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
A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tiss...A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly pine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
Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-st...Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.展开更多
A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this ar...A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics (including ampicillin, carbenicillin, cefotaxime, and cefoperazone), meropenem showed the highest activity in suppressing all tested A. tumefaciens strains (minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]). Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.展开更多
The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N+ implantation at the dosage of 7.8 ×1016 ions/cm2. After treatment, watermelon seeds were incubated with 380μg/μl pumpkin (Cucubita, ma...The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N+ implantation at the dosage of 7.8 ×1016 ions/cm2. After treatment, watermelon seeds were incubated with 380μg/μl pumpkin (Cucubita, maxima Duch) DNA solution at 35 ℃ for 5 hours. By two-generations of selection and resistance screening at seedling stage, one transformed material was selected out, whose rind color is similar to that of the donor pumpkin and whose size of seeds is between that of the donor and the receptor. Using AFLP (amplified fragment length polymorphism) technique, two polymorphic DNA fragments were amplified. This primarily testified that the donor DNA fragments/gene were introduced into the receptor cell and integrated into the genomic DNA of the receptor.展开更多
The application of keV ion beam in life science started in China several decades ago. In 1986, researchers initially studied the mutagenic effect of ion beam, and successfully applied it to plant breeding. Nowadays, i...The application of keV ion beam in life science started in China several decades ago. In 1986, researchers initially studied the mutagenic effect of ion beam, and successfully applied it to plant breeding. Nowadays, ion beam implantation technique has been extensively applied to many biological fields. This paper mainly introduces one of its important applications: genetic transformation mediated by keV ion beam.展开更多
Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression f...Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.展开更多
The plants of hybrid wheatgrass (A. cristatum×A. desertorum cv. Hycrest-Mengnong) were directly induced from embryogenic callus regenerated from immature inflorescence. Immature inflorescence was cultured on im...The plants of hybrid wheatgrass (A. cristatum×A. desertorum cv. Hycrest-Mengnong) were directly induced from embryogenic callus regenerated from immature inflorescence. Immature inflorescence was cultured on improved MS medium containing 2.0-3.0 mg L^-1 2,4-D to regenerate callus. The calli were then transferred to hormone-free MS medium for differentiation and 1/2 MS medium for rooting. Results showed that callus initiation frequency was 83.4% and plant regeneration frequency was 59.6%. Phosphinothricin acetyltransferase (bar) gene was transformed into the hybrid wheatgrass by particle bombardment. Resistant callus was obtained using selecting agent, herbicide glufosinate of 0.5 mg L^-1, and some transgenic plants were recovered in vitro. The transgenic plants were identified by PCR and Southern blot analysis and these plants developed normally in the glufosinate medium, whereas the nontransgenic plants did not. The results demonstrated that bar cDNA integrated into the genomic DNA of the transgenic plants. The transgenic frequencies of bar gene were 1.1%.展开更多
Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major...Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.展开更多
Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural ...Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands.Molecular genetic techniques in particular,associated with rapid technological advancements,provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance,quality,stress tolerance and disease resistance.To date,several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation,and transgenic grape lines have been obtained using established regeneration systems.Nevertheless,a wide range of factors,including genotype,explant source and culture medium,have been shown to affect the efficiency of plant regeneration.Moreover,the selection and use of acceptor materials,bacterial strain and cell density,selectable markers and selection methods also influence transformation efficiency.This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors,and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.展开更多
The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression ofAgrobacterium tumefaciens in Agrobacterium-mediated gene...The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression ofAgrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L^-1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L^-1; however, 300 mg·L^-1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% (control) to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L^-1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L^-1 Timentin. Considering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300 mg·L^-1 is the preferred application in .4. tumefaciens-mediated transformation ofP acerifolia.展开更多
In this study, DERB1A transcription factor and stress-induced promoter rd29A were isolated respectively and amplified from Arabidopsis thaliana, se- quenced and analyzed by DNAsis. In addition, the stress-induced prom...In this study, DERB1A transcription factor and stress-induced promoter rd29A were isolated respectively and amplified from Arabidopsis thaliana, se- quenced and analyzed by DNAsis. In addition, the stress-induced promoter rd29A was utilized to construct the plant expression vector of DERB1A, which was transformed into Agrobacterium tumefaciens. Furthermore, the transgenic regeneration system of fresh-cut chrysanthemum from callus to plantlets was established successfully. On this basis, chrysanthemum leaf-disc explants were genetically transformed with Agrobacterium-mediated method. Two positive transgenie plantlets were obtained in vitro. Based on PCR detection, DREB1A transcription factor was integrated into chrysanthemum genome, which laid the foundation for breeding new transgenie cultivars of fresh-cut chrysanthemum with high comprehensive stress resistance, good cmalitv and high field.展开更多
Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid(2x) to dodecaploid(12x) with tetraploid and octoploid being the most common. The h...Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid(2x) to dodecaploid(12x) with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow well in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocellulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We first reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efficiency. Finally we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.展开更多
Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(su...Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(such as hybridization)have limited ability to nurture new litchi cultivars.Here,an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in‘Feizixiao’litchi.Transgenic materials were verified using polymerase chain reaction(PCR)analysis,β-glucuronidase(GUS)assay,and green fluorescent protein(GFP)assay.To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/associated protein 9(CRISPR/Cas9)technology in‘Feizixiao’litchi and verify the validity of these transformation systems,the litchi polyphenol oxidase gene(LcPPO,JF926153)was knocked out.Various categories of mutations,covering base insertions,deletions,and substitutions,were found in transgenic materials via sequence analysis.The transformation system achieved high feasibility and efficiency,and the system of CRISPR/Cas9 was successfully employed to edit genes in‘Feizixiao’litchi.This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.展开更多
A novel rice gene OsAPT2,which encodes a putative adenine phosphoribosyl transferase(APRT),was cloned.Its full-length cDNA is 1125bp,composing an ORF encoding 212 amino acid residues and a stop cordon,a 5' UTR of ...A novel rice gene OsAPT2,which encodes a putative adenine phosphoribosyl transferase(APRT),was cloned.Its full-length cDNA is 1125bp,composing an ORF encoding 212 amino acid residues and a stop cordon,a 5' UTR of 123 bp and a 3' UTR of 363 bp.The sequence data have been submitted to the DDBJ/EMBL/GenBank databases(accession number:AY238894).The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously reported APRTs.The genomic OsAPT2 gene contains 7 exons and 6 introns.Its total length is 4758 bp.Then,an antisense expression vector of the full-length OsAPT2 cDNA was constructed and transformed into rice variety Taibei309 by Agrobacterium tumefaciens mediated transformation method.In total,650 T0 transgenic plants were obtained based on both antibiotic screening and specific PCR identification.One hundred individuals of them were selected and planted in Hainan Island.From those 11 male sterile lines with seed-setting rate lower than 3% in bagged spike were obtained.Results suggest that OsAPT2 is involved in male sterility.Nine of the 11 male sterile lines were constitutive sterile lines;two of the 11 male sterile lines were thermo-sensitive genic male sterile lines,which may be useful in hybride rice breeding.展开更多
In this paper, several factors that affect the efficiency of in vitro adventitious bud regeneration and Agrobacterium tumefaciens-mediated transformation of F. vesca were studied. The results showed that F. vesca see...In this paper, several factors that affect the efficiency of in vitro adventitious bud regeneration and Agrobacterium tumefaciens-mediated transformation of F. vesca were studied. The results showed that F. vesca seeds germination rate was the highest while seeds were cultured in water, and the germination rate was the lowest while seeds were cultured on MS medium supplemented with hormone; the germination rates that seeds cultured on two and three layers filter paper were higher than that seeds cultured on four and five layers filter paper. In vitro adventitious regeneration efficiency was affected by different explants types. The significant difference was existed between petioles and leaves. When using the same type explants, in vitro adventitious buds regeneration rate and the average number of buds per explant between Ruegen (RE) and Yellow Wonder (YW) had no significant difference. RE to Agrobacterium tumefaciens was more sensitive than YW. Using seedling leaves of RE and YW as materials, an efficient Agrobacterium-mediated transformation system was developed. In this system, the concentration of bacteria was OD600=0.5, the explants were immersed in bacteria broth for 9 min, the co-cultured time was 2 days, and had no pre-cultured time. The percentage of explants with resistant buds of RE and YW was compared. The putative transformed plants were confirmed by PCR.展开更多
The development of transgenic cotton varieties resistant to bollworms has been a major success of applying plant genetic engineering technology to agriculture,evidenced by phenomenal increase in
In order to promote the research of transgenic insect-resistant maize,the target gene were transferred into maize material Hi-Ⅱ by Agrobacterium-mediated genetic transformation of maize embryos,and maize plants with ...In order to promote the research of transgenic insect-resistant maize,the target gene were transferred into maize material Hi-Ⅱ by Agrobacterium-mediated genetic transformation of maize embryos,and maize plants with CryNGc insect-resistant genes were cultured by explant infection,co-culture and differentiation screening to study the genetic expression and resistance of exogenous genes in the offspring.The results showed that the infection effect was the best when the size of young maize embryo was 1.2-1.8 mm.Ten positive transformed plants with CryNGc insect-resistant genes were successfully obtained,and the transformation efficiency was 1.428‰.展开更多
[Objectives]This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the re...[Objectives]This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene,optimization experiments were carried out in terms of infection time and method,co-cultivation time and method,and PCR detection technology.[Results]The transformation effect was better under the conditions of shaking Agrobacterium liquid,infection time of 1-2 h,and co-cultivation on sterilized filter paper for 2 d.After co-cultivation,the recipient material was first subjected to recovery culture,and then used for Hyg gradient screening,which was conducive to obtaining resistant transformants.The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants,and the proportion of transgenic plants was 16.7%.[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.展开更多
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.
文摘A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly pine
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the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.
文摘A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics (including ampicillin, carbenicillin, cefotaxime, and cefoperazone), meropenem showed the highest activity in suppressing all tested A. tumefaciens strains (minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]). Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.
基金This work was supported by National Key Project (96-538-01-01).
文摘The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N+ implantation at the dosage of 7.8 ×1016 ions/cm2. After treatment, watermelon seeds were incubated with 380μg/μl pumpkin (Cucubita, maxima Duch) DNA solution at 35 ℃ for 5 hours. By two-generations of selection and resistance screening at seedling stage, one transformed material was selected out, whose rind color is similar to that of the donor pumpkin and whose size of seeds is between that of the donor and the receptor. Using AFLP (amplified fragment length polymorphism) technique, two polymorphic DNA fragments were amplified. This primarily testified that the donor DNA fragments/gene were introduced into the receptor cell and integrated into the genomic DNA of the receptor.
文摘The application of keV ion beam in life science started in China several decades ago. In 1986, researchers initially studied the mutagenic effect of ion beam, and successfully applied it to plant breeding. Nowadays, ion beam implantation technique has been extensively applied to many biological fields. This paper mainly introduces one of its important applications: genetic transformation mediated by keV ion beam.
文摘Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.
文摘The plants of hybrid wheatgrass (A. cristatum×A. desertorum cv. Hycrest-Mengnong) were directly induced from embryogenic callus regenerated from immature inflorescence. Immature inflorescence was cultured on improved MS medium containing 2.0-3.0 mg L^-1 2,4-D to regenerate callus. The calli were then transferred to hormone-free MS medium for differentiation and 1/2 MS medium for rooting. Results showed that callus initiation frequency was 83.4% and plant regeneration frequency was 59.6%. Phosphinothricin acetyltransferase (bar) gene was transformed into the hybrid wheatgrass by particle bombardment. Resistant callus was obtained using selecting agent, herbicide glufosinate of 0.5 mg L^-1, and some transgenic plants were recovered in vitro. The transgenic plants were identified by PCR and Southern blot analysis and these plants developed normally in the glufosinate medium, whereas the nontransgenic plants did not. The results demonstrated that bar cDNA integrated into the genomic DNA of the transgenic plants. The transgenic frequencies of bar gene were 1.1%.
基金financially supported by grants from the Biogreen 21 Program, RDA, Korea (PJ00810304)the Agricultural Science and Technology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences (2014–2015)the Beijing Municipal Education Commission, China (KM200910011001)
文摘Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.
基金the National Natural Science Foundation of China(U1603234)the 948 Project from the Ministry of Agriculture of China(2012-S12)+1 种基金the Project for the Key Science and Technology Innovation Team of Shaanxi Province,China(2013KCT-25)the Key Research and Development Plan of Ningxia Hui Autonomous Region,China(2019BEF02005)。
文摘Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands.Molecular genetic techniques in particular,associated with rapid technological advancements,provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance,quality,stress tolerance and disease resistance.To date,several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation,and transgenic grape lines have been obtained using established regeneration systems.Nevertheless,a wide range of factors,including genotype,explant source and culture medium,have been shown to affect the efficiency of plant regeneration.Moreover,the selection and use of acceptor materials,bacterial strain and cell density,selectable markers and selection methods also influence transformation efficiency.This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors,and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.
基金This research is partially supported by the National Natural Science Foundation of China (Grant No. 30371015) the Bureau of Science and Technology of Wuhan. The kindness of doctor Lin Yong-jun in providing Agrobacterium tumefaciens strains EHA 105, harboring the binary vector pCAMBI2301 for this experiment is greatly appreciated. We thank all the colleagues in our laboratory, especially doctor Gao Li-ping, for constructive discussion and technical support.
文摘The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression ofAgrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L^-1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L^-1; however, 300 mg·L^-1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% (control) to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L^-1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L^-1 Timentin. Considering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300 mg·L^-1 is the preferred application in .4. tumefaciens-mediated transformation ofP acerifolia.
基金Supported by Natural Science Foundation of Yunnan Province(2007C213M)Provincial Key Discipline of Landscape Plant&Ornamental Horticulture of Yunnan Province+1 种基金Provincial Key Lab of Colleges and Universities in Landscape Plants and Ornamental Horticulture of Yunnan ProvinceLarge Apparatuses Sharing Platform of Southwest Forestry University
文摘In this study, DERB1A transcription factor and stress-induced promoter rd29A were isolated respectively and amplified from Arabidopsis thaliana, se- quenced and analyzed by DNAsis. In addition, the stress-induced promoter rd29A was utilized to construct the plant expression vector of DERB1A, which was transformed into Agrobacterium tumefaciens. Furthermore, the transgenic regeneration system of fresh-cut chrysanthemum from callus to plantlets was established successfully. On this basis, chrysanthemum leaf-disc explants were genetically transformed with Agrobacterium-mediated method. Two positive transgenie plantlets were obtained in vitro. Based on PCR detection, DREB1A transcription factor was integrated into chrysanthemum genome, which laid the foundation for breeding new transgenie cultivars of fresh-cut chrysanthemum with high comprehensive stress resistance, good cmalitv and high field.
基金supported by a grant from the Bill Melinda Gates FoundationNational Institute of Food and Agriculture of the United States Department of Agriculture for support (Award number 2013-33522-21091)
文摘Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid(2x) to dodecaploid(12x) with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow well in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocellulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We first reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efficiency. Finally we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.
基金supported by grants from the National Key R&D Program of China(Grant No.2019YFD1000900)the Hainan Province Science and Technology Special Fund(Grant No.ZDYF2022XDNY253)the earmarked fund for CARS(Grant No.CARS-32-01)。
文摘Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(such as hybridization)have limited ability to nurture new litchi cultivars.Here,an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in‘Feizixiao’litchi.Transgenic materials were verified using polymerase chain reaction(PCR)analysis,β-glucuronidase(GUS)assay,and green fluorescent protein(GFP)assay.To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/associated protein 9(CRISPR/Cas9)technology in‘Feizixiao’litchi and verify the validity of these transformation systems,the litchi polyphenol oxidase gene(LcPPO,JF926153)was knocked out.Various categories of mutations,covering base insertions,deletions,and substitutions,were found in transgenic materials via sequence analysis.The transformation system achieved high feasibility and efficiency,and the system of CRISPR/Cas9 was successfully employed to edit genes in‘Feizixiao’litchi.This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.
文摘A novel rice gene OsAPT2,which encodes a putative adenine phosphoribosyl transferase(APRT),was cloned.Its full-length cDNA is 1125bp,composing an ORF encoding 212 amino acid residues and a stop cordon,a 5' UTR of 123 bp and a 3' UTR of 363 bp.The sequence data have been submitted to the DDBJ/EMBL/GenBank databases(accession number:AY238894).The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously reported APRTs.The genomic OsAPT2 gene contains 7 exons and 6 introns.Its total length is 4758 bp.Then,an antisense expression vector of the full-length OsAPT2 cDNA was constructed and transformed into rice variety Taibei309 by Agrobacterium tumefaciens mediated transformation method.In total,650 T0 transgenic plants were obtained based on both antibiotic screening and specific PCR identification.One hundred individuals of them were selected and planted in Hainan Island.From those 11 male sterile lines with seed-setting rate lower than 3% in bagged spike were obtained.Results suggest that OsAPT2 is involved in male sterility.Nine of the 11 male sterile lines were constitutive sterile lines;two of the 11 male sterile lines were thermo-sensitive genic male sterile lines,which may be useful in hybride rice breeding.
基金Supported by the Scientific Research fund of the Guangxi Zhuang Autonomous Region(GXNYRKS201601)
文摘In this paper, several factors that affect the efficiency of in vitro adventitious bud regeneration and Agrobacterium tumefaciens-mediated transformation of F. vesca were studied. The results showed that F. vesca seeds germination rate was the highest while seeds were cultured in water, and the germination rate was the lowest while seeds were cultured on MS medium supplemented with hormone; the germination rates that seeds cultured on two and three layers filter paper were higher than that seeds cultured on four and five layers filter paper. In vitro adventitious regeneration efficiency was affected by different explants types. The significant difference was existed between petioles and leaves. When using the same type explants, in vitro adventitious buds regeneration rate and the average number of buds per explant between Ruegen (RE) and Yellow Wonder (YW) had no significant difference. RE to Agrobacterium tumefaciens was more sensitive than YW. Using seedling leaves of RE and YW as materials, an efficient Agrobacterium-mediated transformation system was developed. In this system, the concentration of bacteria was OD600=0.5, the explants were immersed in bacteria broth for 9 min, the co-cultured time was 2 days, and had no pre-cultured time. The percentage of explants with resistant buds of RE and YW was compared. The putative transformed plants were confirmed by PCR.
文摘The development of transgenic cotton varieties resistant to bollworms has been a major success of applying plant genetic engineering technology to agriculture,evidenced by phenomenal increase in
基金Supported by Strategic Leading Science and Technology Project of Chinese Academy of Sciences(XDA28130504)Special Project of Agricultural Science and Technology Innovation Leaping Project of Heilongjiang Academy of Agricultural Sciences(HNK2019CX14)Scientific Research Fund Project of Heilongjiang Provincial Scientific Research Institutes(CZKYF2021C008)。
文摘In order to promote the research of transgenic insect-resistant maize,the target gene were transferred into maize material Hi-Ⅱ by Agrobacterium-mediated genetic transformation of maize embryos,and maize plants with CryNGc insect-resistant genes were cultured by explant infection,co-culture and differentiation screening to study the genetic expression and resistance of exogenous genes in the offspring.The results showed that the infection effect was the best when the size of young maize embryo was 1.2-1.8 mm.Ten positive transformed plants with CryNGc insect-resistant genes were successfully obtained,and the transformation efficiency was 1.428‰.
文摘[Objectives]This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene,optimization experiments were carried out in terms of infection time and method,co-cultivation time and method,and PCR detection technology.[Results]The transformation effect was better under the conditions of shaking Agrobacterium liquid,infection time of 1-2 h,and co-cultivation on sterilized filter paper for 2 d.After co-cultivation,the recipient material was first subjected to recovery culture,and then used for Hyg gradient screening,which was conducive to obtaining resistant transformants.The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants,and the proportion of transgenic plants was 16.7%.[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.