Manipulating photosynthesis

The levels of individual photosynthetic proteins can be independently decreased by theAgrobacterium-mediated transformation of plants with antisens RNA constructs. Protocols for the introduction of such constructs intoAgrobacterium, theAgrobacterium-mediated transformation of tobacco leaf disks, and the screening and analysis of the transgenic plants produced are described.     

An improved procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of trifoliate orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.) via indirect organogenesis

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.     

Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation

The effect of several β-lactam antibiotics on shoot regeneration, growth and rooting of carnation (Dianthus caryophyllus L.), and their use in combination with kanamycin in Agrobacterium-mediated genetic transformation studies, was determined. Carbenicillin, cefotaxime and ticarcillin increased the regeneration rate when added alone in non-inoculated explants; whereas, with inoculated explants, this effect was only observed in ticarcillin-containing medium. Cefotaxime inhibited root growth in both transgenic and non-transgenic shoots. Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin. The different antibiotics used, alone or in combination, did not prevent the occurrence of false positive shoots, but it was possible to select transgenic shoots when rooting was induced in a kanamycin + ticarcillin-containing medium. Regenerated transformed shoots were free of Agrobacterium after culturing in rooting medium, as was proven by the PCR analysis for the nptI gene, the antibiogram and the culture of tissue pieces of transgenic shoots on LB broth. The use of kanamycin and timentin with or without carbenicillin, was very useful in the transformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the selection of transgenic versus false-positive shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

T-DNA structure in transgenic tobacco plants with multiple independent integration sites

Summary Transgenic tobacco plants were produced by inoculation of leaf disks withAgrobacterium tumefaciens harboring a disarmed binary vector containing soybean leghemoglobin Lbc3 and glycinin G2 genes. Physical and genetic characterization of these plants indicated that one to six copies of DNA from the vector were transferred and maintained in the plant genome. Approximately 30% of the copies transferred were found to be incomplete or rearranged and in some cases joined as inverted repeats. The transferred DNA was found at multiple genetic loci in five of the six cases examined. In one plant, kanamycin-resistance traits were at four independent chromosomal positions, although two were genetically linked at about 3 centimorgans. Thus,Agrobacterium-mediated DNA transfer to plants has some characteristics in common with “natural” systems in animals, such as retroviral or P-element derived systems, some characteristics in common with “artificial” systems, such as microinjection, electroporation, or calcium phosphate coprecipitation techniques, and some novel characteristics.     

Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant pretreatment medium and the co-culture medium improved the transformation efficiency by decreasing the escape frequency. More than 90% rooting frequency of transformed citrange shoots was achieved by two-step culture: first on media supplemented with auxins, and then on media without hormones. Inclusion of 20 mg l^–1 kanamycin in rooting medium efficiently discriminated transformed shoots from non-transgenic escaped shoots. Shoot grafting in vitro was used to regenerate transformed plants, due to the slow growth of most sweet orange shoots.     

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.     

Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants

For the development of anAgrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing -glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenic shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulentAgrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way.     

Transformation of plants via the shoot apex

Summary We have transformed petunia byAgrobacterium tumefaciens containing genes for kanamycin resistance and beta-glucuronidase using isolated shoot apices from seedling tissue. Regeneration of transformed plants in this model system was rapid. The technique of shoot apex transformation is an alternative for use inAgrobacterium-mediated transformation of dicotyledonous crop species for which a method of regeneration via protoplasts, leaf disks, or epidermal strips does not exist. This approach offers direct and rapid regeneration of plants and low risk of tissue-culture-induced genetic variation. Texas Agricultural Experiment Station Technical Article No. 23317.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

Development of anAgrobacterium-mediated transformation system for regenerating garland chrysanthemum (Chrysanthemum coronarium L.)

Although efficient shoot regeneration and selection are essential for genetic transformation mediated byAgrobacterium, success has been limited with the garland chrysanthemum (Chrysanthemum coronarium L.). In this study, we developed a useful protocol for shoot regeneration with leaf disk explants. The optimal concentrations of NAA and BA were 0.2 mg L⁻¹ and 0.5 mg L⁻¹, respectively. To optimize the selection system for regenerating plants from genetically transformed tissues, we tested the effects of four antibiotics (kanamycin, hygromycin, carbenicillin, and cefotaxime). Among them, 5 mg L^-1 hygromycin proved adequate as a selectable marker, whereas 500 mg L^-1 carbenicillin was effective in eliminating excessiveAgrobacterium after co-cultivation. Transgenic plants were obtained by first co-culturing garland chrysanthemum leaf disks withA. tumefaciens strain EHA105, which harbors plasmid pRCVII containing the hygromycin resistance (hpt) and β-glucuronidase (GUS) genes. After the transgenic plants were confirmed via Southern analysis, they were rooted in soil and appeared phenotypically normal. Our report is the first to describe the optimum conditions for producing transgenic plants of this species.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

Cross-protection and selectable marker genes in plant transformation

Summary Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.     

Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.