Agrobacterium Tumefaciens-mediated Genetic Transformation Research Articles

Plant regeneration and Agrobacterium-mediated transformation of the miracle tree Neolamarckia cadamba

Neolamarckia cadamba is a miracle tree species with considerable economic potential used as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish an efficient protocol of plant regeneration and Agrobacterium-mediated genetic transformation for N. cadamba. Greenish callus were induced from different type explants when cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) and kinetin (KT). The callus could differentiate into somatic embryos or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. It was found that MS medium supplemented with 2 mg/L TDZ, 0.2 mg/L KT provided the most suitable medium for leaf explants to induce adventitious shoot, delivering a maximum regeneration frequency with a mean of 8.2 shoots per explant. The micro-shoots developed roots in MS supplemented with 0.05 mg/L α-naphthaleneacetic acid (NAA), and could be successfully acclimatized to a greenhouse with more than 90% survival. Based on this regeneration protocol, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for N. cadamba. Genetic transformation was obtained through the infection of leaf with A. tumefaciens strain GV3101 harboring a modified binary vector pCAMBIA1305 containing the marker genes β-glucuronidase (GUS) and fluorescent protein YFP. The presence of GUS in transformed plants was confirmed using PCR and histological stain, while the expression of YFP was confirmed through fluorescent microscopy and RT-PCR. Out of 276 putative transformants, 15 plants were found to be positive transgenic. Nevertheless, by adopting this protocol, positive transformed plants could be obtained within three months.

Biotechnological advances in litchi (Litchi chinensisSonn.): present scenario and future prospects in crop improvement – a review

Crop improvement program in litchi is mainly concerned with the development of cultivars adapted to a wide range of climatic conditions, extended harvesting season and quality fruits with improved shelf life. Conventional breeding methods alone do not hold much promise in crop improvement due to the complications related to long juvenile phase, narrow genetic base, high genetic heterozygosis, lack of information about inheritance pattern of specific traits and polygenic nature of several important traits. In addition to this, in intervarietal and interspecific hybridization, the requirement of repeated backcrossing to homogenize and fix desired traits as well as additional breeding required to eliminate the unwanted traits that come along during such hybridization makes conventional breeding more cumbersome and time-consuming. Thus it becomes inevitable to evaluate the potential of biotechnological approaches for complementing and expediting the conventional breeding methods. In litchi, the use of biotechnological approaches for crop improvement is still in its infancy. However, in the past few years several studies involving in vitro regeneration, micro-propagation, genetic transformation, and marker assisted selection, etc. has been conducted at various centers around the world. Somatic embryogenesis and plant regeneration has been claimed in various genotypes of litchi. Agrobacterium tumefaciens-mediated genetic transformation is also reported. Cloning of ACC oxidase gene to enhance the storage life of fruit has been attempted. A high density molecular linkage map constructed for litchi based on AFLP and RAPD markers can provide a strong foundation for QTL location or molecular-assisted breeding. Use of molecular markers for identification and detection of cultivars and hybrids, respectively, can help in reducing the breeding cycle, hence making it more efficient. Production of synthetic seeds in litchi by encapsulating somatic embryos, shoot buds, etc. can be helpful in propagation, short-term storage and germplasm exchange and needs intensive research. In vitro conservation of embryogenic calluses has been identified as one of the methods to conserve the germplasm of litchi, thus reducing the space requirement and labor cost. The main objective of this review is to investigate the biotechnological advances made in litchi till date and their future implications in improvement of this crop.

Embryogenic Callus as Target for Efficient Transformation of Cyclamen persicum Enabling Gene Function Studies.

Cyclamen persicum is an ornamental plant with economic relevance in many parts of the world. Moreover, it can be regarded as an applied model for somatic embryogenesis, since transcriptomic, proteomic, and metabolomic comparisons have revealed insights into this regeneration process on the molecular level. To enable gene function analyses, the aim of this study was to establish an efficient Agrobacterium tumefaciens-mediated genetic transformation protocol for C. persicum. For the first time, embryogenic callus cultures were used as a target material. The advantages of embryogenic callus are the defined and known genotype compared to seedlings, the high regeneration potential and the stability of the regenerated plants. A. tumefaciens strains EHA105 and LBA4404 were most efficient for transformation, resulting in transformation efficiencies of up to 43 and 20%, respectively. In regenerated plants, the presence of the transgenes was verified by PCR, Southern hybridization, and a histochemical GUS assay. The protocol was applied successfully to two C. persicum genotypes. Moreover, it served to transfer two reporter constructs, the auxin-responsive promoter DR5 driving the gus gene and the redox sensor roGFP2_Orp1, to the C. persicum genotypes, allowing the localization of high auxin concentrations and reactive oxygen species in order to study their roles in somatic embryogenesis in the future. For success in transformation, we regard the following factors as important: highly embryogenic cell lines, the use of Silwet® L-77 as a surfactant during co-culture, a genotype-specific appropriate selection schedule with hygromycin, and A. tumefaciens strains EHA105 and LBA4404.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.

Establishment of an Efficient Plant Regeneration Culture Protocol and Achievement of Successful Genetic Transformation in Jatropha curcas L.

An efficient and reproducible protocol is described for shoot-bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of J. curcas. Treating the explants with high concentrations (5–120 mg/L) of TDZ for short durations (5–80 min) before inoculation culture increased significantly the regeneration frequency and improved the quality of the regenerated buds. The highest shoot-buds induction rate (87.35%) was achieved when petiole explants were treated with 20 mg/L TDZ solution for 20 min and inoculated on hormone-free MS medium for 30 days. Regenerated shoots of 0.5 cm or a little longer were isolated and grafted to seedling stocks of the same species, and then the grafted plantlets were planted on half-strength MS medium containing 0.1 mg/L IBA and 2 mg/L sodium nitroprusside (SNP). This grafting strategy was found to be very effective, to obtain that healthy grafted plantlets ready for acclimatization within 20 days. By the above mentioned protocol and with general Agrobacterium–mediated genetic transformation methods only 65 days were needed to obtain intact transgenic plants.

Cloning of fatty acid desaturase-coding sequence (Lufad3) from flax and its functional validation in rice

Flax contains very high levels of alpha-linolenic acid (57%) and a fatty acid desaturase 3-coding sequence (Lufad3) of flax has been amplified from the RNA isolated from developing seeds. The deduced amino acid sequence of LuFAD3 showed the presence of three histidine motifs, six membrane spanning domains and an endoplasmic reticulum (ER) retention signal KSK, indicating its plausible localization into the ER. Flax is not amenable for genetic transformation and not suitable for functional validation of Lufad3 gene. Hence, rice with well-developed genetic transformation has been selected as heterologous host system. Coding sequence of Lufad3 driven by maize Ubi1 promoter has been introduced into indica rice by Agrobacterium tumefaciens-mediated genetic transformation. Southern analysis of putative transformants (T 0) revealed signals at variable lengths at >5 kbp indicating random integration of transgene into the genomes of different transformants. The Mendelian segregation observed for selectable marker gene hyg in both T1 and T2 generations confirmed stable inheritance and single-site integration of transgenes. As compared to untransformed control (UC), homozygous transgenic rice expressing Lufad3 showed higher levels of essential α-linolenic acid in leaves and seeds validating its functionality.

Standardization of Agrobacterium tumefaciens-mediated genetic transformation protocol in Punica granatum L. cv. Kandhari Kabuli

A protocol for Agrobacterium tumefaciens-mediated genetic transformation in Punica granatum cv. Kandhari Kabuli has been developed using cotyledon and hypocotyl explants via both direct and indirect organogenesis pathways. Agrobacterium tumefaciens strain LBA 4404 harboring gus and npt-II genes in a binary vector pBI 121 had been used for genetic transformation studies. Pre-culturing of explants for 48 h (2 days) on regeneration medium before agrobacterial infection significantly increased the transformation frequency. Co-cultivation period of 48 h (2 days) was found to be optimum for genetic transformation. Cefotaxime in a concentration of 500 mg/l was found optimal for controlling the excessive bacterial growth and transformed shoots were selected using 50 mg/l kanamycin. Only the transformed cells were able to grow on the selective regeneration medium. Transgenic shoots were rooted on half-strength MS medium supplemented with 0.10 mg/l NAA, 0.02% activated charcoal, 50 mg/l kanamycin and 500 mg/l cefotaxime and subsequently acclimatized to the culture room conditions. Stable transformation and expression were confirmed on the basis of GUS histochemical and spectrophotometric assays. This genetic transformation protocol will provide a platform for genetic manipulation of pomegranate for incorporation of genes governing various horticulturally important traits in future.

Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex.

Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex.

English

An efficient method for Agrobacterium tumefaciens-mediated genetic transformation of Brussels sprouts was developed. We tested two strains, LBA4404 and EHA105 both with the binary vector p35SGUSINT containing the NPT11 gene for kanamycin resistance. Explants were derived from in vitro-grown seedlings of two commercial cultivars, Winter Pick and Troika. Transformation and regeneration efficiency were based on transient GUS expression and the ability of the explants to grow on media supplemented with kanamycin. Factors influencing GUS expression and shoot regeneration included the plant genotype, the A. tumefaciens strain, and the explant type. Winter Pick had significantly (P ≤ 0.05) higher GUS expression, up to 44.3% in leaf sections compared to 25.6% in Troika, and significantly (P ≤ 0.05) higher shoot regeneration. Agrobacterium strain LBA4404 was easier to control in culture and it achieved significantly (P ≤ 0.05) higher infection rate, 46.1 %, than EHA105 (22.0%), ultimately achieving higher shoot regeneration. The length and temperature of co-cultivation did not significantly affect GUS expression, but co-cultivation for 2 d at either 21°C or 24°C resulted in more and healthier shoots as opposed to the vitreous shoots obtained from 3 d and 5 d co-cultivation periods. Compared to hypocotyl and petiole explants, leaf sections were the best, with highest GUS expression (44.8%) and moderate shoot regeneration of 45.8% on MS medium with 2.0 mg L -1 BAP, 5.0 m L -1 AgNO3 and 50 mg L -1 kanamycin. This optimised protocol can be used to introduce the thaumatin gene for sweetness to mask the bitter taste in Brussels sprouts.

A simple and efficient Agrobacterium tumefaciens-mediated plant transformation of Brassica rapa ssp. pekinensis.

The present study aims to investigate the numerous factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Chinese cabbage (Brassica rapa ssp. pekinensis). Factors affecting transformation efficiency, such as age of explants, Agrobacterium concentration, and effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were examined. The pre-cultured hypocotyls from young seedlings prior to exposure to Agrobacterium showed higher shoot regeneration. The plant transformation with the modest A. tumefaciens concentrations (0.8 OD) and the 3 days co-cultivation periods increased transformation efficiency. Plant growth hormones [1-naphthyl acetic acid (NAA) and 6-benzyl amino purine (BAP)] were essential for callus and shoot formation. Root formation was effective in half strength MS medium without supplementation of root-inducing hormones. To maintain selection pressure, plant subculture was carried out every 2 weeks with selective antibiotics. The putative transgenic plants were acclimatized in the greenhouse. Polymerase chain reaction was performed to confirm the integration of T-DNA into the genome of transgenic plants. A transformation efficiency of 15 % was obtained. This protocol allows effective transformation and indirect regeneration of Brassica rapa.

Agrobacterium-mediated transformation and plant regeneration in Chinese dwarf cherry [Cerasus humilis (Bge.) Sok

ABSTRACTChinese dwarf cherry [Cerasus humilis (Bge.) Sok] is a small deciduous shrub which produces fruit that contain high concentrations of nutritional and medicinal compounds. It is also highly drought and cold tolerant, so it has been exploited for soil and water conservation purposes. Using mature leaves as initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system has been developed for Chinese dwarf cherry. Leaf segments from cultivar ‘99-02’ were successfully transformed with Agrobacterium strain LBA4404 harbouring the vector prolC1 which contained the rolC gene, the neomycin phosphotransferase gene (nptII), and the β-glucuronidase gene (gus). Transformation was confirmed using a histochemical β-glucuronidase (GUS) assay, by polymerase chain reaction (PCR) amplification, and by reverse transcription-PCR of the rolC gene. Transgenic shoots of Lines 1 and 2 showed significant increases in root numbers and root lengths compared to non-transformed control shoots on medium without any added auxin or with a low concentration of auxin. This optimised genetic transformation procedure will provide a tool for future molecular breeding of Chinese dwarf cherry.

Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops

Steady increase in global population poses several challenges to plant science research, including demand for increased crop productivity, grain yield, nutritional quality and improved tolerance to different environmental factors. Transgene-based approaches are promising to address these challenges by transferring potential candidate genes to host organisms through different strategies. Agrobacterium-mediated gene transfer is one such strategy which is well known for enabling efficient gene transfer in both monocot and dicots. Due to its versatility, this technique underwent several advancements including development of improved in vitro plant regeneration system, co-cultivation and selection methods, and use of hyper-virulent strains of Agrobacterium tumefaciens harbouring super-binary vectors. The efficiency of this method has also been enhanced by the use of acetosyringone to induce the activity of vir genes, silver nitrate to reduce the Agrobacterium-induced necrosis and cysteine to avoid callus browning during co-cultivation. In the last two decades, extensive efforts have been invested towards achieving efficient Agrobacterium-mediated transformation in cereals. Though high-efficiency transformation systems have been developed for rice and maize, comparatively lesser progress has been reported in other graminaceous crops. In this context, the present review discusses the progress made in Agrobacterium-mediated transformation system in rice, maize, wheat, barley, sorghum, sugarcane, Brachypodium, millets, bioenergy and forage and turf grasses. In addition, it also provides an overview of the genes that have been recently transferred to these graminaceous crops using Agrobacterium, bottlenecks in this technique and future possibilities for crop improvement.

2-Acetyl-1-pyrroline augmentation in scented indica rice (Oryza sativa L.) varieties through Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) gene transformation.

2-Acetyl-1-pyrroline (2AP) has been identified as a principal aroma compound in scented rice varieties. Δ(1)-Pyrroline-5-carboxylate synthetase (P5CS) gene is reported to regulate the proline synthesis in plants and acts as the precursor of 2AP. Two scented indica rice varieties, namely Ambemohar 157 and Indrayani, were subjected to Agrobacterium tumefaciens-mediated genetic transformation containing P5CS gene. Overexpression of P5CS led to a significant increase in proline, P5CS enzyme activity and 2AP levels in transgenic calli, vegetative plant parts, and seeds over control in both the varieties. 2AP level increased more than twofold in transgenic seeds in both varieties. This is the first report of enhancement in 2AP content through overexpression of using P5CS gene, indicating the role of proline as a precursor amino acid in the biosynthesis of 2AP in scented rice.

Overexpression of NrCN improved TMV resistance in selection marker-free tobacco generated by Gene-Deletor system

Two expression cassettes, the Nicotiana tabacum Ubi.U 4 promoter-driven tobacco mosaic virus (TMV)-resistant gene NrCN element and Arabidopsis thaliana senescence-expressive gene SAG 12 promoter-driven LoxP/FRT site-specific recombinase gene FLP expression fragment, were both introduced into the TMV-sensitive tobacco variety K326 via Agrobacterium tumefaciens-mediated genetic transformation. In T0 transgenic plants inoculated TMV, the hypersensitive response (HR) and systemic HR (SHR) occurred at the inoculated leaf and the upper non-inoculated leaf, respectively. The chlorophyll, H2O2 and salicylic acid (SA) content but malonyldialdehyde (MDA) content in transgenic inoculation plants was significantly higher than non-transgenic plants. Meanwhile, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were exactly significantly higher than non-transgenic ones at the early stage after TMV inoculation, but the CAT activity in the former declined and was significantly lower than the latter at the later stage. In addition, the gene relative expression analysis of TMV-resistance-related protein components showed that the expressions of NrCN, NTF6, and PR-1a in genetically modified tobacco (GMT) plants were significantly upregulated after inoculation compared with wild-type tobaccos. Analysis of foreign gene deletion efficiency in transgenic plants indicated the foreign genes (e.g., recombinase gene FLP and screening reporter gene Bar::GUS) deletion events occurred in determinated GMT plant leaf with the development and maturity of the leaves. As a result, the transgenic plants became sensitive to herbicides. Therefore, the introduction of a plant expression vector (Gene-Deleter system with site-specific sequences LoxP/FRT and NrCN-contianing) into TMV-sensitive tobacco variety K326 enhanced the TMV resistance of sensitive tobacco plants. This study provides a basis for screening and acquisition of new varieties of marker-free and safe transgenic antiviral tobacco.

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation in asakura-sanshoo (Zanthoxylum piperitum (L.) DC. F. inerme Makino) an important medicinal plant.

Context:Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo.Aims:To establish a regeneration and Agrobacterium tumefaciens-mediated genetic transformation system in Asakura-sanshoo, which could be used for cultivar improvement and gene function analysis.Settings and Design:The various combinations of indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) were explored for the optimal plant regeneration from petiole and stem of Asakura-sanshoo. The half-strength woody plant medium (WPM) with different concentrations of NAA and IBA was used to induce root. For genetic transformation, A. tumefaciens strain EHA-105 harboring the plasmid pBin-Ex-H-ipt which carries the isopentenyl transferase (ipt) gene, β-glucuronidase (GUS) gene and kanamycin resistance gene neomycin phosphotransferase II (NPTII) were used. The transformation efficiency was detected by the kanamycin resistant frequency.Materials and Methods:Petioles and stems were obtained from the in vitro cultured Asakura-sanshoo. The petiole and stem segments were precultured for 3 days, and then inflected using the bacterium at the concentration of OD600 0.5–0.8 for 10 min, followed by 3 days co-cultivation. Selection of the transgenic plants was carried out after 7 days the regeneration using gradient kanamycin at 30 mg/L and 50 mg/L, respectively. Successful transformed plants were confirmed by GUS histochemical assays, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and Southern blotting analysis.Results:The highest shoots regeneration was obtained on WPM supplement with 0.5 mg/L BA and 0.2 mg/L NAA. The optimal rooting medium was half strength macro-element WPM. The kanamycin resistant frequency of petiole and stem was 24.66% and 25.93%, respectively. Thirty-five shoots in thousands adventitious buds were confirmed through GUS histochemical assays, PCR, RT-PCR, and Southern blotting. The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants.Conclusions:Individual transgenic Asakura-sanshoo lines were obtained. In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.

Inhibition of isoflavone biosynthesis enhanced T-DNA delivery in soybean by improving plant–Agrobacterium tumefaciens interaction

Soybean is a difficult crop to manipulate through Agrobacteriumtumefaciens-mediated genetic transformation. Plant–bacterium interaction plays an important role in the transformation process. Being rich with isoflavones, soybean may have an adverse effect on the A. tumefaciens-mediated genetic transformation. To investigate whether high content of endogenous isoflavones in soybean is a serious obstacle in achieving high efficient Agrobacterium-mediated transformation in soybean, a series of experiments on inhibition of isoflavone biosynthesis were conducted to improve upon soybean transformation efficiency. Results indicated that soybean isoflavones inhibited A. tumefaciens growth and respiration, the transformation efficiency [β-glucuronidase (GUS) transient expression] was negatively correlated with the phenylalanine ammonia-lyase activity and isoflavones content. The biosynthesis of soybean isoflavones was partially inhibited by sonication treatment and applying antagonists in co-culture medium and thereby decreased the adverse effects of isoflavones on Agrobacterium infection. A discernible improvement in transformation efficiency was achieved when sonication at 40 kHz for 3 min was applied along with Agro-infection and the explants were cultured on co-culture medium containing 20 μM α-aminooxyacetic acid (AOA), with the percentage of GUS transient expression as 41.4 %, being 3.6 times higher than that not sonicated and co-cultured on medium without AOA. Sonication was found not only to simply make micro-wounds for Agrobacterium to penetrate or releasing phenolic compounds for induced Agrobacterium vir gene expression; it disturbed the biosynthesis of isoflavones at the transcription level and decreased the adverse effects of isoflavones on soybean transformation, and thereby improving soybean transformation efficiency.

Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls

An Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for white ash (Fraxinus americana) using hypocotyls as the initial explants. Hypocotyls isolated from mature embryos germinated on Murashige and Skoog (MS) medium supplemented with 22.2 µM 6-benzyladenine (BA) and 0.5 µM thidiazuron (TDZ) were transformed using A. tumefaciens strain EHA105 harboring the binary vector pq35GR containing a fusion gene between neomycin phosphotransferase (nptII) and gusA, as well as an enhanced green fluorescent protein (EGFP). Explants were transformed in a bacterial suspension with 100 µM acetosyringone using 90 s sonication and 10 min vacuum infiltration. Putative transformed shoots representing seven independent lines were selectively regenerated on MS medium with 22.2 µM BA, 0.5 µM TDZ, 50 mg L−1 adenine sulfate, 10 % coconut water, 30 mg L−1 kanamycin, and 500 mg L−1 timentin. Timentin at 500 mg L−1 was optimal for controlling excess bacterial growth, and transformed shoots were selected using 30 mg L−1 kanamycin. The presence of GUS (β-glucuronidase), nptII, and EGFP in transformed plants was confirmed by polymerase chain reaction (PCR). Reverse transcription-PCR and fluorescence microscopy confirmed the expression of EGFP. Transgenic microshoots were rooted (80 %) on woody plant medium supplemented with 4.9 µM indole-3-butyric acid, 2.9 µM indole-3-acetic acid, and 500 mg L−1 timentin, and subsequently acclimatized to the culture room. This transformation protocol provides the framework for future genetic modification of white ash to produce plant material resistant to the emerald ash borer.

Protoplast: A more efficient system to study nucleo-cytoplasmic interactions

Agrobacterium tumefaciens-mediated genetic transformation is a powerful tool for plant research, but it can be labor-intensive and time-consuming. Here, we report a protoplast-based approach to study nucleo-cytoplasmic interactions, such as cytoplasmic male sterility/fertility restoration (CMS/Rf) and organellar RNA editing. To test the system, we transfected the fertility restorer gene Rf5, which is involved in the rice HL-CMS/Rf system, into rice protoplasts prepared from the HL-CMS line. As the Rf5 protein accumulated in the transformed protoplasts, the CMS-associated transcripts were endonucleolytically cleaved. There were much lower levels of the CMS-associated protein ORFH79 in the transfected protoplasts than in the mock-transfected protoplasts. Next, we used a dsRNA-mediated gene silencing approach to down-regulate the pentatricopeptide protein gene MPR25, which participates in RNA editing of the organellar transcript nad5. The editing efficiency of mitochondrial transcripts of nad5 at nucleotide 1580 was much lower in the transfected protoplasts than in the mock-transfected protoplasts. Together, these results show that protoplast is a simple and efficient system to study interactions between the nucleus and organelles.

Stable expression of mtlD gene imparts multiple stress tolerance in finger millet.

Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet.