in plants is involved in various cellular processes mediated during abiotic stress. However, the Pro-regulatory mechanisms are unclear. We used a suppressor mutation technique to isolate novel genes involved in the regulation of Pro metabolism in Arabidopsis. Using atrzf1 as a parental plant for T-DNA tagging mutagenesis, we identified a suppressor mutant, termed proline content alterative 21 (pca21), that displayed reduced Pro contents compared with the atrzf1 under osmotic stress conditions. Genomic Thermal Asymmetric Interlaced (TAIL)-PCR revealed pca21 harbored an inserted T-DNA in the region of At2g36620 that encodes Ribosomal Protein L24A. In general, the pca21 mutant partially suppressed the insensitivity of atrzf1 to osmotic stress and abscisic acid during seed germination and early seedling stage. Additionally, the pca21 mutant had increased MDA content and lower expression of several Pro biosynthesis-related genes than the atrzf1 mutant during drought condition. These results suggest that pca21 acts as partial suppressor of atrzf1 in the osmotic stress response through the Pro-mediated pathway.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants

A promoter trap mutant line of Arabidopsis carrying a promoterless β-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (−594/−30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5′ and 3′ promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence −594/−30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

Identification of root-preferential transcription factors in rice by analyzing GUS expression patterns of T-DNA tagging lines

T-DNA tagging lines are useful for analyzing the functions of genes and regulatory elements. We have previously generated approximately 100,000 insertional mutants in japonica rice (Oryza sativa), using T-DNA vectors carrying the promoter-less GUS reporter gene. In this study, we conducted GUS assays of seedlings from 430 lines in which TDNA was inserted into transcription factor genes. Among the 75 lines that showed GUS signals, nine displayed an endospermpreferential expression pattern; two lines demonstrated GUS signals in both endosperm and roots; 21 lines had GUS expression mainly in leaves; 19 lines showed GUS signal in both leaves and roots; and 24 lines expressed GUS predominantly in the roots. Co-segregation analyses of 49 homozygous lines indicated that the GUS expression patterns observed from 38 lines were due to the T-DNA insertion. We also identified fusion transcripts between tagged genes and the GUS reporter in six lines. Quantitative RT-PCR confirmed that the GUS expression patterns of those tagged lines indeed represent organ- and tissue-preferential expression of the tagged genes. The GUS-tagged transcription factor lines identified here will be useful for functional analysis of these candidates.

Ectopic Expression of OsSta2 Enhances Salt Stress Tolerance in Rice.

Salt stress can severely reduce crop yields. To understand how rice (Oryza sativa) plants respond to this environmental challenge, we investigated the genes involved in conferring salt tolerance by screening T-DNA tagging lines and identified OsSta2-D (Oryza sativa Salt tolerance activation 2-Dominant). In that line, expression of OsSta2 was enhanced by approximately eightfold when compared with the non-transformed wild type (WT). This gene was highly expressed in the callus, roots, and panicles. To confirm its role in stress tolerance, we generated transgenic rice that over-expresses OsSta2 under a maize ubiquitin promoter. The OsSta2-Ox plants were salt-tolerant at the vegetative stage, based on our calculations of chlorophyll fluorescence (Fv/Fm), fresh and dry weights, chlorophyll concentrations, and survival rates. Under normal paddy field conditions, the Ox plants were somewhat shorter than the WT control but had improved agronomic traits such as higher total grain yield. They were also more tolerant to osmotic stress and hypersensitive to abscisic acid. Based on all of these results, we suggest that OsSta2 has important roles in determining yields as well as in conferring tolerance to salt stresses.

Misexpression of AtTX12 encoding a Toll/interleukin-1 receptor domain induces growth defects and expression of defense-related genes partially independently of EDS1 in Arabidopsis.

In this study, a tissue-specific GAL4/UAS activation tagging system was used for the characterization of genes which could induce lethality when ubiquitously expressed. A dominant mutant exhibiting stunted growth was isolated and named defective root development 1-D (drd1-D). The T-DNA tag was located within the promoter region of AtTX12, which is predicted to encode a truncated nucleotide-binding leucine-rich repeat (NLR) protein, containing a Toll/interleukin-1 receptor (TIR) domain. The transcript levels of AtTX12 and defense-related genes were elevated in drd1-D, and the misexpression of AtTX12 recapitulated the drd1-D phenotypes. In the presence of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), a key transducer of signals triggered by TIR-type NLRs, a low-level of AtTX12 misexpression induced strong defective phenotypes including seedling lethality whereas, in the absence of EDS1, a high-level of AtTX12 misexpression induced weak growth defects like dwarfism, suggesting that AtTX12 might function mainly in an EDS1-dependent and partially in an EDS1-independent manner. [BMB Reports 2016; 49(12): 693–698]

Gene tagging in Physcomitrella patens: an addition to the genetic toolbox.

This article is a commentary on Vives et al., 212: 759–769.

Development of an Effective PCR Technique for Analyzing T-DNA Integration Sites in Brassica Species and Its Application

Abstract: Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage ( Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was desi gned by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linke d sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapawere designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identificatio n of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments T.his method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis scree ns.Additional key words:

Identification of AtWRKY75 as a transcriptional regulator in the defense response to Pcc through the screening of Arabidopsis activation-tagged lines

The necrotrophic pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc) causes soft rot in a broad range of plant hosts. Approximately 60,000 independent seeds from Arabidopsis activation tagging lines were inoculated with Pcc and screened for resistant mutants. An Rpe1 (resistance protein to Pectobacterium 1) mutant, which had more resistance to Pcc than wild-type (WT) plants, was selected for further study. The T-DNA inserting locus in Rpe1 was located on the middle of chromosome V by flanking sequence analysis. Through expression analysis with several genes adjacent to the T-DNA tagging region, AtWRKY75 gene was highly up-regulated in the Rpe1 mutant compared to the WT plant. The up-regulation of AtWRKY75 gene was shown to be correlated on the induction of the PDF1.2, VSP1 and PR1 genes compared to the WT plant. AtWRKY75 over-expression lines exhibited reduced Pcc bacterial growth compared to WT. Taken together, our data suggest that AtWRKY75 should be a positive regulator in the JA- or SA-mediated defense signaling responses to Pcc.

Analysis of the early-flowering mechanisms and generation of T-DNA tagging lines in Kitaake, a model rice cultivar.

As an extremely early flowering cultivar, rice cultivar Kitaake is a suitable model system for molecular studies. Expression analyses revealed that transcript levels of the flowering repressor Ghd7 were decreased while those of its downstream genes, Ehd1, Hd3a, and RFT1, were increased. Sequencing the known flowering-regulator genes revealed mutations in Ghd7 and OsPRR37 that cause early translation termination and amino acid substitutions, respectively. Genetic analysis of F2 progeny from a cross between cv. Kitaake and cv. Dongjin indicated that those mutations additively contribute to the early-flowering phenotype in cv. Kitaake. Because the short life cycle facilitates genetics research, this study generated 10 000 T-DNA tagging lines and deduced 6758 flanking sequence tags (FSTs), in which 3122 were genic and 3636 were intergenic. Among the genic lines, 367 (11.8%) were inserted into new genes that were not previously tagged. Because the lines were generated by T-DNA that contained the promoterless GUS reporter gene, which had an intron with triple splicing donors/acceptors in the right border region, a high efficiency of GUS expression was shown in various organs. Sequencing of the GUS-positive lines demonstrated that the third splicing donor and the first splicing acceptor of the vector were extensively used. The FST data have now been released into the public domain for seed distribution and facilitation of rice research.

알라닌 아미노기전이효소가 상실된 벼(Oryza sativa L.) 돌연변이체의 고염 스트레스에 대한 반응

An AlaAT knock-out mutant (alaat) of rice (Oryza sativa L.) was isolated from T-DNA tagging lines and the genotypes of its progeny were determined with AlaAT1-specific primers. The alaat phenotypes showed decreased growth and grain yield when compared with control plants. The activity of AlaAT1 in the mutant plants was practically undetectable. The responses of alaat plants to growth under salt stress were compared with those of control plants by measuring chlorophyll fluorescence and the activities and mRNA expression of antioxidant enzymes. All abiotic stresses tested (salt, drought, and chilling) caused a similar decrease in chlorophyll fluorescence in both alaat and wild type plants. The activity of peroxidase (POX), an antioxidant enzyme, decreased following salt treatment of alaat plants, while control plant showed an increased activity. The mRNA levels for cAPX (cytosolic ascorbate peroxidase), POX2, and AlaAT were determined by RT-PCR following salt stress. No AlaAT1 mRNA was detected in alaat plants. The POX2 mRNA showed a slightly increased level in the wild type but was not detected in alaat plants, in agreement with the activity assays. The levels of cAPX mRNA were greatly increased in both the wild type and alaat plants. The salt stress effects on rice plant growth are therefore proposed to reflect a loss of function of AlaAT, which alters the activity and synthesis of antioxidant enzymes (especially peroxidases), rather than a direct effect on photosynthesis.

Utilization of T-DNA tagging lines in rice

We have previously developed more than 100,000 T-DNA insertion mutant populations in japonica rice. These include simple knockouts as well as those for activation tagging. T-DNA insertion sites have been determined from more than 50,000 lines. The database for insertion positions is now open to the public, and these tagging lines are widely distributed to members of the rice research community. To utilize these genetic resources more efficiently, we are summarizing the important features of these tagging vectors, rice varieties, and flanking sequences. We also provide methods for handling such materials.

Effort and Contribution of T‐DNA Insertion Mutant Library for Rice Functional Genomics Research in China: Review and Perspective

With the completion of the rice (Oryza sativa L.) genome-sequencing project, the rice research community proposed to characterize the function of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.