Genetic Transformation Technology Research Articles

Advances in and Perspectives on Transgenic Technology and CRISPR-Cas9 Gene Editing in Broccoli

Broccoli, a popular international Brassica oleracea crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an Agrobacterium-mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.

Improving Sedum plumbizincicola genetic transformation with the SpGRF4-SpGIF1 gene and the self-excision CRE/LoxP system.

S. plumbizincicola genetic transformation was optimized using a self-excision molecular-assisted transformation system by integrating the SpGRF4/SpGIF1 gene with XVE and Cre/loxP. Sedum plumbizincicola, despite being an excellent hyperaccumulator of cadmium and zinc with significant potential for soil pollution phytoremediation on farmland, has nonetheless trailed behind other major model plants in genetic transformation technology. In this study, different explants and SpGRF4-SpGIF1 genes were used to optimize the genetic transformation of S. plumbizincicola. We found that petiole and stem segments had higher genetic transformation efficiency than cluster buds. Overexpression of SpGRF4-SpGIF1 could significantly improve the genetic transformation efficiency and shorten the period of obtaining regenerated buds. However, molecular assistance with overexpression of SpGRF4-SpGIF1 leads to abnormal morphology, resulting in plant tissue enlargement and abnormal growth. Therefore, we combined SpGRF4-SpGIF1 with XVE and Cre/loxP to obtain DNA autocleavage transgenic plants induced by estradiol, thereby ensuring normal growth in transgenic plants. This study optimized the S. plumbizincicola genetic transformation system, improved the efficiency of genetic transformation, and established a self-excision molecular-assisted transformation system. This work also established the basis for studying S. plumbizincicola gene function, and for S. plumbizincicola breeding and germplasm innovation.

Genetic Transformation of Forest Trees and Its Research Advances in Stress Tolerance

Forests represent a vital natural resource and play a crucial role in climate regulation and maintaining biodiversity. However, the growth and development of forest trees are increasingly challenged by rising environmental pressures, particularly detrimental abiotic stressors. To address these challenges, genetic transformation technologies have emerged as effective solutions. Despite various difficulties in genetic transformation for forest trees, including prolonged life cycles, genetic diversity, interspecies variations, and complex regeneration systems, significant research progress has been achieved in tree gene editing, transgenic technology, and methods for delivering exogenous molecules. These technologies have the potential to enhance tree quality, increase productivity, and improve resistance to abiotic stress. This review provides an overview of the main methods and transformation receptors in tree genetic transformation. Additionally, we summarize several novel techniques, such as nanoparticle-mediated gene transformation, advanced gene editing technology, various novel delivery carriers, and non-genetically modified protein function interference through peptide aptamer. Notably, we also place emphasis on several referable genes from forest trees and common crops, together with their potential function for improving abiotic stress responses. Through this research, we aspire to achieve sustainable utilization and conservation of tree resources, thereby providing substantial support for future livelihoods and economic development.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis.

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Optimization of High-Efficiency Tissue Culture Regeneration Systems in Gray Poplar.

A series of tissue culture regeneration protocols were conducted on gray poplar (P. tremula × P. alba) to select the most efficient callus induction medium, adventitious shoot differentiation medium, shoot elongation medium and rooting medium, which laid the foundation for the optimization of genetic transformation technology for gray poplar. The results showed that the Woody Plant Medium (WPM) supplemented with 0.10 mg L-1 kinetin (KT) and 1.00 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) was the most suitable medium for callus induction. The callus induction rates of different tissues were greater than 85.7%. The optimal adventitious shoot differentiation medium was the WPM supplemented with 0.02 mg L-1 thidiazuron (TDZ), and the adventitious shoot differentiation rates of young tissues were 22.2-41.9%. The optimal direct differentiation medium was the Murashige and Skoog (MS) medium supplemented with 0.20 mg L-1 6-benzylaminopurine (6-BA), 0.10 mg L-1 indole butyric acid (IBA) and 0.001 mg L-1 TDZ, and the differentiation rate of adventitious shoots was greater than 94%. The best shoot elongation medium for adventitious shoots was the MS medium with 0.10 mg L-1 naphthylacetic acid (NAA). After 45 days of cultivation in the MS medium with 0.10 mg L-1 NAA, the average plant height was 1.8 cm, and the average number of elongated adventitious shoots was 11 per explant. The 1/2 MS medium with 0.10 mg L-1 NAA showed the best performance for rooting, and later, shoot growth. The direct shoot induction pathway can induce adventitious shoots much faster than the indirect adventitious shoot induction pathway can, and the time cost via the direct adventitious shoot induction pathway can be shortened by 2-6 weeks compared to that of the indirect shoot induction pathway.

Technological Development and Application of Plant Genetic Transformation.

Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. Agrobacterium-mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (Bbm, Wus2, and GRF-GIF) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.

Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops

Cucurbits (Cucurbitaceae) include major horticultural crops with high nutritional and economic value that also serve as model plants for studying plant development and crop improvement. Conventional breeding methods have made important contributions to the production of cucurbit crops but have led to a breeding bottleneck because of the narrow genetic bases and low variation rates of these crops. With the development of molecular techniques, innovations in germplasm development through transgenesis and gene editing have led to breakthroughs in horticultural crop breeding. Although the development of genetic transformation and gene editing techniques for cucurbit crops has lagged behind that for other major crops, great progress has been made in recent years. Here, we summarize recent advances in improving the genetic transformation efficiency of cucurbit crops, including the screening of germplasm and the application of physical treatments, morphogenic genes, and selection markers. In addition, we review the application of gene editing technology to cucurbit crops, including CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9)-mediated gene knockout and base editing. This work provides a reference for improving genetic transformation efficiency and gene editing technology for cucurbit crops.

Acetyltransferase OsACE2 acts as a regulator to reduce the environmental risk of oxyfluorfen to rice production

The constant use of the pesticide oxyfluorfen (OFF) in farmland contaminates the soil, posing threats to crop growth and human health. To avoid the contamination of food crops with OFF, it is critically important to understand its absorption and degradation mechanisms. In this study, we characterized a new functional locus encoding an acetyltransferase (OsACE2) that can facilitate OFF degradation in rice. OsACE2 was drastically induced by OFF at 0.04–0.2 mg L−1 for 6 days and the rice growth was significantly inhibited. To demonstrate the regulatory role of OsACE2 in resistance to OFF toxicity, we generated OsACE2 overexpression (OE) and knockout mutant using genetic transformation and gene-editing technologies (CRISPR/Cas9). The OE plants grown in the hydroponic medium showed improved growth (plant elongation and biomass), increased chlorophyll content, and reduced OFF-induced oxidative stress. The OsACE2-improved growth phenotypes of rice were attributed to the significantly lower OFF accumulation in OE plants. Conversely, knocking out OsACE2 resulted in compromised growth phenotypes compared to the wild-type (WT). Using LC-LTQ-HRMS/MS, five mono-metabolites and eleven conjugates of OFF were characterized through various canonical pathways, such as hydrolysis, oxidation, reduction, glycosylation, acetylation, malonylation, and interaction with amino acids. These metabolites increased in the OE plants, and five acetylated conjugates were reported for the first time. Collectively, OsACE2 plays a primary role in catabolizing OFF residues in rice through multiple degradation pathways and reducing OFF in its growth environment.

Highly efficient hairy root genetic transformation and applications in citrus.

Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2-8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining "hard-to-get" transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

Recent Progress in the Regeneration and Genetic Transformation System of Cucumber

Cucumber (Cucumis sativus L.), belonging to the gourd family (Cucurbitaceae), is one of the major vegetable crops in China. Conventional genetic breeding methods are ineffective for improving the tolerance of cucumber to various environmental stresses, diseases, and pests in the short term, but bio-engineering technologies can be applied to cucumber breeding to produce new cultivars with high yield and quality. Regeneration and genetic transformation systems are key technologies in modern cucumber breeding. Compared with regeneration systems, genetic transformation systems are not yet fully effective, and the low efficiency of genetic transformation is a bottleneck in cucumber cultivation. Here, we systematically review the key factors influencing the regeneration and genetic transformation of cucumber plants, including the selection of genotype, source of explants and forms of exogenous hormones added to the medium, the methods of transgene introduction and co-cultivation, and selection methods. In addition, we also focus on recent advances in the study of molecular mechanisms underlying important agronomic traits using genetic transformation technology, such as fruit length, fruit warts, and floral development. This review provides reference information for future research on improvements in cucumber varieties.

Expression of CYP76C6 Facilitates Isoproturon Metabolism and Detoxification in Rice

Agricultural chemical residues in farmland and crops is one of the serious public issues that constantly threatens crop production, food security, and human health. Understanding their decay mechanism in crops for accelerating their degradative metabolism is important. In this study, a rice uncharacterized cytochrome P450 gene encoding CYP76C6 was functionally identified in rice exposed to isoproturon (IPU). To verify the role of CYP76C6 in rice resistance to IPU toxicity, CYP76C6 overexpression (OEs) and knockout mutant rice by CRISPR/Cas9 were generated through genetic transformation and gene-editing technologies. Assessment of growth and physiological responses revealed that the growth of OE lines was improved, the IPU-induced cellular damage was attenuated, and IPU accumulation was significantly repressed, whereas the Cas9 lines displayed a contrasting phenotype compared to the wild-type. Both relative contents of IPU metabolites and conjugates in OE lines were reduced and those in Cas9 line were increased, suggesting that CYP76C6 plays a critical role in IPU degradation. Our study unveils a new regulator, together with its mechanism for IPU decay in rice crops, which will be used in reality to reduce environmental risks in food safety and human health.

The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation.

Although great progress has been achieved regarding wheat genetic transformation technology in the past decade1-3, genotype dependency, the most impactful factor in wheat genetic transformation, currently limits the capacity for wheat improvement by transgenic integration and genome-editing approaches. The application of regeneration-related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency4-11. In the present study, we found that overexpression of the wheat gene TaWOX5 from the WUSCHEL family dramatically increases transformation efficiency with less genotype dependency than other methods. The expression of TaWOX5 in wheat calli prohibited neither shoot differentiation nor root development. Moreover, successfully transformed transgenic wheat plants can clearly be recognized based on a visible botanic phenotype, relatively wider flag leaves. Application of TaWOX5 improved wheat immature embryo transformation and regeneration. The use of TaWOX5 in improvement of transformation efficiency also showed promising results in Triticum monococcum, triticale, rye, barley and maize.

Functional Verification of Two Genes Related to Stripe Rust Resistance in the Wheat-Leymus mollis Introgression Line M8664-3.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most widespread and destructive fungal diseases of wheat worldwide. The cultivation and growth of resistant wheat varieties are the most economical, effective, and environmental friendly methods to control stripe rust. Therefore, it is necessary to use new resistance genes to breed resistant wheat varieties. A single dominant gene temporarily designated as YrM8664-3, from a wheat-Leymus mollis introgression line M8664-3 highly resistant to Chinese predominant Pst races, is a potentially valuable source of stripe rust resistance for breeding. Herein, based on previous YrM8664-3 chromosome location results (bin 4AL13-0.59-0.66 close to 4AL12-0.43-0.59) and expression change information of candidate genes and bioinformatics analysis, several candidate genes with significantly different expression changes were then selected and verified by virus-induced gene silencing (VIGS). Two of the candidate genes temporarily designated as TaFBN [containing plastid lipid-associated proteins (PAP)_fibrillin domain in its protein] and Ta_Pes_BRCT [containing Pescadillo and breast cancer tumour suppressor protein C-terminus (BRCT) domain in its protein], produced the most significant resistance changes in the wheat-Pst interaction system after silencing. These two genes were further verified by Agrobacterium-mediated wheat genetic transformation technology. According to the identification of disease resistance, the resistance function of the candidate gene TaFBN was further verified. Then, the expression of TaFBN under hormone treatment indicated that TaFBN may be related to the salicylic acid (SA) and abscisic acid (ABA) signaling pathways. Combined with the expression of TaFBN in response to environmental stress stimulation, it can be reasonably speculated that TaFBN plays an important role in the resistance of wheat to Pst and is involved in abiotic stress pathways.

Nanocarrier-Mediated Delivery of miRNA, RNAi, and CRISPR-Cas for Plant Protection: Current Trends and Future Directions

Current trends in plant genetic transformation technologies, i.e., designing and applying molecules like miRNA, RNAi, and CRISPR-Cas, largely enable researchers to target specific sites in the plant genome to avert the growing biotic and abiotic threats to plants. However, the delivery of these molecules through conventional techniques brings an array of drawbacks such as low efficiency due to the cell wall barrier, tissue damage that leads to browning or necrosis, degradation of these biomolecules by physiological conditions (high temperature, harsh pH, and light), and plant-specific protocols. The advancements in nanotechnology offer an excellent alternative for the safe and highly efficient delivery of biomolecules such as miRNA, CRISPR-Cas, and RNAi without damaging the plant tissues. Nanoparticle (polymeric, metallic, magnetic, silica, carbon, etc.)-based delivery of biomolecules can be efficiently utilized especially for plant protection applications. Herein, we present a comprehensive overview of current trends (with a focus on the previous five years) in nanoparticle-based delivery of miRNA, RNAi, CRISPR-Cas and simillar biomolecules for plant protection applications. In addition, a future perspective focuses on the research gaps and unexplored potentials of nanoparticles for the delivery of biomolecules.

The use of biotechnology in hop (Humulus lupulus L.) improvement

Hop (Humulus lupulus L.) is a clonally propagated, dioecious, perennial, climbing plant used commercially for their secondary metabolites. The resins containing α- and β-acids, and essential oils produced by the lupulin glands, present on the female flowers are used to add bitterness, aroma and flavour to beer. Recently, flavonoids, including chalcones and flavanones, of hops have been shown to exert a variety of biological activities, including oestrogenic and anticancerogenic characteristics. In this review, we provide a overview of the techniques and opportunities presented by the integration of plant biotechnology into hop improvement. The use of tissue culture techniques such as micropropagation, meristem culture, in vitro storage, adventitious shoot induction, callus culture and cell suspension culture in hops are briefly reviewed. The usefullness of genetic transformation technology to introduce novel traits into hop is also discussed.

Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L. pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

The miR156/SPL module regulates apple salt stress tolerance by activating MdWRKY100 expression.

SummarySalt stress dramatically impedes plant growth and development as well as crop yield. The apple production regions are reduced every year, because of the secondary salt damage by improper fertilization and irrigation. To expand the cultivation area of apple (Malus domestica) and select salt‐resistant varieties, the mechanism of salt tolerance in apple is necessary to be clarified. The miR156/SPL regulatory module plays key roles in embryogenesis, morphogenesis, life cycle stage transformation, flower formation and other processes. However, its roles in the mechanisms of salt tolerance are unknown. In order to elucidate the mechanism of 156/SPL regulating salt stress in apple, we performed RLM‐5’ RACE and stable genetic transformation technology to verify that both mdm‐MIR156a and MdSPL13 responded to salt stress in apple and that the latter was the target of the former. MIR156a overexpression weakened salt resistance in apple whereas MdSPL13 overexpression strengthened it. A total of 6094 differentially expressed genes relative to nontransgenic apple plants were found by RNA‐Seq analysis of MdSPL13OE. Further verification indicated that MdSPL13 targeted the MdWRKY100 gene promoter. Moreover, MdWRKY100 overexpression enhanced salt tolerance in apple. Our results revealed that the miR156/SPL module regulates salt tolerance by up‐regulating MdWRKY100 in apple. This study is the first to elucidate the mechanism underlying the miRNA network response to salt stress in apple and provides theoretical and empirical bases and genetic resources for the molecular breeding of salt tolerance in apple.

Utilization of sucrose during cocultivation positively affects Agrobacterium-mediated transformation efficiency in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is one of the most important industrial crops throughout world. With the availability of suitable genetic transformation technologies, the yield, quality, and stress tolerance of sugar beet could be improved significantly. However, low transformation efficiencies seriously limit the application of molecular technologies to the genetic improvement of sugar beet. With the aim of improving gene transfer techniques for sugar beet, the effect of different sucrose concentrations during cocultivation on the initial Agrobacterium-mediated transformation efficiencies in sugar beet was tested. To develop an efficient experimental system through which the effect of sucrose could be tested, first, a prolific regeneration system was optimized by testing the effect of different plant growth regulators on in vitro regeneration and rooting efficiencies from sugar beet cotyledonary node explants. The highest mean number of regenerated shoots per explant was obtained when the cotyledonary node explants excised from young seedlings were grown on MS medium supplemented with 1.0 mg/L 6-benzylaminopurine. Using this regeneration system, the effect of different concentrations of sucrose included in the cocultivation medium on the initial genetic transformation efficiencies observed in T0 plants was tested using an Agrobacterium tumefaciens strain carrying the pBin19/35S:GUS-INT construct. The inclusion of 4.5% sucrose in the cocultivation medium resulted in significantly higher transformation (34.09%) and expression efficiencies (22.72%), confirmed by polymerase chain reaction and β-glucuronidase assays, respectively, in regenerated T0 seedlings. If translated into stably inherited transformation efficiencies, these findings could contribute to the success of genetic transformation studies in sugar beet and other crops recalcitrant to Agrobacterium-mediated transformation.

Recent developments and applications of genetic transformation and genome editing technologies in wheat.

Wheat (Triticum aestivum) is a staple crop across the world and plays a remarkable role in food supplying security. Over the past few decades, basic and applied research on wheat has lagged behind other cereal crops due to the complex and polyploid genome and difficulties in genetic transformation. A breakthrough called as PureWheat was made in the genetic transformation of wheat in 2014 in Asia, leading to a noticeable progress of wheat genome editing. Due to this great achievement, it is predicated that wheat biotechnology revolution is arriving. Genome editing technologies using zinc finger nucleases, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats-associated endonucleases (CRISR/Cas) are becoming powerful tools for crop modification which can help biologists and biotechnologists better understand the processes of mutagenesis and genomic alteration. Among the three genome editing systems, CRISR/Cas has high specificity and activity, and therefore it is widely used in genetic engineering. Generally, the genome editing technologies depend on an efficient genetic transformationsystem. In this paper, we summarize recent progresses and applications on genetic transformation and genome editing in wheat. We also examine the future aspects of genetic transformation and genome editing. We believe that the technologies for wheat efficient genetic engineering and functional studies will become routine with the emergence of high-quality genomic sequences.

Molecular tagging of biocontrol fungus Trichoderma asperellum and its colonization in soil.

To conduct molecular tagging of the biocontrol fungus Trichoderma asperellum strain T4 and elucidate its colonization patterns in soil. We constructed an expression vector harbouring a hygromycin B-resistant gene (hph) and an efficient green fluorescent protein (egfp) gene. By applying Agrobacterium AGL-1-mediated genetic transformation technology, we conducted molecular tagging of T. asperellum and monitored the colonization dynamics of T. asperellum in soil. The results of tracking five independent transformants of T. asperellum indicated that its expansion rates ranged from 4·7 to 6·8cmweek-1 . After inoculation in soil, the quantities of T. asperellum could be maintained at over 10×104 CFU per gram soil in the first year. In the third year after inoculation, the quantities of T. asperellum in soil were still higher than 1×103 CFU per gram soil. In addition, molecularly tagged T. asperellum in soil in the second year (i.e. 12months) after inoculation could still reach the biocontrol effect on cucumber Rhizoctonia rot by more than 74%. Trichoderma asperellum strain T4 is capable of effectively colonizing in soil and surviving for more than 1year. This study has provided the scientific basis for applying T. asperellum as the biocontrol fungus for prevention and control of plant diseases.