Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene trans...Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0-1.5mm in size, C58c1 of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production,shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptⅡ ELISA (nptⅡ enzyme assay), PCR, Southern blot and leaf bleach,29 positive plants were identified from two genotypes of Bobwhite and Yanglmai 10, with an average transformation efficiency of 0.82 %. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.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 importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Ni...The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.展开更多
Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus ...Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.展开更多
Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for r...Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.展开更多
We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated...We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13?and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.展开更多
This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a s...This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).展开更多
Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK...Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.展开更多
Phytohormone, ethylene plays an important role in plant growth and development including fruit ripening and flower senescence. The synthesis of 1-aminocylo-propane-1- carboxylate (ACC), the immediate precursor of ethy...Phytohormone, ethylene plays an important role in plant growth and development including fruit ripening and flower senescence. The synthesis of 1-aminocylo-propane-1- carboxylate (ACC), the immediate precursor of ethylene, from S-adenosyl-methionine is catalyzed by ACC synthase;and which is also a rate limiting step in the ethylene biosynthetic pathway. Therefore, it plays a key role in ethylene biosynthesis and the genes that code for ACC synthase are of special interest. Moreover, in zonal geraniums, ethylene bursts released from cuttings can have profound impact on the viability of explants for plant propagation. Biotechnological approach involving genetic modification that may reduce ethylene levels has potential for increasing the shelf-life of cuttings for plant propagation. These considerations have led us to clone several cDNA of ACC synthase genes from Pelargonium x hortorum cv. ?‘Sincerity’. To transform geranium cells with Agrobacterium tumefaciens an in vitro regeneration? system was developed using very young petiole explants. An Antisense construct of ACC synthase cDNA (PHSacc41) ligated into binary vector pAM696 was introduced into A. tumefaciens EHA 105 cells. Petiole explants were incubated with the Agrobacterium for 15 min and then co-cultivated for several days on MS medium containing 5 mM BAP and ?1 mM IAA in? the dark without the antibiotics. Selection for transformants was carried out in the presence of kanamycin and timentin. Transgenic plantlets generated were examined for inserted gene cassette by PCR and Southern blotting. Recovery of positive transformants that survived selection suggested that it is possible to transform and introduce genes via transformation in hybrid geraniums for genetic modification.展开更多
Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification...Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.展开更多
Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increa...Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.展开更多
Brassica rapa L.is cultivated globally and consumed in many areas worldwide.Using the transgenic Agrobacterium-mediated transformation method,which is a reproducible and efficient technique,genes can be transferred in...Brassica rapa L.is cultivated globally and consumed in many areas worldwide.Using the transgenic Agrobacterium-mediated transformation method,which is a reproducible and efficient technique,genes can be transferred into various B.rapa species.This review summarizes the processes involved in Agrobacterium-mediated transformation of B.rapa,including surface seed sterilization,co-cultivation with A.tumefaciens,induction of callus/shoot/root formation,and confirmation of transgenic plants.In addition,factors such as the Agrobacterium strain,plant genotype,explant age,transformation efficiency of the hybrid or inbred line,and the concentrations of N6-benzyl amino purine and naphthalene acetic acid,are discussed.And this review shows clearly how to do it,what to do,and what not to do in the transgenic Agrobacterium-mediated in Brassica rapa.The information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic B.rapa production,thereby benefiting both basic and applied research.展开更多
The development of plant gene engineering requires a simple, rapid and efficient system for genetic transformation. It is recently demonstrated that certain compounds, secreted by host plant cells, are capable of elic...The development of plant gene engineering requires a simple, rapid and efficient system for genetic transformation. It is recently demonstrated that certain compounds, secreted by host plant cells, are capable of eliciting the activation of Agrobacterium and Ti plasmid genes which are involved in promoting T-DNA transfer from the bacterium to the plant cell,展开更多
A system for transformation and regeneration of Lycium barbarum L. , an important Chinese medical plant, has been established. Young stem segments from Lycium barbarum L. were infected with Agrobacterium tumefaciens C...A system for transformation and regeneration of Lycium barbarum L. , an important Chinese medical plant, has been established. Young stem segments from Lycium barbarum L. were infected with Agrobacterium tumefaciens C58cl(pGV3850: :ineo1130), and the transformed calli selected from the callus induction medium containing 50 fig/ml kanamycin could regenerate buds on differentiation medium containing 25 μg/ml kanamycin. 30% of the regenerated buds were normal in morphology. The normal buds could develop into whole plantlets after they were transferred to the rooting medium to induce roots. Nopa-line detection, NPT-Ⅱ enzyme activity assay and Southern blotting hybridization indicated that the foreign genes had been integrated into the genome of Lycium barbarum L. and expressed in the plant. In the processes of experiments, it was found that (i) after the pre-processes, the explants which formed callus quickly were easy to transform ; (Ⅱ) the rate of normal regenerated plants from transgenic calli was展开更多
So far there is no suitable vector system for the genetic engineering of monocotyledonous plants, especially cereal crops. It is first reported in this paper that Agrobacterium tumefaciens strains T37, A208 and B6 can...So far there is no suitable vector system for the genetic engineering of monocotyledonous plants, especially cereal crops. It is first reported in this paper that Agrobacterium tumefaciens strains T37, A208 and B6 can transform some varieties of Triticum aestivum and Hordeum vulgare to form swellings and tumors. Itis shown in the experiments that phenolic compound acetosyringone may promote the transformation process. In addition, inoculating agrobacteria on appropriate plant tissues is also proved to be the key step in achieving successful transformation. The host range of A. tumefaciens and the possibility of utilizing Ti plasmid as the vector for the gene transfer of cereal crops are discussed.展开更多
A simple and effective system for the transformation and regeneration of a leguminous plant has been developed. The cotyledon explants of Lotus corniculatus (var. Leo) were infected by Agrobacterium tumefaciens that c...A simple and effective system for the transformation and regeneration of a leguminous plant has been developed. The cotyledon explants of Lotus corniculatus (var. Leo) were infected by Agrobacterium tumefaciens that contained a non-oncogenic Ti plasmid vector. The vector carried a chimaeric npt-Ⅱ gene and a nopaline synthase gene (nos). On the selective medium that contained kanamycin, 40% of the explants formed buds within 3 weeks. Developed shoots were cut off and transferred to rooting medium. Normal-looking plants were recovered and grew well after being transplanted into soil, bloomed and set seeds. Foreign genes were integrated onto the L. corniculatus genome, expressed, and inherited through sexual reproduction, which was proven by nopaline detection, NPT-Ⅱ enzyme activity detection and DNA hybridization test of the transformed plants and the nopaline detection of the progeny.展开更多
文摘Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0-1.5mm in size, C58c1 of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production,shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptⅡ ELISA (nptⅡ enzyme assay), PCR, Southern blot and leaf bleach,29 positive plants were identified from two genotypes of Bobwhite and Yanglmai 10, with an average transformation efficiency of 0.82 %. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.
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 importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.31902052 and 31972450)the National Key Research and Development Program of China(Grant No.2016YFC0500300)+1 种基金the Natural Science Foundation of Heilongjiang Province of China(Grant No.C2018021)the‘Academic backbone’Project of Northeast Agricultural University of China(Grant No.18XG08).
文摘Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.
基金The authors thank Dr.Manzhu Bao(Huazhong Agricultural University,Wuhan,China),Dr.Hibrand-Saint Oyant L.(INRA,Agrocampus-Ouest,Universitéd’Angers,Beaucouzé,France)and Dr.Fabrice Foucher(INRA,78026 Versailles Cedex,France)for their excellent suggestions.We are also grateful to Dr.Wenxue Li and Dr.Hongqiu Wang(Chinese Academy of Agricultural Sci-ences,Beijing,China)for assistance with the experiments.This work was supported by grants from National Natural Science Foundation of China(Grant No.31522049)Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects(Grant No.CEFF-PXM2019_014207_000032).
文摘Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.
文摘We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13?and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.
文摘This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).
文摘Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.
文摘Phytohormone, ethylene plays an important role in plant growth and development including fruit ripening and flower senescence. The synthesis of 1-aminocylo-propane-1- carboxylate (ACC), the immediate precursor of ethylene, from S-adenosyl-methionine is catalyzed by ACC synthase;and which is also a rate limiting step in the ethylene biosynthetic pathway. Therefore, it plays a key role in ethylene biosynthesis and the genes that code for ACC synthase are of special interest. Moreover, in zonal geraniums, ethylene bursts released from cuttings can have profound impact on the viability of explants for plant propagation. Biotechnological approach involving genetic modification that may reduce ethylene levels has potential for increasing the shelf-life of cuttings for plant propagation. These considerations have led us to clone several cDNA of ACC synthase genes from Pelargonium x hortorum cv. ?‘Sincerity’. To transform geranium cells with Agrobacterium tumefaciens an in vitro regeneration? system was developed using very young petiole explants. An Antisense construct of ACC synthase cDNA (PHSacc41) ligated into binary vector pAM696 was introduced into A. tumefaciens EHA 105 cells. Petiole explants were incubated with the Agrobacterium for 15 min and then co-cultivated for several days on MS medium containing 5 mM BAP and ?1 mM IAA in? the dark without the antibiotics. Selection for transformants was carried out in the presence of kanamycin and timentin. Transgenic plantlets generated were examined for inserted gene cassette by PCR and Southern blotting. Recovery of positive transformants that survived selection suggested that it is possible to transform and introduce genes via transformation in hybrid geraniums for genetic modification.
基金This work was supported by Jiangsu Seed Industry Revitalization Project,China[JBGS(2021)071]Fundamental Research Funds for the Central Universities,China(YDZX2023019)+3 种基金the National Natural Science Foundation of China(32172579)the earmarked fund for Jiangsu Agricultural Industry Technology System,China[JATS(2023)421]the Jiangsu Postgraduate Scientific Research Innovation Plan,China(KYCX21_0610-2021)the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD).
文摘Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.
基金financial assistance provided by the High-End Foreign Expert Recruitment Program(G2022051003L)National Natural Science Foundation of China(32201878)+3 种基金Hainan Yazhou Bay Seed Lab(B21HJ0215)Agricultural Science and Technology Innovation Program of CAAS(CAASZDRW202002,CAAS-ZDRW202201)Hebei Natural Science Foundation(C2021205013)Long Mao is also a“Yellow River Delta Scholar”in Sino-Agro Experimental Station for Salt Tolerant Crops(SAESSTC),Dongying,Shandong,China.
文摘Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.
基金supported by funding from the China Agricultural Research System(CARS-25-A-01)the National Key Research and Development Program of China(2017YFD0101802)
文摘Brassica rapa L.is cultivated globally and consumed in many areas worldwide.Using the transgenic Agrobacterium-mediated transformation method,which is a reproducible and efficient technique,genes can be transferred into various B.rapa species.This review summarizes the processes involved in Agrobacterium-mediated transformation of B.rapa,including surface seed sterilization,co-cultivation with A.tumefaciens,induction of callus/shoot/root formation,and confirmation of transgenic plants.In addition,factors such as the Agrobacterium strain,plant genotype,explant age,transformation efficiency of the hybrid or inbred line,and the concentrations of N6-benzyl amino purine and naphthalene acetic acid,are discussed.And this review shows clearly how to do it,what to do,and what not to do in the transgenic Agrobacterium-mediated in Brassica rapa.The information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic B.rapa production,thereby benefiting both basic and applied research.
基金Present address: Biotechnology Research Center, Zhongshan University, Guangzhou 510275, PRC
文摘The development of plant gene engineering requires a simple, rapid and efficient system for genetic transformation. It is recently demonstrated that certain compounds, secreted by host plant cells, are capable of eliciting the activation of Agrobacterium and Ti plasmid genes which are involved in promoting T-DNA transfer from the bacterium to the plant cell,
文摘A system for transformation and regeneration of Lycium barbarum L. , an important Chinese medical plant, has been established. Young stem segments from Lycium barbarum L. were infected with Agrobacterium tumefaciens C58cl(pGV3850: :ineo1130), and the transformed calli selected from the callus induction medium containing 50 fig/ml kanamycin could regenerate buds on differentiation medium containing 25 μg/ml kanamycin. 30% of the regenerated buds were normal in morphology. The normal buds could develop into whole plantlets after they were transferred to the rooting medium to induce roots. Nopa-line detection, NPT-Ⅱ enzyme activity assay and Southern blotting hybridization indicated that the foreign genes had been integrated into the genome of Lycium barbarum L. and expressed in the plant. In the processes of experiments, it was found that (i) after the pre-processes, the explants which formed callus quickly were easy to transform ; (Ⅱ) the rate of normal regenerated plants from transgenic calli was
文摘So far there is no suitable vector system for the genetic engineering of monocotyledonous plants, especially cereal crops. It is first reported in this paper that Agrobacterium tumefaciens strains T37, A208 and B6 can transform some varieties of Triticum aestivum and Hordeum vulgare to form swellings and tumors. Itis shown in the experiments that phenolic compound acetosyringone may promote the transformation process. In addition, inoculating agrobacteria on appropriate plant tissues is also proved to be the key step in achieving successful transformation. The host range of A. tumefaciens and the possibility of utilizing Ti plasmid as the vector for the gene transfer of cereal crops are discussed.
文摘A simple and effective system for the transformation and regeneration of a leguminous plant has been developed. The cotyledon explants of Lotus corniculatus (var. Leo) were infected by Agrobacterium tumefaciens that contained a non-oncogenic Ti plasmid vector. The vector carried a chimaeric npt-Ⅱ gene and a nopaline synthase gene (nos). On the selective medium that contained kanamycin, 40% of the explants formed buds within 3 weeks. Developed shoots were cut off and transferred to rooting medium. Normal-looking plants were recovered and grew well after being transplanted into soil, bloomed and set seeds. Foreign genes were integrated onto the L. corniculatus genome, expressed, and inherited through sexual reproduction, which was proven by nopaline detection, NPT-Ⅱ enzyme activity detection and DNA hybridization test of the transformed plants and the nopaline detection of the progeny.