Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T₁ transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T₂ generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.     

Expression of ipt gene controlled by an ethylene and auxin responsive fragment of the LEACO1 promoter increases flower number in transgenic Nicotiana tabacum

Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO1_{0.821 kb}-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25°C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO1_{0.821 kb}-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO1_{0.821 kb} promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO1_{0.821 kb} promoter fragment. The LEACO1_{0.821 kb}-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit.     

Construction of a GFP-BAR plasmid and its use for switchgrass transformation

 A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l^–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T₁ plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene. Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs

The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to obtain RNA-mediated resistance at high efficiency. To test this observation as a possible method for high frequency induction of CMV resistance, Nicotiana benthamiana plants were transformed with transgenes designed to produce double strand RNA molecules of CMV RNA 2 or coat protein (CP) gene sequences. Seventy-five percent of the tested R₀ plants transformed with an RNA 2-derived inverted repeat construct (1534 nt CMV sequence) showed extreme resistance to CMV, while a lower percentage of resistance (30%) was observed in R₀ lines transformed with a similar construct of a shorter viral RNA 2 sequence (490 nt). The resistance level conferred by CP sequences was also efficient by using a dsRNA construct, reaching a level of 50%. Self-pollinated (S₁) progenies obtained from most resistant R₀ plants all showed resistance levels of 100%, perfectly correlating with the expression of transgenic siRNAs. The results indicate that the use of inverted repeat viral transgenes is a highly efficient approach to obtain CMV resistant transgenic plants. Consequently, only a handful of transgenic plants will have to be generated using such constructs for successful resistance, which enables the implementation of this protocol for crops that are difficult to transform, such as ornamental plants in which CMV is an important pathogen.     

Antimicrobial peptides fromMirabilis jalapa andAmaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

The cDNAs encoding the seed antimicrobial peptides (AMPs) fromMirabilis jalapa (Mj-AMP2) andAmaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolleet al., Plant Mol Biol 28:713–721 (1995) and 22:1187–1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195–1206 (1991)]; an Ac-AMP2wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. Thein vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with eitherBotrytis einerea orAlternaria longipes.     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

 A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T₀, T₁, and T₂ transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants. Received: 11 August 1998 / Accepted: 26 August 1998     

Decreased peroxidase activity in transgenic tobacco and its effect on lignification

An antisense gene construct of a peroxidase gene (Shpx6a) from a tropical pasture legume Stylosanthes humilis was transferred into tobacco cells via Agrobacterium tumefaciens to test whether peroxidase activity could be decreased and what effect this would have on lignification. A large number of tobacco cell lines were regenerated on selective media and stable integration of the transgene was confirmed in randomly selected putative transformants. Analyses of the primary transgenic plants and their progeny (T ₁) demonstrated that the total peroxidase activity was significantly decreased (up to 36%) as compared to that measured in untransformed control plants. Importantly, reduction in peroxidase activity is accompanied by decreases (up to 23%) in lignin content in several transgenic lines.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

The DefH9-iaaM-containing construct efficiently induces parthenocarpy in cucumber

Parthenocarpy (seedless fruits) is a desirable trait that has been achieved in many plant cultivars. We generated parthenocarpic cucumber fruits by introducing the chimeric DefH9-iaaM construct into the cucumber genome using an Agrobacterium tumefaciens-mediated protocol. The construct consists of the DefH9 promoter from Antirrhinum majus and the iaaM coding sequence from Pseudomonas syringae. Transgenic plants were obtained from nine independent transformation events: half of these were tetraploid and did not produce seeds following self-pollination, while the remaining half were capable of displaying parthenocarpy in the subsequent reproductive generation. Of the fruits produced by the transgenic lines, 70–90% were parthenocarpic. The segregation of the marker gene in the transgenic T₁ progeny indicated single gene inheritance. The seed set in the transgenic lines and their F₁ hybrids was lower than in the non-transgenic control plants. Some of the methodological details and the practical significance of the results are discussed. This paper is dedicated to Prof. Juergen Grunewaldt from Hannover on the occasion of his retirement.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

Antisense RNA approach targeting Rep gene of Mungbean yellow mosaic India virus to develop resistance in soybean

The Yellow mosaic disease is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Yellow mosaic disease (YMD) is a major constraint to the production of soybean in South-East Asia. In India, yield losses of 10–88% had been reported due to YMD of soybean. An effort has been made to generate resistant soybean plants, by a construct targeting replication initiation protein (Rep) gene sequences of MYMIV. A construct containing the sequences of Rep gene (566?bp) in antisense orientation was used to transform cotyledonary node explants of three soybean cultivars (JS 335, JS 95-60 and NRC 37). Transformation efficiencies of 0.2, 0.21 and 0.24% were obtained with three soybean cultivars, JS 335, JS 95-60 and NRC 37, respectively. The presence of transgene in T₁ plants was confirmed by polymerase chain reaction (PCR) and sequence analysis. The level of resistance was observed by challenge inoculation with the virus in T₁ lines. The inheritance of transgene showed classical Mendelian pattern in six transgenic lines.     

The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S ₂ -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S ₂ RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S ₂ -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S ₂ polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S ₂ -RNase protein in pollen.     

Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R₁ kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R₁ kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R₃ seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R₃ seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.