Rapid,efficient production of homozygous transgenic tobacco plants withagrobacterium tumefaciens: A seed-to-seed protocol

An optimized complete protocol was developed forAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cultivar SR1), producing T₁ flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T₂ seedlings in 6 to 7 months from the time of cocultivation withAgrobacterium. Previous protocols require up to 9 to 12 months to obtain similar results. Procedures unique and important to this protocol include; a modified “whole-leaf” transformation coupled with a long duration of cocultivation, resulting in high rates of transformation, high levels of kanamycin in selection media resulting in few escapes, and extensive rooting of regenerants prior to a greenhouse hardening procedure. Once in the greenhouse, primary regenerants were maintained in small containers with long day photoperiod and high light levels, greatly shortening the time to seed set. Flowers from primary transformants were bagged to allow self pollination, and seed capsules harvested and dried prior to normal maturation on the plant. T₁ and T₂ seeds were plated and selected on kanamycin media by an improved seed plating technique which eliminates the need for the placement of individual seeds, saving time and improving selection homogeneity. Using this protocol, over 130 independent tobacco lines from six separate gene constructs have been generated in a very short time period. Of these 130, nearly 60 percent segregated 3∶1 for kanamycin resistance: susceptibility, indicating single transgene insertion events.     

Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks

An improved method for genetic transformation of cucumber (Cucumis sativus L. cv. Shinhokusei No. 1) was developed. Vacuum infiltration of cotyledonary explants with Agrobacterium suspension enhanced the efficiency of Agrobacterium infection in the proximal regions of explants. Co-cultivation on filter paper wicks suppressed necrosis of explants, leading to increased regeneration efficiency. Putative transgenic plants were screened by kanamycin resistance and green fluorescent protein (GFP) fluorescence, and integration of the transgene into the cucumber genome was confirmed by genomic polymerase chain reaction (PCR) and Southern blotting. These transgenic plants grew normally and T₁ seeds were obtained from 7 lines. Finally, stable integration and transmission of the transgene in T₁ generations were confirmed by GFP fluorescence and genomic PCR. The average transgenic efficiency for producing cucumbers with our method was 11.9 ± 3.5 %, which is among the highest values reported until date using kanamycin as a selective agent.     

Genetic characterization of a double-flowered tobacco plant obtained in a transformation experiment

Summary A leaf-disk transformation experiment was performed with tobacco (Nicotiana tabacum L.) using a binary vector and a strain of Agrobacterium tumefaciens that carried a wild-type Ti-plasmid, pTiBo542. Although the majority of kanamycin-resistant, transgenic plants was morphologically normal, one of the plants was double-flowered and had a slightly wavy stem and leaves whose edges were bent slightly upwards. The abnormal morphology was controlled by a single, dominant Mendelian gene. Young plants that carried this gene were distinguishable from normal plants at the stage of cotyledons. The homozygotes, with respect to this gene, were more seriously deformed than the heterozygotes. DNA segments derived from the binary vector and from the T_L-and T_R-DNA of pTiBo542 were detected in the double-flowered plant, but the T-DNA genes involved in biosynthesis of phytohormones were absent from the plant. The abnormal morphology, the resistance to kanamycin, and the segments of foreign DNA were genetically linked, and the linkage was very tight, at least between the abnormal morphology and the resistance to kanamycin; the meiotic recombination frequency was less than 0.02%, if recombination occurred at all.     

Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene

Transgenic broccoli plants expressing a Trichoderma harzianum endochitinase gene were obtained by Agrobacterium tumefaciens-mediated transformation. PCR and Southern blot analysis confirmed the presence of the gene in plants initially selected via resistance to kanamycin. Primary transformants (T₀) and selfed progeny (T₁) were examined for expression of the endochitinase gene using a fluorometric assay and for their resistance to the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum. All transgenic plants with elevated endochitinase activity had the expected 42 kDa endochitinase band in western blot analysis, whereas no such band was detected in the non-transgenic control. Leaves of most mature T₀ plants had 14–37 times higher endochitinase activity than controls; mature T₁ plants had higher endochitinase activity (100–200 times that in controls), in part because of lower control values. T₀ plantlets in vitro or young plants in soil had higher absolute and relative endochitinase activity. When detached leaves of T₀ plants were inoculated with A. brassicicola, lesion size showed a significant negative correlation with endochitinase levels. After inoculation of two-month old T₀ plants with A. brassicicola, all 15 transgenic lines tested showed significantly less severe disease symptoms than controls. In contrast, lesion size on petioles of T₀ and T₁ plants inoculated with S. sclerotiorum was not statistically different from controls.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

Expression of the cry1EC gene in castor (Ricinus communis L.) confers field resistance to tobacco caterpillar (Spodoptera litura Fabr) and castor semilooper (Achoea janata L.)

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T₀ plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T₄ generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T₁–T₄ generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

转大肠杆菌过氧化氢酶基因棉花的抗旱生理研究

以导入大肠杆菌过氧化氢酶基因KatE的T3代转基因棉花为供试材料,经卡那霉素检测和PCR鉴定,将筛选出的阳性转基因植株与对照棉花进行整个生育期的持续水分胁迫处理直至收获,比较材料间的生理生化指标的差异,鉴定转基因植株的耐旱能力。结果显示:(1)干旱胁迫持续至初蕾期时,转基因棉花与对照植株间各项抗旱生理指标差异均未达到显著水平。(2)水分胁迫持续至盛蕾和盛花期时,转基因棉花叶片相对含水量、光系统Ⅱ最大光化学效率(Fv/Fm)、CAT活性,以及叶片的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均显著或极显著高于对照植株,叶绿素含量也都明显高于对照植株。干旱胁迫持续至吐絮期时,转基因棉花的株高、果枝数和铃数均显著或极显著高于对照植株,且转基因棉花和对照的籽棉产量分别比正常灌溉处理降低57.5%和60.1%,全生育期的水分胁迫严重影响了棉花籽棉产量,但转基因棉花的籽棉产量仍显著高于对照。研究表明,在新疆石河子当地自然降水(干旱胁迫)条件下,转KatE基因棉花表现出了较好的生理和生长优势,KatE基因有助于提高棉花的抗旱性。     

Expression of GUS in primary transformants and segregation patterns of GUS,TL- and TR-DNA in the T1 generation of hairy root transformants ofLotus corniculatus

Agrobacterium rhizogenes was assessed as a vehicle for transformation ofLotus corniculatus. Plants were co-transformed usingA. rhizogenes strain LBA 9402 harbouring the bacterial plasmid pRi1855 and the binary transformation vector pJit 73. pRi 1855 transfers both T_L and T_R sequences, while pJit 73 encodes β-glucuronidase (GUS) and also two selectable marker genes giving resistance to the antibiotics kanamycin and hygromycin. Three primary transformants (lines 1,6 and 12) were subjected to detailed morphological and biochemical analysis and lines 6 and 12 were also analysed at the molecular level. Tissues of both lines 6 and 12 were resistant to hygromycin and expressed GUS. Analysis of various tissues of each line showed a significantly lower GUS activity in line 6 than in line 12. Genetical analysis of progeny produced between control plants and lines 6 and 12 indicated that line 6 had one dose of theuid gene while line 12 had two or more independently segregating doses of the gene. Both lines 6 and 12 contained multiple copies of T_L-DNA, while only line 6 was T_R positive. In the progeny of lines 6 and 12 there was no evidence for linkage of T_L-DNA withuid, while in the progeny of line 6, T_R-DNA was under-represented. GUS-positive progeny which were free of both T_L and T_R sequences were identified from both lines.     

Transformation of tomato using an Ri plasmid vector

An intermediate vector, pAMNeo10, was constructed containing the replication origin and carbenicillin-resistance gene of pBR322, an homology region to allow insertion into the T_L-DNA of pRiA4 in Agrobacterium A4T, and a chimaeric kanamycin-resistance gene (nop. neoΔ) for identification of T_L-DNA::pAMNeo10 transformed roots. Roots produced by inoculating stem explants of Lycopersicon esculentum, L. hirsutum × L. esculentum (KNVF Rootstock) and L. peruvianum with an exconjugant stain, A4T (pRiA4::pAMNeo10), were resistant to kanamycin at levels that completely inhibit the growth of transformed roots produced with wild-type A4T. When transformed by the exconjugant strain, roots of the three tomato hosts were resistant to different levels of kanamycin, and, in the case of L. peruvianum, regenerated plants were tolerant to much higher levels (10×) of kanamycin than the transformed roots from which they were derived. Kanamycin-resistant transformed roots expressed aminoglycoside phosphotransferase activity, and Southern blotting confirmed the presence of the intermediate vector sequence in transformed roots and in shoots of regenerated plants. T_R-DNA was shown to be present in most transformed roots and regenerated shoots by testing for agropine and mannopine. The application of Ri plasmid vectors to the study of foreign gene expression in plants is discussed.     

Development of cotton transgenics with antisense <Emphasis Type="Italic">AV2</Emphasis> gene for resistance against cotton leaf curl virus (CLCuD) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Cotton transgenics for resistance against cotton leaf curl disease using antisense movement protein gene (AV2) were developed in an Indian variety (F846) via Agrobacterium-mediated transformation using the protocol developed previously. A binary vector pPZP carrying the antisense AV2 (350 bp) gene along with the nptII gene was used. Transgenic nature of the putative transgenics was confirmed by molecular analysis. Shoots were induced on selection medium and subcultured on rooting medium containing IBA and 75 mg l^–1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Preliminary analysis of the field-established plantlets was conducted by PCR. T₁ plants were obtained from T₀ seeds, the presence of the AV2 and nptIIgenes in the transgenic plants was verified by PCR and integration of T-DNA with AV2 into the plant genome of putative transgenics was further confirmed by Southern blot analysis. Several T₁ lines were maintained in the greenhouse. Progeny analysis of these plants by PCR analysis showed a classical Mendelian pattern of inheritance.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta

Stable genetic transformation of Arabidopsis thaliana was achieved by simple in planta inoculation of Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBl121. The transformation procedure, which we call in planta transformation, involves severing of apical shoots at their bases, inoculation with Agrobacterium at the severed sites, and in planta generation of shoots from the severed sites. On average, 5.5% of the newly formed shoots produced transformed progenies. These progenies (T₂ generation) contained T-DNA in the genome as examined by assaying the T-DNA encoded β-glucuronidase and kanamycin resistance and by genomic Southern blot analysis, the copy number of the T-DNAs in the Arabidopsis genome being single (33%) or multiple. The genetic behavior of the transformants examined at the T₃ and T₄ generations or with the F₂ progenies of the outcrosses between transformants and wild-type plants showed that most of the inserted T-DNA are inherited in a Mendelian fashion. This procedure provides a new approach for simple and efficient transformation of A. thaliana, obviating the need for plant regeneration from tissue explants in vitro.     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.     

Genetic engineering of Treponema pallidum subsp. pallidum,the Syphilis Spirochete

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kan^R) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan^R gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kan^R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan^R cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl₂-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan^R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan^R message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprA^ko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.