Altered Tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) Fruit Cuticle Biomechanics of a Pleiotropic Non Ripening Mutant

Tomato (Lycopersicon esculentum Mill.) fruit ripening involves multiple metabolic changes resulting in softening and pigmentation. We investigated the mechanics and morphology of the enzymatically isolated cuticular membrane (CM) of cv. Ailsa Craig wild-type (wt) and nonripening mutant (nor) at three developmental stages. Cuticle thickness and degree of cutinization increased significantly from immature to fully ripe fruits for both wt and nor without differences between them. Mechanical characterization was carried out on dry and fully hydrated samples in uni-axial tension to determine their modulus of elasticity, stress, and strain at failure. Corresponding stress-strain diagrams were biphasic and showed yield for virtually all dry CM samples, while that of hydrated CM displayed considerable differences between wt and nor fruits. Concerning the mechanical properties, the CM of wt fruits was characterized by increasing stiffness and strength during fruit growth and maturation in both dry and hydrated states, whereas the CM of nor fruits was significantly less stiff and weaker at full maturity. Hydration generally caused lower moduli of elasticity and strength, while breaking strain was significantly affected only for the CM of ripe nor fruits. This plasticizing effect of water increased towards full maturity for both wt and nor, and may be related to fiber content in the CM matrix and hydration state of the cuticle. Comparative analysis of two additional wild-type tomato cultivars supported the ripening-related stiffening of the CM of Ailsa Craig wt and the altered mechanical properties of the nor mutant, as well as the plasticizing effect of water.     

Activity of an atypical <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pectin methylesterase

An Arabidopsis thaliana pectin methylesterase that was not predicted to contain any signaling sequence was produced in E. coli and purified using a His tag added at its N-terminus. The enzyme demethylesterified Citrus pectin with a Km of 0.86 mg/ml. The enzyme did not require salt for activity and was found to be relatively temperature-sensitive. The precipitation of enzyme-treated pectin by CaCl2 suggested that the enzyme had a blockwise mode of pectin demethylesterification. A purified kiwi (Actinidia chinensis) pectin methylesterase inhibitor had no effect on the activity of the enzyme whereas it strongly inhibited a flax pectin methylesterase. A model of the protein structure revealed that an extra amino acid sequence in this particular Arabidopsis pectin methylesterase could form a ss-strand outside the core structure, which might be preventing the inhibitor from binding the protein.     

Induced androgenesis in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.). III. Characterization of the regenerants

We present data on the morphological, cytological, biochemical and genetic characteristics of tomato regenerants obtained through anther culture. As a result of induced androgenesis, more than 6,000 rooted regenerants were developed that differed both from the donor plants and among each other with respect to habitus and leaf, flower and inflorescence morphology. Cytological analysis revealed a great variability in chromosome number in the cells of the regenerated plants. While most of the regenerants were mixoploid, the majority of the cells had a haploid chromosome number. R₁ and R₂ progenies were tested for their resistance to Clavibacter michiganense subsp. michiganense (Cmm 7). Some of the regenerants were resistant to the pathogen. A biochemical analysis of fruit from R₃ and R₄ plants showed a higher content of dry matter, sugars and vitamin C in the regenerant plants obtained from the hybrids than in those from the cultivars and control plants. The values of the parameters of hybrid regenerants grown in the greenhouse were about 1.5-fold higher than those of the hybrid regenerants grown in the field, and this trend is clearly expressed in all of the hybrid regenerants. The results obtained suggest that induced androgenesis and gametoclonal variation may be used as an additional tool to create a large range of new forms. The application of the latter in breeding programs would accelerate the development of tomato lines and varieties that would be more productive, disease-resistant, highly nutritive and flavour-acceptable.Abbreviations BAP N⁶-Benzylaminopurine - Cmm Clavibacter michiganense subsp. michiganense - cfu Colony-forming units - GA ₃ Gibberellic acid - IBA Indole-3-butyric acid - ms Male sterility - PDA Potato dextrose agar Communicated by H. Lörz     

Analysis of RNA Silencing in Agroinfiltrated Leaves of <Emphasis Type="Italic">Nicotiana Benthamiana</Emphasis> and <Emphasis Type="Italic">Nicotiana Tabacum</Emphasis>

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Pleiotropic effect of the insertion of the <Emphasis Type="Italic">Agrobacterium rhizogenes rolD</Emphasis> gene in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

The Agrobacterium rhizogenes rolD gene, coding for an ornithine cyclodeaminase involved in the biosynthesis of proline from ornithine, has been inserted in Lycopersicon esculentum cv Tondino with the aim of studying its effects on plant morphological characters including pathogen defense response. The analysis of plants transgenic for rolD did not show major morphological modifications. First generation transgenic plants however were found to flower earlier, and showed an increased number of inflorescences and higher fruit yield. Transformed plants were also analysed for parameters linked to pathogen defense response, i.e. ion leakage in the presence of the toxin produced by the fungus Fusarium oxysporum f. sp. lycopersici, and expression of the pathogenesis-related PR-1 gene. All the plants harbouring the rolD gene were shown to be more tolerant to the toxin in ion leakage experiments, with respect to the untransformed regenerated controls and the cv Tondino. PR-1 gene expression was quantitated by means of real-time PCR both at the basal level and after treatment with salicylic acid, an inducer of Systemic Acquired Resistance. In both cases the amount of PR-1 mRNA was higher in the transgenic plants. It seems therefore that the transformation of tomato plants with rolD could lead to an increased competence for defense response, as shown by toxin tolerance and increased expression of the Systemic Acquired Resistance marker gene PR-1. The results are finally discussed in view of their possible economic relevance.Communicated by G. Wenzel     

Analysis of RNA cleavage by RNA polymerases from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase beta subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20 degrees C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2-3-fold) increase in the cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.     

Evaluation of methods for celery (<Emphasis Type="Italic">Apium Graveolens</Emphasis> L.) transformation using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the <Emphasis Type="Italic">bar</Emphasis> gene as selectable marker

Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery. Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l⁻¹). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l⁻¹; calluses formed only with 0, 0.35 and 0.5 mg·l⁻¹ GS. All growing calluses from 0 and 0.35 mg·l⁻¹ and a few from 0.5 mg·l⁻¹, were divided and placed back on C + GS 0.35–0.5 mg·l⁻¹ for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l⁻¹. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected, but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine 2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l⁻¹ glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line.     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Accumulation of cell wall-bound phenolic metabolites and their upliftment in hairy root cultures of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

Alkaline hydrolysis of cell wall material of tomato hairy roots yielded ferulic acid as the major phenolic compound. Other phenolics were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. The content of phenolics was much higher at the early stage of hairy root growth. The ferulic acid content decreased up to 30 days and then sharply increased to 360 microg/g at 60 days of growth. Elicitation of hairy root cultures with Fusarium mat extract (FME) increased ferulic acid content 4-fold after 24 h. As the pathogen-derived elicitors have specific receptors in plants, FME may thus be used for inducing resistance against Fusarium oxysporum f. sp. lycopersici.     

The ploidy level of transgenic plants in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato cotyledons (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.Mill.) is genotype and procedure dependent

A protocol avoiding the feeder-layer cell system was optimized for Agrobacterium-mediated transformation of tomato cotyledonary explants. Over 500 transgenic plants from five tomato cultivars were regenerated in 15 independent experiments. Depending on both genotype and procedure, transformation frequencies ranged from 1.8% to 11.3%. The optimal transformation rate was obtained by inoculating explants with a bacterial suspension in exponential growth ( D(600) = 10(2)-10(3) cells/ml) and transferring cotyledon explants to fresh selective regeneration medium every 3 weeks. The ploidy level of both tomato genotypes used as explant source and primary transformants, was studied by flow cytometry. The inbred lines and cultivars were diploid but a polysomatic pattern in the cotyledon explant was confirmed. The rate of tetraploid transgenic plants ranged from 24.5% to 80% and depended on both the genotype and the transformation procedure. Surprisingly, the percentages of transformed plants with higher ploidy levels were not related to the proportion of 4C and 8C nuclei in the cotyledonary tissue. For some genotypes the optimisation of the transformation rate resulted in an increase of tetraploid transgenic plants. Results obtained in this work indicate the convenience of checking the ploidy level of the primary transformants before performing basic studies or introducing tomato transgenic material in a breeding program.     

Morphology and colonization preference of arbuscular mycorrhizal fungi in <Emphasis Type="Italic">Clethra barbinervis</Emphasis>, <Emphasis Type="Italic">Cucumis sativus</Emphasis>, and <Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>

Clethra barbinervis (Ericales), Cucumis sativus, and Lycopersicon esculentum were grown in soils collected from six different vegetation sites (cedar, cypress, larch, red pine, bamboo grass, and Italian ryegrass), and morphology and colonization preference of arbuscular mycorrhizal (AM) fungi were investigated by microscopic observation and PCR detection. C. barbinervis consistently formed Paris-type AM throughout the sites. C. sativus formed both Arum- and Paris-type AM with high occurrence of Arum-type AM. L. esculentum also formed both Arum- and Paris-type AM but with high occurrence of Paris-type AM. AM diversity within the same plant species was different among the sites. Detected AM diversity from AM spores in different site soils did not consistently reflect AM fungal diversity seen in test plants. Detected families were different, depending on test plants grown even in the same soil. AM fungi belonging to Glomaceae were consistently detected from roots of all test plants throughout the sites. Almost all the families were detected from roots of C. barbinervis and L. esculentum. On the other hand, only two or three families of AM fungi (Archaeosporaceae and/or Paraglomaceae and Glomaceae) but not two other families (Acaulosporaceae and Gigasporaceae) were detected from roots of C. sativus, indicating strong colonization preference of AM fungi to C. sativus among test plants. This study demonstrated that host plant species strongly influenced the colonization preference of AM fungi in the roots.     

Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

Cadmium stress induces changes in the lipid composition and biosynthesis in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) leaves

The effects of cadmium (Cd) stress on lipid composition and biosynthesis were investigated in young leaves of ten-day-old tomato seedlings (Lycopersicon esculentum Mill. cv. Ibiza F1). Cd was found to be mainly accumulated in roots, but a severe inhibition of biomass production occurred in leaves, even at its low concentration (1.0 μM). Seven days after Cd treatment, the membrane lipids were extracted and separated on silica-gel thin layer chromatography (TLC). Fatty acid methyl esters were analyzed by FID-GC on a capillary column. Our results showed that Cd stress decreased the quantities of all lipids classes (phospholipids, galactolipids and neutral lipids). Likewise, there was also a decline in the levels of tri-unsaturated fatty acids, such as linolenic (C18:3) and hexadecatrienoic (C16:3) acids. The linolenic acid (C18:3) decreased in monogalactosyldiacylglycerol (MGDG) and all phospholipids, while hexadecatrienoinic acid (C16:3) declined mainly in MGDG. Moreover, Cd at high concentrations (25.0 and 50.0 μM) significantly enhanced the levels of lipid peroxides. Radiolabelling experiments were carried out by laying down microdroplets of [1-¹⁴C]acetate–a major precursor of lipid biosynthesis–on attached leaves of the control and Cd-treated plants. After incubation for 1, 2, 12 and 24 h, the leaves were harvested and lipids extracted and analysed. Cd stress was found to decrease the incorporation of [1-¹⁴C]acetate in total lipids. The biosynthesis of total lipids was altered with 25.0 and 50.0 μM Cd. The decline in the incorporation of [1-¹⁴C]acetate due to Cd stress was observed in all lipid classes. There was also a substantial decline in the incorporation of [1-¹⁴C]acetate in tri-unsaturated fatty acids. The results indicate that Cd treatment induces an oxidative stress by inhibiting the chloroplastic and extrachloroplastic lipid-biosynthesis pathways as well as lipid peroxidation.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

Nitric Oxide is Involved in the <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>-induced Lateral Root Formation in Tomato

Azospirillum spp. is a well known plant-growth-promoting rhizobacterium. Azospirillum-inoculated plants have shown to display enhanced lateral root and root hair development. These promoting effects have been attributed mainly to the production of hormone-like substances. Nitric oxide (NO) has recently been described to act as a signal molecule in the hormonal cascade leading to root formation. However, data on the possible role of NO in free-living diazotrophs associated to plant roots, is unavailable. In this work, NO production by Azospirillum brasilense Sp245 was detected by electron paramagnetic resonance (6.4 nmol. g^–1 of bacteria) and confirmed by the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). The observed green fluorescence was significantly diminished by the addition of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Azospirillum-inoculated and noninoculated tomato (Lycopersicon esculentum L.) roots were incubated with DAF-2 DA and examined by epifluorescence microscopy. Azospirillum-inoculated roots displayed higher fluorescence intensity which was located mainly at the vascular tissues and subepidermal cells of roots. The Azospirillum-mediated induction of lateral root formation (LRF) appears to be NO-dependent since it was completely blocked by treatment with cPTIO, whereas the addition of the NO donor sodium nitroprusside partially reverted the inhibitory effect of cPTIO. Overall, the results strongly support the participation of NO in the Azospirillum-promoted LRF in tomato seedlings.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation using embryogenic suspension cultures in sweetpotato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis. β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in quantity of independent events per transformation experiment in sweetpotato.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.