Litchi has great economic significance as a global fruit crop.However,the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation.Here,we prese...Litchi has great economic significance as a global fruit crop.However,the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation.Here,we present an efficacious Agrobacterium tumefaciens-mediated transformation system in somatic embryos of‘Heiye'litchi.This system was developed through the optimization of key variables encompassing explant selection,A.tumefaciens strain delineation,bacterium concentration,infection duration,and infection methodology.The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1,resulting in the generation of transgenic calli.However,the differentiation of transgenic calli into somatic embryos encountered substantial challenges.To delineate the intricate molecular underpinnings of LcMYB1's inhibitory role in somatic embryo induction,a comprehensive transcriptome analysis was conducted that encompassed embryogenic calli(C),globular embryos(G),and transgenic calli(TC).A total of 1,166 common differentially expressed genes(DEGs)were identified between C-vs.-G and C-vs.-TC.Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that these common DEGs were mostly related to plant hormone signal transduction pathways.Furthermore,RT-qPCR corroborated the pronounced down-regulation of numerous genes that are associated with somatic embryo induction within the transgenic calli.The development of this transformation system provides valuable support for functional genomics research in litchi.展开更多
Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional ...Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.展开更多
[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was...[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was transformed by G418 resistant DNA plasmid using PEG/CaCl2 method. [Result] The growth phenotype and PCR detection showed that resistance gene had integrated into A.tenuissima genome. The transformation efficiency of this method reached per μg DNA 3-4 transformants. After subculture thrice under nonselective condition, G418 resistance could still inherit stably. [Conclusion] The transformation system of A.tenuissima was successfully established, which laid basis for studying of the gene function of Alternaria tenuissima.展开更多
The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this ...The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.展开更多
Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are...Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium(MS basal salts, 30 g Lsucrose, 8 g Lagar, 5 mg L6-benzylaminopurine(6-BA), 2 mg Lthidiazuron(TDZ), and 1 mg L1-naphthlcetic acid(NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime(150 mg L) and kanamycin(15 mg L). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.展开更多
[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-...[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.展开更多
[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants...[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.展开更多
[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used...[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used as the explant,and the Agrobacterium mediation method was used to optimize its genetic transformation efficiency so as to establish the efficient Agrobacterium mediated genetic transformation system of tomato cotyledon.[Result] The highest transformation efficiency was obtained when the explants were cultivated for 2 d on MS ＋ 2.0 mg/L 6-BA＋ 0.5 mg/L IAA medium and then infected with Agrobacterium EHA105（OD = 0.4）for 5 min;it was proved by PCR analysis that the target nptII gene had been integrated into the genome of regenerated plants.[Conclusion] The result in this study had provided basis for the transfer of valuable genes into tomato Meifen No.1.展开更多
The development and wide application of genetic transformation for cotton improvement are restrained by the unresolved problem of genotype dependence in regeneration in vitro.High embryogenic and regenerative potentia...The development and wide application of genetic transformation for cotton improvement are restrained by the unresolved problem of genotype dependence in regeneration in vitro.High embryogenic and regenerative potential have been obtained for limited number of Coker type genotypes。展开更多
Accelerating the transformation of agricultural scientific and technological achievements is a key link in implementing innovation-driven development strategy and rural revitalization strategy,and improving developmen...Accelerating the transformation of agricultural scientific and technological achievements is a key link in implementing innovation-driven development strategy and rural revitalization strategy,and improving development quality and core competitiveness.How to build a scientific and systematic transformation system of scientific and technological achievements and improve the overall management level of scientific and technological achievements transformation of agricultural scientific research institutes is one of the key tasks to measure how a scientific research institute supports industry and serves society.Taking the Chinese Academy of Tropical Agricultural Sciences as an example,this paper explores the construction and practice of its scientific and technological achievements transformation system since the 13 th Five-Year Plan period.By arranging the current situation of resource elements for the transformation of scientific and technological achievements,analyzing the progress of the construction of the scientific and technological achievements transformation system,summarizing the practical results of the scientific and technological achievements transformation system,this paper puts forward 10 strategies and measures(implementing key projects for the transformation and application of scientific and technological achievements;striving to promote the transformation and application of on-duty scientific and technological achievements;accelerating the development and utilization of advantageous and characteristic resources;strengthening the use and protection of intellectual property rights;actively expanding cooperation activities between government,industry and research;increasing special financial support for the transformation of scientific and technological achievements;innovating state-owned asset management to accelerate scientific and technological development;piloting equity incentives to expand scientific and technological development channels to increase income;striving to create a relaxed environment for the transformation of scientific and technological achievements,effectively create scientific and technological value,enhance the development strength of the institute,and promote high-quality industrial development)in order to provide a useful reference for the transformation of scientific and technological achievements of agricultural research institutes.展开更多
Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is genera...Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.展开更多
Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested ...Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.展开更多
To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transf...To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.展开更多
Tremellafuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy ...Tremellafuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 （containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B） and plasmid pLg-hph （containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph） were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL^-1 hygromycin. The putative transformants were obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL^-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub- culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.展开更多
Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the ...Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.展开更多
Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation ...Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.展开更多
The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon,and a fusion gene expression vector of insulin combined with green fluorescent protein(GFP)was constructed.The op...The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon,and a fusion gene expression vector of insulin combined with green fluorescent protein(GFP)was constructed.The optimization of the flax callus culturing was undertaken,and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved.The critical concentration values of hygromycin on the flax hypocotyl development,as well as on its differentiated callus,were explored by the method of antibiotic gradient addition,and the application of antibiotic screening for the verification of positive calluses was assessed.The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed,as confirmed through polymerase chain reaction and Western blotting.In conclusion,we have established a flax callus culture system suitable for insulin expression.By optimizing the conditions of the flax callus induction,transformation,screening,and verification of a transgenic callus,we have provided an effective way to obtain insulin.Moreover,the herein-employed flax callus culture system could provide a feasible,cheap,and environmentally friendly platform for producing bioactive proteins.展开更多
Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to e...Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum,hexaploid triticale,and rye(Secale cereale L.)using their immature embryos.Transgenic plants were efficiently generated.During the transformation process,the Agrobacterium infection efficiency was assessed by histochemical staining forβ-glucuronidase(GUS).Finally,the transgenic nature of regenerated plants was verified by polymerase chain reaction(PCR)-based genotyping for the presence of the GUS and bialaphos resistance(bar)genes,histochemical staining for GUS protein,and the QuickStix strip assay for bar protein.The transformation efficiency of T.monococcum genotype PI428182 was 94.4%;the efficiencies of four hexaploid triticale genotypes Lin456,ZS3297,ZS1257,and ZS3224 were 52.1,41.2,19.4,and 16.0%,respectively;and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.Fluorescence in situ hybridization(FISH)and genomic in situ hybridization(GISH)analyses indicated that the GUS transgenes were integrated into the distal or near centromere(proximal)regions of the chromosomes in transgenic T.monococcum and hexaploid triticale plants.In the transgenic hexaploid triticale plants,the foreign DNA fragment was randomly integrated into the AABB and RR genomes.Furthermore,the transgene was almost stably inherited in the next generation by Mendel’s law.The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.展开更多
Graph transformation systems have become a general formal modeling language to describe many models in software development process.Behavioral modeling of dynamic systems and model-to-model transformations are only a ...Graph transformation systems have become a general formal modeling language to describe many models in software development process.Behavioral modeling of dynamic systems and model-to-model transformations are only a few examples in which graphs have been used to software development.But even the perfect graph transformation system must be equipped with automated analysis capabilities to let users understand whether such a formal specification fulfills their requirements.In this paper,we present a new solution to verify graph transformation systems using the Bogor model checker.The attributed graph grammars(AGG)-like graph transformation systems are translated to Bandera intermediate representation(BIR),the input language of Bogor,and Bogor verifies the model against some interesting properties defined by combining linear temporal logic(LTL) and special-purpose graph rules.Experimental results are encouraging,showing that in most cases our solution improves existing approaches in terms of both performance and expressiveness.展开更多
Cotton large-scale transformation methods system was established based on innovation of cotton transformation methods.It obtains 8000 transgenic cotton plants per year by combining Agrobacterium tumefaciens-mediated,p...Cotton large-scale transformation methods system was established based on innovation of cotton transformation methods.It obtains 8000 transgenic cotton plants per year by combining Agrobacterium tumefaciens-mediated,pollen-tube pathway and biolistic methods together efficiently.More展开更多
基金supported by the National Natural Science Foundation of China(31872066 and 32272663)the Science and Technology Planning Project of Guangzhou,China(2023B01J2002)+2 种基金the Key Research and Development Program of Hainan,China(ZDYF2023XDNY052)the Seed Industry Engineering Project of Department of Agriculture and Rural Affairs of Guangdong,China(2022-NPY-00-004 and 2022-NBH00-001)the Litchi Industry Science and Technology Special Mission of Yunnan,China(202204BI090021)。
文摘Litchi has great economic significance as a global fruit crop.However,the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation.Here,we present an efficacious Agrobacterium tumefaciens-mediated transformation system in somatic embryos of‘Heiye'litchi.This system was developed through the optimization of key variables encompassing explant selection,A.tumefaciens strain delineation,bacterium concentration,infection duration,and infection methodology.The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1,resulting in the generation of transgenic calli.However,the differentiation of transgenic calli into somatic embryos encountered substantial challenges.To delineate the intricate molecular underpinnings of LcMYB1's inhibitory role in somatic embryo induction,a comprehensive transcriptome analysis was conducted that encompassed embryogenic calli(C),globular embryos(G),and transgenic calli(TC).A total of 1,166 common differentially expressed genes(DEGs)were identified between C-vs.-G and C-vs.-TC.Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that these common DEGs were mostly related to plant hormone signal transduction pathways.Furthermore,RT-qPCR corroborated the pronounced down-regulation of numerous genes that are associated with somatic embryo induction within the transgenic calli.The development of this transformation system provides valuable support for functional genomics research in litchi.
基金supported by Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (NO.2023ZD04041)the Project of China Agriculture Research System (Grant No. CARS-15-06)+2 种基金Natural Science Foundation of Henan Province (Grant No. 232300421041 and 222300420382)National Natural Science Foundation of China (Grant No. U21 A20213)the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162023017 and 1610162023028)。
文摘Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.
文摘[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was transformed by G418 resistant DNA plasmid using PEG/CaCl2 method. [Result] The growth phenotype and PCR detection showed that resistance gene had integrated into A.tenuissima genome. The transformation efficiency of this method reached per μg DNA 3-4 transformants. After subculture thrice under nonselective condition, G418 resistance could still inherit stably. [Conclusion] The transformation system of A.tenuissima was successfully established, which laid basis for studying of the gene function of Alternaria tenuissima.
基金supported by the grants from the National Natural Science Foundation of China(Grant Nos.30621005 and 31030003)the Ministry of Science and Technology of China(Grant No.2009CB118905)
文摘The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.
基金supported by the National Key Research and Development Program of China(2019YFD1001403-4)the Key S&T Special Projects of Shaanxi Province,China(2020zdzx03-01-02)the earmarked fund for the China Agriculture Research System of MOF and MARA(CARS-27)。
文摘Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium(MS basal salts, 30 g Lsucrose, 8 g Lagar, 5 mg L6-benzylaminopurine(6-BA), 2 mg Lthidiazuron(TDZ), and 1 mg L1-naphthlcetic acid(NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime(150 mg L) and kanamycin(15 mg L). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.
文摘[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.
文摘[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.
基金Supported by Open Subjects in State Key Laboratory of Plant Physiology and Biochemistry(SKLPPBKF09011)~~
文摘[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used as the explant,and the Agrobacterium mediation method was used to optimize its genetic transformation efficiency so as to establish the efficient Agrobacterium mediated genetic transformation system of tomato cotyledon.[Result] The highest transformation efficiency was obtained when the explants were cultivated for 2 d on MS ＋ 2.0 mg/L 6-BA＋ 0.5 mg/L IAA medium and then infected with Agrobacterium EHA105（OD = 0.4）for 5 min;it was proved by PCR analysis that the target nptII gene had been integrated into the genome of regenerated plants.[Conclusion] The result in this study had provided basis for the transfer of valuable genes into tomato Meifen No.1.
文摘The development and wide application of genetic transformation for cotton improvement are restrained by the unresolved problem of genotype dependence in regeneration in vitro.High embryogenic and regenerative potential have been obtained for limited number of Coker type genotypes。
基金Supported by Natural Science Foundation of Hainan Province(721QN0938).
文摘Accelerating the transformation of agricultural scientific and technological achievements is a key link in implementing innovation-driven development strategy and rural revitalization strategy,and improving development quality and core competitiveness.How to build a scientific and systematic transformation system of scientific and technological achievements and improve the overall management level of scientific and technological achievements transformation of agricultural scientific research institutes is one of the key tasks to measure how a scientific research institute supports industry and serves society.Taking the Chinese Academy of Tropical Agricultural Sciences as an example,this paper explores the construction and practice of its scientific and technological achievements transformation system since the 13 th Five-Year Plan period.By arranging the current situation of resource elements for the transformation of scientific and technological achievements,analyzing the progress of the construction of the scientific and technological achievements transformation system,summarizing the practical results of the scientific and technological achievements transformation system,this paper puts forward 10 strategies and measures(implementing key projects for the transformation and application of scientific and technological achievements;striving to promote the transformation and application of on-duty scientific and technological achievements;accelerating the development and utilization of advantageous and characteristic resources;strengthening the use and protection of intellectual property rights;actively expanding cooperation activities between government,industry and research;increasing special financial support for the transformation of scientific and technological achievements;innovating state-owned asset management to accelerate scientific and technological development;piloting equity incentives to expand scientific and technological development channels to increase income;striving to create a relaxed environment for the transformation of scientific and technological achievements,effectively create scientific and technological value,enhance the development strength of the institute,and promote high-quality industrial development)in order to provide a useful reference for the transformation of scientific and technological achievements of agricultural research institutes.
基金the National key research and Development Program(Grant No.2017YFD0101802)the National Natural Science Foundation of China(Grant Nos.31772326 and 31701930)China Postdoctoral Science Foundation(Grant Nos.2016M601345 and 2019T120219).
文摘Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.
基金supported by the National Key Project of transgenic varieties breeding(2009ZX08003-003B)the Light of West Talent Training Project of China(2010-2011)the Project of Sichuan Province Finance Genetic Engineering,China(2011JYGC01-002)
文摘Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.
基金supported by a ‘Special Fund for Agro-scientific Research in the Public Interest’ from the Ministry of Agriculture of China(Grant No.nyhyzx3-16)
文摘To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.
基金funded by the National Hi-Tech Research and Development Program of China (863 Program,2006AA10Z301)the National Natural Science Foundation of China (30371000, 30671457)
文摘Tremellafuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 （containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B） and plasmid pLg-hph （containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph） were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL^-1 hygromycin. The putative transformants were obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL^-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub- culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.
基金Foundation project： This paper was supported by Zhejiang Provincial Science and Technology Plan of China （Grant No. 2003C30053） and Zhejiang Provincial Natural Science Foundation of China （Grant No.Y504076）.
文摘Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.
基金funded by National Natural Science Foundation of China (Grant No.32072575)Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant No.KYCX20_0588)National Vegetable Industry Technology System (Grant No.CARS-23-A16)。
文摘Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.
基金funded by Hebei Development and Reform Commission of China,Hebei Engineering Laboratory,grant number(2021)1157 and the Innovation Fund Project of Hebei University of Engineering(114/SJ2401002149).
文摘The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon,and a fusion gene expression vector of insulin combined with green fluorescent protein(GFP)was constructed.The optimization of the flax callus culturing was undertaken,and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved.The critical concentration values of hygromycin on the flax hypocotyl development,as well as on its differentiated callus,were explored by the method of antibiotic gradient addition,and the application of antibiotic screening for the verification of positive calluses was assessed.The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed,as confirmed through polymerase chain reaction and Western blotting.In conclusion,we have established a flax callus culture system suitable for insulin expression.By optimizing the conditions of the flax callus induction,transformation,screening,and verification of a transgenic callus,we have provided an effective way to obtain insulin.Moreover,the herein-employed flax callus culture system could provide a feasible,cheap,and environmentally friendly platform for producing bioactive proteins.
基金supported by grants from the National Natural Science Foundation of China (31971946)the Technology Innovation Program of the Chinese Academy of Agricultural Sciences, China (2060302-2-23 and ASTIP-2060302-2-19).
文摘Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum,hexaploid triticale,and rye(Secale cereale L.)using their immature embryos.Transgenic plants were efficiently generated.During the transformation process,the Agrobacterium infection efficiency was assessed by histochemical staining forβ-glucuronidase(GUS).Finally,the transgenic nature of regenerated plants was verified by polymerase chain reaction(PCR)-based genotyping for the presence of the GUS and bialaphos resistance(bar)genes,histochemical staining for GUS protein,and the QuickStix strip assay for bar protein.The transformation efficiency of T.monococcum genotype PI428182 was 94.4%;the efficiencies of four hexaploid triticale genotypes Lin456,ZS3297,ZS1257,and ZS3224 were 52.1,41.2,19.4,and 16.0%,respectively;and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.Fluorescence in situ hybridization(FISH)and genomic in situ hybridization(GISH)analyses indicated that the GUS transgenes were integrated into the distal or near centromere(proximal)regions of the chromosomes in transgenic T.monococcum and hexaploid triticale plants.In the transgenic hexaploid triticale plants,the foreign DNA fragment was randomly integrated into the AABB and RR genomes.Furthermore,the transgene was almost stably inherited in the next generation by Mendel’s law.The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.
文摘Graph transformation systems have become a general formal modeling language to describe many models in software development process.Behavioral modeling of dynamic systems and model-to-model transformations are only a few examples in which graphs have been used to software development.But even the perfect graph transformation system must be equipped with automated analysis capabilities to let users understand whether such a formal specification fulfills their requirements.In this paper,we present a new solution to verify graph transformation systems using the Bogor model checker.The attributed graph grammars(AGG)-like graph transformation systems are translated to Bandera intermediate representation(BIR),the input language of Bogor,and Bogor verifies the model against some interesting properties defined by combining linear temporal logic(LTL) and special-purpose graph rules.Experimental results are encouraging,showing that in most cases our solution improves existing approaches in terms of both performance and expressiveness.
文摘Cotton large-scale transformation methods system was established based on innovation of cotton transformation methods.It obtains 8000 transgenic cotton plants per year by combining Agrobacterium tumefaciens-mediated,pollen-tube pathway and biolistic methods together efficiently.More
