Hygromycin Phosphotransferase Gene Research Articles

Development of transgenic Brassica napus plants with AtATM3 gene to enhance cadmium and lead tolerance

Four popular genotypes of Brassica napus, cv. BARI Sarisha-8, BARI Sarisha-13, Binasarisha-4, and Binasarisha-8 were subjected to cadmium (Cd) and lead (Pb) stress to identify a genotype with enhanced tolerance of Cd and Pb. The goal was to use the selected genotype for genetic engineering, thereby improving traits related to heavy metal tolerance and accumulation to remediate polluted agricultural soils. The genotype, BARI Sarisha-8 with superior tolerance to both Cd and Pb stresses, was chosen for genetic engineering with Arabidopsis thaliana ABC transporter of the mitochondrion 3 (AtATM3) gene. The transformation process involved coculturing the cotyledon explants with Agrobacterium strain GV3101 carrying a binary vector containing the hygromycin phosphotransferase (HPT) gene as a selectable marker and the AtATM3 gene. Transformation efficiency was significantly enhanced with a two-day co-cultivation period on shoot induction medium composed of MS medium, α-naphthaleneacetic acid (0.5 mg/L), and 6-benzyladenine (3 mg/L), supplemented with acetosyringone (20 mg/L), along with a four-day delay in exposing the explants to the selective agent hygromycin. Hygromycin-resistant shoots were obtained by employing a three-step selection process. The refined protocol resulted in a transformation efficiency of 15.38%. Polymerase chain reaction (PCR) analysis confirmed the integration of AtATM3 and HPT genes into the host genome in all recombinant plants. Transgenic B. napus plants expressing the AtATM3 gene exhibited notable improvements in tolerance, demonstrating a 1.4- to 1.7fold increase in Cd tolerance and a 1.3- to 1.5-fold increase in Pb tolerance compared to the wild type (nontransformed) under both Cd and Pb stress conditions.

CRISPR/Cas9-mediated deletion of large chromosomal segments identifies a minichromosome modulating the Colletotrichum graminicola virulence on maize

Colletotrichum graminicola causes anthracnose on maize, an economically significant disease worldwide. To decipher how the pathogen controls its virulence/pathogenicity on maize at the minichromosomal level, we sequenced the genome and transcriptome of the C. graminicola strain T1-3-3. The 61.91 Mb genome contains three transcriptionally repressed, full-length strain-specific minichromosomes (<1 Mb; Chr11 through Chr13). A CRISPR/Cas9-based system was developed to knock out large chromosomal segments; it involved the generation of multiple simultaneous DNA double-strand breaks across a targeted genomic region, followed by homology-directed replacement thereof with a donor DNA template carrying the selectable marker hygromycin phosphotransferase gene flanked by homologous sequence arms of the targeted region. Using this system, we obtained distinct mutants functionally nullisomic for individual minichromosomes. Only the ΔChr12 mutant lacking the 498.44 Kb genomic region carrying all of the 31 genes of Chr12 exhibited attenuated virulence on maize and was indistinguishable from T1-3-3 in fungal growth and conidiation, indicating that Chr12 is a conditionally dispensable minichromosome and imparts full virulence to C. graminicola on maize. The CRISPR/Cas9-mediated genome editing system developed in this study will enable the determination of the biological functions of minichromosomes or large chromosomal segments in fungal plant pathogens.

Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified Organisms Based on the Microbial-Infection-Linked HPT Gene

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

Exploratory and confirmatory molecular approaches to determine genetically modified status in different crops

BackgroundOne of the parameters required for the assessment of food and feed safety is detection and identification of genetically modified organisms. Legislation in some countries necessitates detection and quantification of modification in food and feed samples. Scientists have raised concern about safety of antibiotic resistance marker (ARM) genes used for transformation of crops intended for human and animal consumption. In the present work two molecular approaches have been adopted: one exploratory; for detection and quantification of ARM genes in tested plant samples and the other confirmatory; to determine the specificity/reliability of the obtained results.ResultsResults revealed that primers for neomycin phosphotransferase (nptII) and aminoglycoside 3″ adenyl-transferase (aadA) were amplified in the majority of the 36 DNA screened samples. Melting curve analysis using hygromycin phosphotransferase (aphIV) gene as target sequence for the fluorescent-based detection approach was performed to ensure reliability and specificity of this procedure and to confirm results obtained by using conventional polymerase chain reaction (PCR). Quantitative RT-PCR results and validation analysis followed, revealed that all of the tested DNA samples were not violating the European legislation for GMOs labeling (0.9%).ConclusionsThe results fully demonstrated the reproducibility, sensitivity/specificity of the adopted approaches for detection and quantification of even traces of GMO contents. Applying measurement uncertainty (MU) procedures presented in this work will help decision makers to ensure compliance with International Legislation and Regulations. This in its turn will facilitate and enhance trading with countries having compelling labeling regulations.

Establishment of Agrobacterium tumefaciens – mediated genetic transformation of apple pathogen Marssonina coronaria using marker genes under the control of CaMV 35S promoter

Premature leaf fall of apple caused by Marssonina coronaria is economically very important apple disease and all the commercially available apple cultivars are susceptible to this disease. The non-availability of an efficient transformation system for this fungus hinders the functional genomics research. Herein, we report for the first time, the successful Agrobacterium-mediated transformation in apple leaf blotch fungus M. coronaria by transferring T-DNA harbouring the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA) and green fluorescent protein (gfp) under the control of CaMV 35S promoter. The key factors that affect the transformation efficiency including type of recipient fungal material, acetosyringone concentration, the conditions for co-cultivation, Agrobacterium concentration, Agrobacterium strains and membrane types as support were investigated. The present results have recommended that 250 μM concentration of acetosyringone, 24 °C temperature and 48 h time, 0.5 OD600 of A. tumefaciens, EHA105 Agrobacterium strain and Whatman filter paper were the optimal co-cultivation conditions for the transformation of M. coronaria by using fragmented mycelia suspension and mycelial plugs. We observed that conidia were tedious to transform as compared to the fragmented mycelia and mycelial plugs of this slow growing fungus. These optimized parameters yielded 54 and 70 average transformants per 60 mycelial plugs and 104 fragmented mycelia, respectively. Fungal transformants were analysed for T-DNA integration, gus gene expression and gfp gene expression. Strong gus histochemical staining and green fluorescence expression indicated that the CaMV 35S promoter can drive gene expression in M. croronaria. Some mutants showed difference in the morphology of the colony as compared to the wild type control. This report will be very useful to inspect molecular basis of apple-M. coronaria interactions by deciphering the functional roles of various genes in this pathogenic fungus.

Efficiency of Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L.) with rice OsNAC6 gene

Crop improvement by genetic engineering requires efficient plant regeneration derived from cultured cells or tissues. The success of cell or tissue culture regeneration depends on reliable explant source. In this study, three different types of explant (callus, stem, and leaf) of tobacco (Nicotiana tabacum L.) were evaluated to determine the most effective explant source for Osnac6 gene transformation. OsNAC6 encodes a transcription factor belonged to NAC transcription factor family in rice (Oryza sativa L.). OsNAC6 transcription factor has been reported in some studies to confer tolerance to drought stress. Agrobacterium tumefaciens strain EHA 105 harboring pC1305 plasmid containing the OsNAC6 gene driven by CaMV 35S promoter (pARNAC6) was used for transformation. The T-DNA contains hygromycin phosphotransferase (hpt) gene as a selectable marker. PCR confirmed the presence of the hpt gene in the T0 generation of the transformed tobacco plants. The PCR result showed that a total of 50 tobacco plants were positive for hpt gene which 18, 20, and 12 of them derived from callus, stem, and leaf explants respectively. The highest transformation efficiency was obtained by using the stem as explant material, i.e. 17%. Meanwhile, the obtained transformation efficiency by using callus and leaf was 15% and 10% respectively.

Agrobacterium tumefaciens-Mediated Transformation of Rice by Hygromycin Phosphotransferase (hptII) Gene Containing CRISPR/Cas9 Vector.

The CRISPR/Cas9 technique for rice genome engineering is gaining momentum and requires a precise gene delivery system. For rice and other crop plants, Agrobacterium tumefaciens-mediated transformation (AMT) is considered a suitable gene transformation method. The AMT for indica-type rice is a challenge because it is less efficient in tissue culture response than japonica-type rice. Here is a protocol of the AMT method that we developed for IR64 variety which has been successfully tested in other popular indica-type rice varieties. We used embryogenic calli as explant and an empty gRNA-containing CRISPR/Cas9 vector with hptII (hygromycin phosphotransferase) gene for the transformation. This technique would speed up rice genome editing via CRISPR/Cas9 technology and facilitate to achieve varied application in the future.

Obtaining Wheat (Triticum aestivum L.) Lines with Yeast Genes for Trehalose Biosynthesis

Yeast (Saccharomyces cerevisiae) genes for trehalose biosynthesis (TPS1 and TPS2) were transferred into genomes of several common wheat cultivars using two methods of Agrobacterium-mediated transformation (in vitro and in planta) to enhance drought tolerance. For this purpose, vectors pBract214-TPS1 and pBract214-TPS2 were constructed using the Gateway-cloning technique. Both the vectors contained TPS1 and TPS2 genes under control of the constitutive maize ubiquitin promoter (PUbi) and selectable marker hygromycin-phosphotransferase (hpt) gene. Three- to five-day-old calluses obtained from immature wheat embryos were used as explants for the transformation in vitro. Selection of transgenic plants was carried out on nutrient medium supplemented with 30 mg/L hygromycin (as a selectable agent). Seeds of wheat (transgenic generation T1) were obtained by the in planta method of transformation. Integration and the presence of yeast genes in wheat genomic DNA isolated from transgenic plants were confirmed by PCR analysis using primers specific to TPS1 and TPS2 genes.

Protocol for Agrobacterium-mediated transformation of tall fescue and future perspective on the application of genome editing.

Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l-1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.

Efficient plant regeneration and Agrobacterium-mediated transformation of Begonia semperflorens-cultorum

Begonia semperflorens-cultorum, known as wax begonia, is one of the most popular Begonia species in which variable commercial cultivars have been produced. The genetic transformation technique is required for further modification of this species because introduction of some desired traits such as novel flower colors cannot be achieved by conventional breeding. Here we report the procedures of an efficient plant regeneration from leaf segment and production of transgenic plants using Begonia semperflorens-cultorum. Efficient induction of adventitious shoots was achieved when the explants were cultured on MS medium supplemented with 1.5 mg/l thidiazuron (TDZ), 0.5 mg/l α-naphthaleneacetic acid (NAA), 30 g/l sucrose and 2.5 mg/l gellan gum. Agrobacterium tumefaciens strain EHA101 containing the plasmid pIG121-Hm was used for gene transfer. The transient GUS expression was significantly increased when 10 mM MES was added to the co-cultivation medium. By culturing the infected explants under the selection pressure with 10 mg/l hygromycin (Hm), the Hm-resistant independent shoots were obtained at a frequency of 0.78 per explant. The regenerated Hm-resistant plants showed the integration of hygromycin phosphotransferase (HPT) gene into the genome and stable GUS expression, as the proof of genetic transformation. Consequently, the conditions established in this study enabled the transformation of Begonia semperflorens-cultorum. Production of transgenic plants through Agrobacterium-mediated transformation using an effective system for plant regeneration from leaf segment of Begonia semperflorens-cultorum was achieved.

Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 μg/ml, cefotaxime sodium concentrations with 200 μg/ml, Acetosyringone (AS) concentration at 200 μmol/l, 1 × 106 T. foetida hypha cells/ml, co-cultivation at 22 °C with 24 h and culture was incubated at 16 °C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium tumefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.

Generation of selectable marker-free soft transgenic rice with transparent kernels by downregulation of SSSII-2

The amylose content (AC) of rice endosperm starch varies from 0 to 35%, and is associated with rice cooking and eating quality. Soft rice has low AC, generally between 6% and 15%, and its eating quality is high whether it is consumed hot or cold. However, the appearance quality of current soft rice cultivars needs to be improved, especially opaque endosperm. Conventional genetic engineering has improved some agronomic traits of soft rice varieties, but not endosperm appearance. In the present study, a RNAi construct of the soluble starch synthase II-2 (SSSII-2) and the hygromycin phosphotransferase (HPT) gene were introduced into an elite japonica rice variety, Kangtiaowuyunjing (KWY8) by co-transformation. Several selectable marker-free (SMF) transgenic lines were obtained, and SSSII-2 expression was significantly downregulated in selected transgenic lines, resulting in lower AC of the endosperm. The physicochemical properties of the transgenic rice kernels, including gel consistency (GC) and rapid visco analyzer (RVA) profile, differed significantly from those of wild-type rice and were similar to those of a soft rice variety, Nanjing 46 (NJ46). These findings indicate that the cooking, eating, and processing qualities of transgenic rice are comparable to those of NJ46. However, the transgenic rice endosperm retained a transparent appearance under low-moisture conditions. Thus, SMF SSSII-2 RNAi rice provides a resource for breeding soft rice with transparent endosperm.

Genetic transformation of the white-rot fungus Dichomitus squalens using a new commercial protoplasting cocktail

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.

Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

The Ascocenda flavanone 3-hydroxylase (AcF3H) gene was successfully transformed into Dendrobium 5N white orchid plants using Agrobacterium-mediated gene transformation. In this study, for the first time, we report the construction of a plant expression vector harboring the AcF3H gene using the Gateway cloning system. Protocorm-like bodies (PLBs) were cocultivated with the Agrobacterium tumefaciens strain AGL1 harboring the plant expression vector pGWB5-AcF3H, which contained the hygromycin phosphotransferase (hpt) gene as a selectable marker. The highest transformation efficiency (10.13%) was achieved when PLBs were cocultivated with Agrobacterium cells for 15 min. Three months after the transformation, the plantlets were regenerated, and the transgenic plants were confirmed by polymerase chain reaction (PCR) analysis using specific primers for the hpt gene and 35S promoter region. PCR products of approximately 400 and 500 bp, corresponding to the hpt gene and the 35S promoter, respectively, were detected in the transgenic plants, while no such product was observed in nontransgenic plants, indicating that the AcF3H gene was integrated into the genome of Dendrobium 5N white orchid plants. The transient expression of the AcF3H gene in Dendrobium 5N white and Dendrobium Anna petals was performed using the agroinfiltration technique, and the results demonstrated that no cyanidin content was detected in the Dendrobium 5N white petals after AcF3H infiltration. In contrast, the cyanidin content was increased by approximately 6% in the Dendrobium Anna petals, suggesting that the AcF3H gene was transiently expressed in this orchid.

RNAi-mediated resistance to rice black-streaked dwarf virus in transgenic rice.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Development of resistant varieties by using conventional breeding methods is limited, as germplasm with high level of resistance to RBSDV have not yet been found. One of the most promising methods to confer resistance against RBSDV is the use of RNA interference (RNAi) technology. RBSDV non-structural protein P7-2, encoded by S7-2 gene, is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway. P8, encoded by S8 gene, is the minor core protein that possesses potent active transcriptional repression activity. In this study, we transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8, and obtained plants harboring the target gene constructs and the selectable marker gene, hygromycin phosphotransferase (HPT). From the offspring of these transgenic plants, we obtained selectable marker (HPT gene)-free plants. Homozygous T5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers. Thus, our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.

Development of Marker-Free Insect-Resistant Indica Rice by Agrobacterium tumefaciens-Mediated Co-transformation.

Agrobacterium-mediated co-transformation is an efficient strategy to generate marker-free transgenic plants. In this study, the vectors pMF-2A∗ containing a synthetic cry2A∗ gene driven by maize ubiquitin promoter and pCAMBIA1301 harboring hygromycin phosphotransferase gene (hpt) were introduced into Minghui86 (Oryza sativa L. ssp. indica), an elite indica restorer line. Two independent transformants containing both the cry2A∗ gene and hpt gene were regenerated. Several homozygous marker-free transgenic progenies were derived from family 2AH2, and three of them were selected for further insect bioassay in the laboratory and field. Insect-resistance assays revealed that all the three transgenic lines were highly resistant to striped stem borer (Chilo suppressalis), yellow stem borer (Tryporyza incertulas) and rice leaf folder (Cnaphalocrocis medinalis). The measurement of Cry2A protein concentration showed that Cry2A protein was stably expressed in leaves and stems of homozygous transgenic lines and their hybrids. The yields of the marker-free homozygous transgenic lines and their hybrids were not significantly different from those of their corresponding controls. Furthermore, the results of flanking sequence isolation showed that the T-DNA in line 8-30 was integrated into the intergenic region of chromosome 2 (between Os02g43680 and Os02g43690). These results indicate that the marker-free transgenic rice has the potential for commercial production.

Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte.

Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected “knockoff” endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These ∆EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production.

Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.

Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Synvip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Synvip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (synbar) gene. Marker gene excision was achieved in hybrids with up to 41.18% recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII+cre-synbar), were established in subsequent generation through genetic segregation.

Antibiotic Sensitivity of Grapevine: A Comparison Between the Effect of Hygromycin and Kanamycin on Shoot Development of Transgenic 110 Richter Rootstock (Vi tis B erlandieri x Vi tis rupestris)

The effects of kanamycin and hygromycin on the in vitro development of 110 Richter rootstock (Vitis Berlandieri x V. rupestris) and derived transgenic clones were studied in order to evaluate their suitability as selectable markers for grapevine transformation. Transgenic clones carried both the neomycin phosphotransferase gene (nptll) under the nopaline synthase promoter (pNOS) and the hygromycin phosphotransferase (hpt) gene under the cauliflower mosaic virus 35S promoter (p35S), which confer the ability to grow in the presence of kanamycin and hygromycin, respectively. Even incorporated at low concentrations, both antibiotics strongly inhibited explant development for the control clone, but hygromycin appeared much more toxic. Whereas growth was stopped with 1 μg/mL hygromycin, 16 μg/mL kanamycin was required to block shoot development. Contrary to the control plants, transgenic clones exhibited a greater tolerance to hygromycin than kanamycin. In some transgenic clones the level of resistance conferred by the nptll was not significantly advantageous in terms of their ability to grow in the presence of kanamycin. Conversely, the hpt gene provided a strong level of tolerance to hygromycin in all transgenic clones, some clones developing up to 64 μg/mL hygromycin. Strategies for selection of grapevine putative transformants with these two antibiotics are discussed.

Plant regeneration through indirect organogenesis and genetic transformation of Eucalyptus polybractea R.T. Baker

Eucalyptus polybractea (blue mallee) is the most widely harvested species in Australia for the production of pharmaceutical grade eucalyptus oil. Its use for both oil and biomass production is set to expand because blue mallee grows vigorously on semi-arid land where agricultural crop production is unsustainable. One of the most important tools for improvement of blue mallee, and for dissection of its unique traits, is an efficient method of indirect organogenesis. Our aim was to develop such a method, which is efficient for all genotypes and applicable to adult-derived explants. We also aimed to test whether genetically modified plants could be produced using this protocol. In vitro cultures were developed from three field-grown adult clones and it was found that over 95% of the resultant leaf explants initiated callus in a medium supplemented with thidiazuron and 1-naphthaleneacetic acid. The effect of the cytokinins, 6-benzylaminopurine (BA) and N6-(2-isopentenyl) adenine (2iP) on plant regeneration from the initiated callus was compared. Ninety percent of callus from all three clones regenerated shoots in a medium supplemented with BA, whereas only one clone regenerated shoots at high efficiency (93%) with 2iP. Roots were successfully developed on regenerated shoots with high efficiency (>60%) via exposure to indole-3-butyric acid, and hardened plants were successfully established in soil. Leaf explants from one clone were transformed with Agrobacterium tumefaciens T-DNA containing genes for hygromycin phosphotransferase (Hpt) resistance and green fluorescent protein (GFP), both constitutively expressed using the CaMV35S promoter. Transformants showed GFP fluorescence in calli and plantlet leaves and were successfully rooted and hardened into plants. This work will enable large scale cloning of adult blue mallee plants and, after optimization of the transformation protocol, provide an important platform for further research on the biosynthesis of eucalyptus oils with the potential to dramatically improve blue mallee as a crop.