Generation of genetically stable transformants by <Emphasis Type="Italic">Agrobacterium</Emphasis> using tomato floral buds

Tomato (Solanum lycopersicum) is a model crop plant for the study of fruit ripening and disease resistance. Here we present a systemic study on in planta transformation of tomato with Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA1303 binary vector bearing HPTII as a plant selectable marker and mGFP/GUS fusion as the reporter gene. We attempted the transformation of tomato at different developmental stages viz. during seed germination, seedling growth, and floral bud development. The imbibition of seeds with Agrobacterium suspension led to seed mortality. The vacuum infiltration of seedlings with Agrobacterium suspension led to sterility in surviving plants. Successful transformation could be achieved either by dipping of developing floral buds in the Agrobacterium suspension or by injecting Agrobacterium into the floral buds. Most floral buds subjected to dip as well as to injection either aborted or had arrested development. The pollination of surviving floral buds with pollen from wild-type plants yielded fruits bearing seeds. A transformation efficiency of 0.25–0.50% was obtained on floral dips/floral injections. Transgenic plants were selected by screening seedlings for hygromycin resistance. The presence of the transgene in genomic DNA was confirmed by Southern blot analysis and expression of the reporter gene up to the T₄ generation. The amenability of tomato for in planta transformation simplifies the generation of transgenic tomato plants obviating intervening tissue culture.     

Breeding of a tomato genotype readily accessible to genetic manipulation

A tomato genotype, superior in regenerating plants from cell cultures, was obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. This genotype, MsK93, greatly facilitates genetic manipulation of tomato, as was demonstrated by successful leaf disc transformation using Agrobacterium tumefaciens and by direct gene transer to protoplasts derived from callus.     

Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells

Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis.     

根癌农杆菌介导的乙肝表面抗原基因对烟草的转化

构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于农杆菌的转化;通过冻融法将重组质粒pBRSAg转入根癌农杆菌LBA4404中,利用农杆菌介导法转化烟草叶盘,经筛选培养获得烟草植株。抗性植株经GUS染色和PCR检测为阳性,初步表明乙肝表面抗原基因在烟草中得到表达。     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Fruit-specific Over-expression of LeEXP1 Gene in Tomato Alters Fruit Texture

Expansins are cellular proteins with diverse physiological functions. Expression of fruit-specific expansin gene in tomato is associated with fruit softening — a desirable trait from the processing point of view. In the present study, an expansin gene LeEXP1 was introduced via Agrobacterium tumefaciens in sense orientation under the control of a fruit-specific promoter LeACS4 with nptII gene as selection marker in Indian tomato cv Pusa Uphar. PCR detection and Southern blot analysis confirmed the integration of the transgene in the transformed tomato plants. RT-PCR and northern blot analysis using total RNA isolated from leaves and fruits confirmed over-expression of the LeEXP1 gene in transgenic fruits as compared to the wild type plants. Apart from the visual change in increased red colouration of fruits at different stages of ripening, overexpression of the LeEXP1 gene resulted in enhanced fruit softening, as determined by force required to rupture the fruit pericarp, in the transgenic fruits from breaker stage onwards as compared to the non-transformed wild type fruits. The results thus suggest an improvement in texture of the LeEXP1 over-expressing fruits, which might be useful for tomato processing industry.     

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.     

Induction of Defense-Related Responses in Cf9 Tomato Cells by the AVR9 Elicitor Peptide of Cladosporium fulvum Is Developmentally Regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F₁ seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.     

Antiviral activity of extracts of transgenic chicory and lettuce plants with the human interferon α2b gene

This paper studies the biological activity of protein extracts of the Cichorium intybus L. and Lactuca sativa L. transgenic plants with the human interferon ??2b gene againsf vesicular stomatitis virus. Extracts from transgenic lettuce and chicory roots, which were obtained after A. rhizogenes-mediated transformation, had antiviral activity in the range 1620?C5400 IU/g of weight; extracts from leaves of chicory plants transformed by A. tumefaciens, up to 9375 IU/g. The dependence of the antiviral activity of plant extracts from roots or leaves on the vector used for plant transformation is shown. The extracts of plant roots obtained by A. rhizogenes-mediated transformation had antiviral activity; at the same time, such activity was absent in the extracts from leaves.     

Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Conditions for plant regeneration from explants of tomato (Lycopersiconesculentum) cv. UC82B were studied for optimizing transformationprocedure. The best regeneration rate was obtained from cotyledonexplants from 8–10-d-old seedlings on a modified Murashigeand Skoog medium (1962) with 0·5 mg dm^–3 zeatinand 0·5 mg dm^–3 indolylacetic acid. Tomato cultivars(UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum‘CMV sel. INRA’ were studied. The cultivarUC82Band the wild Lycopersicon species showed an efficient shootregeneration potential. Early events in the transformation of tomato cotyledons wereanalysed using an Agrobacterium tumefaciens strain carryinga binary vector with an nptII (pnos) gene and a reporter GUS-intron(p35S) chimeric gene. Two days after infection, GUS activityappeared specifically at the cut surface. Subepidermal cellswere more susceptible to transformation than epidermal cells.When selection for kanamycin resistance was applied 2 d afterinoculation, transformed cells were efficiently recovered. Preculturewith feeder cells stimulated cell transformation, but reducedregenerationcapacity from transformed cells. The optimal transformationrate was observed witha time of preculture of 1 and 2 d. Transformationevents for two tomato cultivars (UC82B and Monalbo) occurredat the same rate as 55% of the inoculated explants developedkanamycin resistant calli. However, transformed plants wereobtained at different rates of 8% and 14% for cv. Monalbo andcv. UC82B. Key words: Agrobacterium tumefaciens, ß-glucuronid, Lycopersicon esculentum, plant regeneration, transformation     

Constitutive overexpression of small HSP24.4 gene in transgenic tomato conferring tolerance to high-temperature stress

Acquired thermotolerance in plants refers to the ability to cope with lethal high temperatures and it reflects an actual tolerance mechanism that occurs naturally in plants. Tomato (Solanum lycopersicum syn. Lycopersicon esculentum L.) is sensitive to high temperature at all stages of its growth and development. Considering the important role of the heat shock protein gene (sHSP24.4 gene) in imparting tolerance to high temperature stress in the cells and tissues, we isolated small HSP24.4 (MasHSP24.4) cDNA from wild banana (Musa accuminata) and introduced it into the cultivated tomato cv. PKM1 by using Agrobacterium tumefaciens-mediated genetic transformation. Stable integration and expression of the transgene in the tomato genome was demonstrated by Southern, Northern and Western blot analyses. There was no adverse effect of transgene expression on overall growth and development of the transgenic plants. The genetic analysis of the transgenic T2 lines showed that the transgene segregated in a Mendelian ratio. We compared the survival of T2 transgenic lines compared to the control plants after exposure to different levels of high temperature. The gene MasHSP24.4 was expressed in root, shoot and stem tissues under 45 °C treatment and conferred tolerance to high-temperature stress as shown by increased seed germination, healthy vegetative growth and normal fruit and seed setting. The transgenic tomato plants showed significantly better growth performance in the recovery phase following the stress. This thermotolerance appeared to be solely due to overexpression of the sHSP24.4 gene. Thus, the transgenic tomato plants developed during the present investigations can be grown at high temperatures.     

High efficiency transformation of cultured tobacco cells

Tobacco calli were transformed at levels up to 50% by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harboring the binary transfer-DNA vector, pGA472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and, less so, on the expression of the vir genes of the tumor-inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essental factor for efficient transformation of higher plants.     

Efficient production of transgenic tomatoes via Agrobacterium-mediated transformation

Cotyledonary leaves of 9-d-old tomato (Lycopersicon esculentum Mill.) were co-cultivated with Agrobacterium tumefaciens GV 3101 harboring binary vector pBI101 containing kanamycin resistance gene (npt II) as selection marker. Murashige and Skoog (MS) inorganic salts with Gamborg’s B5 vitamins supplemented with optimized concentrations of zeatin riboside and indole-acetic acid resulted in enhanced regeneration efficiency. Under optimized conditions of plant regeneration, transformation frequency in cvs. Pusa Ruby, Pusa Uphar and DT-39 was greater than 37 %. Transformed shoots were selected on kanamycin medium and the presence of the transgene in the primary transformants was confirmed by PCR. Integration of the npt II gene in the tomato genome was further confirmed by Southern blot analysis. RT-PCR analysis using neomycin phospho-transferase (npt II) gene-specific primers confirmed the expression of the transgene in transgenic plants. Transformed plants were successfully transferred to phytotron, where these plants grew to maturity and produced flowers and fruits.     

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.)

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l⁻¹ 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l⁻¹ hygromycin and 200 mg l⁻¹ cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants

Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple.     

根癌农杆菌介导合欢转TaNHX2基因体系的优化

通过对农杆菌菌液浓度、侵染时间、预培养时间和共培养时间等影响转化效率的因素进行优化,建立了合欢农杆菌转化体系。在此基础上,利用农杆菌介导法将TaNHX2基因导入合欢基因组内,获得大量Kan抗性再生植株。常规PCR和实时荧光PCR检测结果表明,外源基因已整合到合欢基因组中,并正常转录。     

Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System

Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were transformed with a kanamycin resistance marker (neomycin phosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant.     

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.