Transgenic tomato plants as supersweet protein thaumatin II producers

Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.     

Accumulation,assembly, and digestibility of amarantin expressed in transgenic tropical maize

An amaranth (Amaranthus hypochondriacus) 11S globulin cDNA, encoding one of the most important storage proteins (amarantin) of the seed, with a high content of essential amino acids, was used in the transformation of CIMMYT tropical maize genotype. Constructs contained the amarantin cDNA under the control of a tissue-specific promoter from rice glutelin-1 (osGT1) or a constitutive (CaMV 35S) promoter with and without the first maize alcohol dehydrogenase intron (AdH). Southern-blot analysis confirmed the integration of the amarantin cDNA, and copy number ranged from one to more than ten copies per maize genome. Western-blot and ultracentrifugation analyses of transgenic maize indicate that the expressed recombinant amarantin precursors were processed into the mature form, and accumulated stably in maize endosperm. Total protein and some essential amino acids of the best expressing maize augmented 32% and 8–44%, respectively, compared to non-transformed samples. The soluble expressed proteins were susceptible to digestion by simulated gastric and intestinal fluids, and it is suggested that they show no allergenic activity. These findings demonstrate the feasibility of using genetic engineering to improve the amino acid composition of grain crops.Communicated by J.W. Snape     

Transgenic tomato plants expressing strawberry <Emphasis Type="ItalicSmallCaps">d</Emphasis>-<Emphasis Type="Italic">galacturonic acid reductase</Emphasis> gene display enhanced tolerance to abiotic stresses

After analyzing tomato plants transformed with GalUR gene for their ascorbic acid contents, it was found that some transgenic lines contained higher levels of ascorbic acid compared to control plants. In the present study, callus induction rate was 50.2 % in the explant and shoot regeneration rate was 51.5 % from the callus with transformation efficiency of 3.0 %. Based on PCR and Southern blot analysis, three independent transformants containing the insert gene were selected. Phenotypic traits of these transgenic progeny were similar to those of control tomatoes. Tomatoes (H15) with high fruit ascorbic acid contents were selected for next generation (GalUR T₃) analysis. Transgenic tomatoes with increased ascorbic acid contents were found to be more tolerant to abiotic stresses induced by viologen, NaCl, or mannitol than non-transformed plants. In leaf disc senescence assay, the tolerance of these transgenic plants was better than control plants because they could retain higher chlorophyll contents. Under salt stress of less than 200 mM NaCl, these transgenic plants survived. However, control plants were unable to survive such high salt stress. Ascorbic acid contents in the transgenic plants were inversely correlated with MDA contents, especially under salt stress conditions. The GalUR gene was expressed in H15 tomatoes, but not in control plants. Higher expression levels of antioxidant genes (APX and CAT) were also found in these transgenic plants compared to that in the control plants. However, no detectable difference in SOD expression was found between transgenic plants and control plants. Results from this study suggest that the increase in ascorbic acid contents in plants could up-regulate the antioxidant system to enhance the tolerance of transgenic tomato plants to various abiotic stresses.     

两个反义基因在番茄工程植株中的生理抑制效应分析

用番茄乙烯形成酶（ＥＦＥ）和多聚半乳糖醛酸酶（ＰＧ）反义ｃＤＮＡ转化番茄子叶,获得两个转基因系统。分别比较了两个基因系统果实和叶片的乙烯生成速率、果实中ＥＦＥ酶活性和果胶酶活性,表明反义ＥＦＥ基因在番茄工程植株中能显著抑制ＥＦＥ酶活性和乙烯生成;反义ＰＧ基因则主要是抑制其ＰＧ酶活性。     

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

Key message

The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.

Abstract

Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.     

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.     

Overexpression of the lemon basil alpha-zingiberene synthase gene increases both mono- and sesquiterpene contents in tomato fruit

alpha-Zingiberene synthase (ZIS), a sesquiterpene synthase gene that was isolated from lemon basil (Ocimum basilicum L.), encodes an enzyme that catalyzes the formation of alpha-zingiberene, and other sesquiterpenes, from farnesyl diphosphate. Transgenic tomato fruits overexpressing ZIS under the control of the fruit ripening-specific tomato polygalacturonase promoter (PG) accumulated high levels of alpha-zingiberene (224-1000 ng g(-1) fresh weight) and other sesquiterpenes, such as alpha-bergamotene, 7-epi-sesquithujene, beta-bisabolene and beta-curcumene, whereas no sesquiterpenes were detected in non-transformed control fruits. The ZIS-transgenic fruits also produced monoterpenes, such as alpha-thujene, alpha-pinene, beta-phellandrene and gamma-terpinene (1-22 ng g(-1) fresh weight), which were either not detected or were found only in minute concentrations in control fruits. Recombinant ZIS overexpressed in Escherichia coli catalyzed the formation of these monoterpenes from geranyl diphosphate. As the ZIS protein apparently lacks a transit peptide, and is localized in the cytosol, the production of monoterpenes in the transgenic tomatoes suggests that a pool of geranyl diphosphate is available in the cytosol. The phenotype of the ZIS-transgenic tomatoes was the same as that for wild-type tomatoes, with regard to plant vigor and shape, but transgenic plants exhibited a small decrease in lycopene content. This study thus showed that the synthesis of both mono- and sesquiterpenes can be enhanced by the ectopic expression of a single transgene in tomato fruit, and it further demonstrated the interconnection between the pools of terpenoid precursors in the plastids and the cytosol.     

Early anther ablation triggers parthenocarpic fruit development in tomato

Fruit set and fruit development in tomato is largely affected by changes in environmental conditions, therefore autonomous fruit set independent of fertilization is a highly desirable trait in tomato. Here, we report the production and characterization of male‐sterile transgenic plants that produce parthenocarpic fruits in two tomato cultivars (Micro‐Tom and Moneymaker). We generated male‐sterility using the cytotoxic gene barnase targeted to the anthers with the PsEND1 anther‐specific promoter. The ovaries of these plants grew in the absence of fertilization producing seedless, parthenocarpic fruits. Early anther ablation is essential to trigger the developing of the transgenic ovaries into fruits, in the absence of the signals usually generated during pollination and fertilization. Ovaries are fully functional and can be manually pollinated to obtain seeds. The transgenic plants obtained in the commercial cultivar Moneymaker show that the parthenocarpic development of the fruit does not have negative consequences in fruit quality. Throughout metabolomic analyses of the tomato fruits, we have identified two elite lines which showed increased levels of several health promoting metabolites and volatile compounds. Thus, early anther ablation can be considered a useful tool to promote fruit set and to obtain seedless and good quality fruits in tomato plants. These plants are also useful parental lines to be used in hybrid breeding approaches.     

Effect of simultaneous down-regulation of pectate lyase and endo-β-1,4-glucanase genes on strawberry fruit softening

Strawberry is a soft fruit with a short postharvest shelf-life. The loss of fruit firmness during ripening is mainly due to the disassembly of parenchyma cell walls mediated by the expression of genes encoding enzymes acting on pectins, such as pectate lyase, or hemicellulose, e.g. endo-β-1,4-glucanase. To determine if the simultaneous down-regulation of FaplC and FaEG3 genes, encoding a pectate lyase and a endo-β-1,4-glucanase, respectively, exerted an additive effect on strawberry softening, transgenic plants expressing tandem antisense sequences of both genes under the control of the constitutive promoter CaMV35S were generated. Fifteen independent transgenic lines were obtained and fruit yields and several quality parameters of transgenic ripe fruit were recorded during two consecutive years. Fruit yield was reduced in most of the lines, especially in the first evaluation period, and five out of 15 lines (33 %) did not set fruit. The expression of FaplC and FaEG3 genes was measured in ripe fruits from six selected lines showing the highest fruit yields. All selected lines showed a high level of FaplC gene silencing, ranging from 97 to 71 %; however, FaEG3 gene expression was only significantly down-regulated in two lines. Fruit colour and soluble solids contents were similar in control and transgenic ripe fruits, while fruit weight was slightly lower than control in some of the lines. In all lines, transgenic fruits were significantly firmer than control, with an increase in firmness ranging from 19 to 32 %. The reduction of fruit softening in transgenic fruits was not correlated with the suppression of FaEG3 gene expression, and lines with the highest simultaneous down-regulation of FaplC and FaEG3 showed similar fruit firmness to lines where only FaplC was suppressed. These results indicate that pectate lyase and endo-β-1,4-glucanase do not act in an additive or synergistic way during strawberry softening, and question the role of glucanases in this process.     

Genetic characterization of chayote [<Emphasis Type="Italic">Sechium edule</Emphasis> (Jacq.) Swartz.] landraces of North Eastern Hills of India and conservation measure

Chayote or chow–chow is an underutilized cucurbit vegetable crop, widely cultivated by farmers in the backyards and Jhum lands for its tender leaves, fruits and tuberous root. In order to initiate crop improvement program in this crop, the present study was undertaken to assess the genetic variations in the 74 chow–chow landraces collected from the North Eastern Hill region of India. Wide variations for fruit colors, fruit length (6.5–21.5 cm), fruit width (4.2–10.7 cm), fruit weight (60–560 g), vitamin-C (2.6–13.8 mg/100 g), reducing sugar (0.18–2.77%), total sugar (1.09–2.94%) and phenol content (0.17–3.85 mg/100 g FW) were recorded among the landraces. All the landraces were also characterized using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. In RAPD analyses, out of 28 primers a total of 198 reproducible amplicons were formed at an average of 7.01 per primer and an overall polymorphism of 88.38%. Eight fragments were specific to landraces with light green fruits. Four fragments were observed to be specific to RCSC-22 (dark green fruits) and another four specific to a RCSC-30 (pale yellow fruits). Out of 30 ISSR, only 5 primers generated a total of 32 reproducible amplicons with an average of 6.4 per primer and overall polymorphism of 62.5%. The pair wise similarity coefficient values ranged from 0.55 to 0.96. The grouping of landraces in cluster analysis was found to be independent of their respective geographic locations. The cuttings of suckers and shoot top (2 months old) treated with indole-3-butyric acid (200 mg l^?1) provide an alternative for the conservation of the diverse genetic materials to the researchers.     

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0 ± 1.43 (ST2) and 36.6 ± 0.99 (35S3) aphids per plant, which corresponds to 24.9–53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future.     

Overexpression of yeast spermidine synthase impacts ripening,senescence and decay symptoms in tomato

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit‐ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8‐ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild‐type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation‐related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd‐mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S‐ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole‐plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post‐harvest senescence and decay.     

An Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <10% of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process.     

Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice

Cholera toxin B (CTB) subunit is a well-characterized antigen against cholera. Transgenic plants can offer an inexpensive and safe source of edible CTB vaccine and may be one of the best candidates for the production of plant vaccines. The present study aimed to develop transgenic tomato expressing CTB protein, especially in the ripening tomato fruit under the control of the tomato fruit-specific E8 promoter by using Agrobacterium-mediated transformation. Transgenic plants were selected using PCR and Southern blot analysis. Exogenous protein extracted from leaf, stem, and fruit tissues of transgenic plants was detected by ELISA and Western blot analysis, showing specific expression in the ripening fruit, with the highest amount of CTB protein being 0.081% of total soluble protein. Gavage of mice with ripe transgenic tomato fruits induced both serum and mucosal CTB specific antibodies. These results demonstrate the immunogenicity of the CTB protein in transgenic tomato and provide a considerable basis for exploring the utilization of CTB in the development of tomato-based edible vaccine against cholera. The rCTB antigen resulted in much lower antibody titers than an equal amount of exgenous CTB in trangenic fruits, suggesting the protective effect of the fibrous tissue of the fruit to the exogenous CTB protein against the degradation of protease in the digestive tracts of mice. Xiao-Ling Jiang and Zhu-Mei He contributed equally to this work.     

Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.