Establishment of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> O. Swartz)

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass with great salinity tolerance. Based on establishment of embryogenic callus induction and regeneration from different mature seeds of ‘Sea Spray’, an Agrobacterium tumefaciens-mediated transformation was established and optimized in this study. Three clones of callus were selected for examining transformation conditions using Agrobacterium tumefaciens strain AGL1 carrying the binary vector pCAMBIA1305.2, containing β-glucuronidase (GUS) as a reporter gene and hygromycin phosphotransferase (HPT) as a selective marker gene. The results showed that a high transient transformation efficiency was observed by using Agrobacterium concentration of OD₆₀₀?=?0.6, 5 min of sonication treatment during Agrobacterium infection, and 2 d of co-cultivation. By using the optimized transformation conditions, transgenic seashore paspalum plants were obtained. PCR and Southern blot analysis showed that T-DNA was integrated into the genomes of seashore paspalum. GUS staining experiments showed that the GUS gene was expressed in transgenic plants. Our results suggested that the transformation protocol will provide an effective tool for breeding of seashore paspalum in the future.     

A modified <Emphasis Type="Italic">in planta</Emphasis> method of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

Plant transformation has emerged as an important tool to integrate foreign genes in the plant genome to modify the plants for desired traits. Though many techniques of plant transformation are available; getting single copy transgenic events and cost associated remains a big challenge. Thus Agrobacterium-mediated transformation remains the method of choice due to multiple advantages. In the present work a tissue culture free protocol of Agrobacterium-mediated transformation was optimized in safflower, an oil seed crop recalcitrant to transformation. As a proof of concept we selected pCAMBIA2300 gene cassette containing Arabidopsis specific delta 15 desaturase (FAD3) downstream to truncated seed specific promoter beta-conglycinin and optimized tissue culture free protocol of Agrobacterium-mediated transformation using embryos as explants. Addition of silwet L-77, sonication treatment, vacuum infiltration in infection medium and use of paper wicks in co-cultivation period increased the transformation efficiency to 19.3%. Further, success in transformation was confirmed via product accumulation in 21 independent transgenic events wherein oil in transformed seeds showed significant accumulation of alpha-linolenic acid (ALA; 18:3; n3) which is generated from linoleic acid (LA; 18:2; n3) in a FAD3 catalyzed reaction. The present protocol can be utilized to produce transgenic safflower with different desired characters.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and direct plant regeneration in four cultivars of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

We have developed an improved Agrobacterium-mediated transformation and rapid regeneration system for four cultivars (‘CO(Ra)-14’, ‘PR-202’, ‘Try-1’ and ‘Paiyur-2’) of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentration of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%) was achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T₀ and T₁ generations to confirm the gene integration. Six events from T₀ had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

In vitro regeneration, <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation,and genetic assay of chalcone synthase in the medicinal plant <Emphasis Type="Italic">Echinacea pallida</Emphasis>

In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.     

Generation of Backbone-Free,Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome

In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.Since the generation of transgenic plants approximately 25 years ago, Agrobacterium tumefaciens has been widely used for introducing genes into plants for purposes of basic research as well as for generation of commercially used transgenic crops. For plant transformation, the gene of interest is placed between the left and right border repeats of Agrobacterium transferred (T)-DNA (Gelvin, 2003). The T-DNA region harboring the transgene is stably integrated into the plant genome by using an appropriate plant transformation protocol. T-DNA originates from the Agrobacterium tumor-inducing (Ti) plasmid. Because Ti plasmids are large and difficult to manipulate, smaller T-DNA binary vectors are currently predominately used for generation of transgenic plants (de Framond et al., 1983; Lee and Gelvin, 2008).Although Agrobacterium has been used for plant transformation for more than two decades, problems using this bacterium remain. Agrobacterium-mediated transformation generally results in lower transgene copy numbers than do other transformation methods such as particle bombardment or polyethylene glycol-mediated transformation (Kohli et al., 1998; Shou et al., 2004). On the other hand, transformation frequently results in unwanted high copy number T-DNA integration events (Jorgensen et al., 1987; Deroles and Gardner, 1988; Shou et al., 2004; De Buck et al., 2009). Multiple integration events, often coupled with inverted repeat T-DNA integration patterns, may affect the stability of transgene expression by silencing mechanisms (Jorgensen et al., 1996). An additional problem with Agrobacterium-mediated transformation is the propensity for DNA sequences outside the T-DNA region to integrate into the plant genome (Kononov et al., 1997; Wenck et al., 1997; Shou et al., 2004). Integration of such vector backbone sequences can occur with high frequency. For example, Kononov et al. (1997) detected backbone sequences in 75% of tested transgenic tobacco (Nicotiana tabacum) plants, and very often the entire vector backbone is introduced into the plant genome (De Buck et al., 2000). T-DNA vector backbones usually harbor bacterial antibiotic resistance genes that can create governmental regulatory concerns.Here we show that launching T-DNA from the A. tumefaciens chromosome reduces integrated transgene copy number and almost eliminates the presence of T-DNA backbone sequences. We describe several plasmids and bacterial strains to facilitate use of this methodology.     

Marker-free PLRV resistant potato mediated by Cre-<Emphasis Type="Italic">loxP</Emphasis> excision and RNAi

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.     

Rapid and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis>) employing standard binary vectors and <Emphasis Type="Italic">bar</Emphasis> gene as a selectable marker

Key message

A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker.

Abstract

Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7–12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40–50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

     

A simple,rapid and systematic method for the developed GM rice analysis

We generated 383 independent transgenic lines that contained the PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes under the control of a constitutive (CaMV 35S) promoter to generate genetically modified (GM) rice. TaqMan copy number assay was performed to determine the copy numbers of inserted T-DNA. Flanking sequence tags (FSTs) were isolated from 203 single copy T-DNA lines of transgenic plants, and their sequences were mapped to the rice chromosomes. Of the 157 flanking sequence tags that were isolated from single copy lines, transgenes were found to be integrated into genic regions in 58 lines (36 %), whereas 97 lines (62 %) contained transgene insertions in intergenic regions. Approximately 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assays. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNA from leaf tissue of homozygous T1 plants with a single copy and an intergenic insertion of T-DNA. The mRNA expression levels of the examined transgenic rice were significantly increased in all transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants displayed higher levels of transgene protein. Using numerical data for the mass production of transgenic plants can reduce the time required to obtain a genetically modified plant. Moreover, the duration, cost, and efforts required for transformation can be deliberately predicted. These results may be useful for the large-scale production of transgenic plants or T-DNA inserted rice mutants.     

High throughput transcriptome analysis of coffee reveals prehaustorial resistance in response to <Emphasis Type="Italic">Hemileia vastatrix</Emphasis> infection

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.     

Development of transgenic pigeonpea using high throughput plumular meristem transformation method

Tissue culture based poor regeneration along with restricted rooting responses are considered to be major hindrances for in vitro transgenic pigeonpea development. Present study was designed to establish a novel method of Agrobacterium tumefaciens mediated plumular meristem transformation in pigeonpea for improvement of transgenic development frequency. Three days old decapitated seedlings of pigeonpea cultivar ICPL 87119 were pricked at plumular meristem region under in vitro conditions. After infecting with Agrobacterium binary vector pBI121, the explants were co-cultivated in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified- Murashige and Skoog medium. Transformed seedling with well-developed tap root system were established in soil. GUS activity as well as PCR based confirmation of transgene presence was demonstrated in transgenic events. Transformation frequency of 72% was achieved for the first time in pigeonpea. Further, kanamycin mediated stringent selection was used for the screening of T₁ seeds. Established T₁ progenies were analysed by PCR and Southern blot, to confirm transgene integration and copy number, respectively. This is the first report of transgenic pigeonpea development, where the combination of culture based Agrobacterium-infection and culture independent plant establishment, coupled with PCR based selection method was found to be most preferable for faster and frequent establishment of transgenic plants. This method will contribute to large scale transgenic pigeonpea development for its improvement and satisfy the requirement of routine transformation experiments for T-DNA insertion mutagenesis.     

Olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) plants transgenic for tobacco osmotin gene are less sensitive to in vitro-induced drought stress

Olive is one of the most important tree crops in the Mediterranean region, because of its ability to grow and produce acceptable yields under limited water availability. In this study, the drought tolerance of an olive cultivar Canino was compared to the performance of its derived transgenic line expressing osmotin gene from tobacco, obtained by Agrobacterium-mediated transformation of Canino cultivar. Shoot cultures of both wild-type (wt) and transgenic lines were exposed to drought stress over a 28-day period, and their differential responses to in vitro-drought stress were investigated. After exposure to PEG, most of the shoots from wt plants resulted in damage and exhibited decreased levels of chlorophyll, while those of transgenic line did not show injuries and showed a normal growth even when exposed to the highest PEG concentration (4%). After preliminary evaluation we characterized Canino AT17-1, by measuring several physiological parameters, including the activities of the antioxidant enzymes (POD and CAT), and the content of malondialdehyde (MDA). Both the activity of catalase and the proline content were higher in the leaves of the transgenic shoots compared to wt plants. Consequently, it was observed that the transgenic line accumulated less MDA indicating that the presence of the osmotin gene protected the cell membrane from damage by lipid peroxidation. Together, these results could suggest that the transgenic line Canino AT17-1 was more efficient in the activation of defense responses against oxidative stress with respect to the Canino wt. The further finding that the transgenic shoots also showed higher proline accumulation supported the hypothesis that the osmotin gene conferred to transgenic shoots increased tolerance to drought stress compared with the wt.     

Genetic transformation and evaluation of two sweet sorghum genotypes for resistance to spotted stemborer, <Emphasis Type="Italic">Chilo partellus</Emphasis> (Swinhoe)

Sweet sorghum is a climate smart crop with multiple uses. The crop is susceptible to attack by the spotted stemborer, Chilo partellus (Swinhoe). This causes deadheart formation, leading to lodging of plants and consequent high economic losses. Lack of stable sources of resistance make any genetic enhancement through breeding difficult. We report a study to build up host plant resistance using transgenic technology by introducing two different classes of Bt genes (cry1Aa and cry1B) into two elite sweet sorghum genotypes of India (SSV84 and RSSV9). We devised tissue culture methods to suit the genotypes of our interest, SSV84 and RSSV9, and employed two methods of genetic transformation: the particle bombardment and in planta method of Agrobacterium. Modification of in vitro culture methods involved subculture every 3 days in the initial stages of culture and the use of precultured embryos as target tissues. For the in planta method, a floral dip for 1 h in Agrobacterium suspension supplemented with l-cysteine and Tween-20 was used. Sixteen transgenic events were generated; inheritance, integration and stable expression of the transgenes till the T₄ generation were confirmed. The amount of Bt Cry1Aa protein at 25–30 days of growth ranged from 24.8 to 72.8 ng/g of fresh leaf tissue. We recorded 78.4 % larval mortality, reduced leaf damage (3.0 out of 9.0) and reduced feeding (41.0 %) over the controls in insect feed assays. Stable inheritance and expression in the in planta-derived transgenics are presented.     

Promotion of artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by overexpression of multiple artemisinin biosynthetic pathway genes

Artemisinin, isolated from an annual herbaceous plant Artemisia annua L., is an effective antimalarial compound. However, artemisinin is accumulated in small amounts (0.01–0.1% leaf dry weight) in A. annua, resulting in constant high artemisinin price. Although metabolic engineering of partial artemisinin metabolic pathway in yeast achieved great success, artemisinin from A. annua is still the important business resource. Here, we report on the generation of transgenic plants with simultaneously overexpressing four artemisinin biosynthetic pathway genes, amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene 12-monooxygenase gene (CYP71AV1), cytochrome P450 reductase gene (CPR), and aldehyde dehydrogenase 1 gene (ALDH1) via Agrobacterium-mediated transformation. The qRT-PCR analysis demonstrated that the introduced four genes of the transgenic lines were all highly expressed. Through high-performance liquid chromatography analysis, the artemisinin contents were increased markedly in transformants, with the highest being 3.4-fold higher compared with non-converter. These results indicate that overexpression of multiple artemisinin biosynthetic pathway genes is a promising approach to improve artemisinin yield in A. annua.     

T-DNA-induced mutations in transgenic plants

The review surveys experimental data on changes of individual traits in genetically modified (transgenic) plants. The attention is focused on mutations induced by T-DNA insertions upon Agrobacterium-induced transformation of dicotyledonous plants. The character of mutation appearance in transgenic plants is examined. The prospects of mutations induced by T-DNA insertions are considered.