Kanamycin-resistant Plants Research Articles

Establishment and application of highly efficient regeneration, genetic transformation and genome editing system for cucurbitacins biosynthesis in Hemsleya chinensis

Background Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis.ResultsHighly efficient calli were induced on MS medium added 1.5 mg·L− 1 6-benzylaminopurine (6-BA) and 0.02 mg·L− 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L− 1 6-BA and 0.1 mg·L− 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants.ConclusionsHere, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism.

Establishment of a high-efficiency transformation and genome editing method for an essential vegetable and medicine Solanum nigrum.

Solanum nigrum, which belongs to the Solanaceae family, is an essential plant for food and medicine. It has many important secondary compounds, including glycoproteins, glycoalkaloids, polyphenolics, and anthocyanin-rich purple berries, as well as many ideal characteristics such as self-fertilization, a short life cycle and a small genome size that make it a potential model plant for the study of secondary metabolism and fruit development. In this study, we report a highly efficient and convenient tissue culture, transformation and genome editing method for S. nigrum using leaf segments after 8 weeks of tissue culture, with a required period from transformation initiation to harvest of about 3.5 months. Our results also show multi-shoot regeneration per leaf segment and a 100% shoot regeneration efficiency in a shoot regeneration medium. Moreover, over 82% of kanamycin-resistant plants exhibited strong green fluorescence marker protein expression, with genetic integration confirmed by PCR results and green fluorescence protein expression in their T1 progeny. Furthermore, we successfully applied this transformation method to achieve an average of 83% genome editing efficiency of SnMYB1, a gene involved in regulating the anthocyanin biosynthetic pathway of S. nigrum in response to missing nutrients. Taken together, the combination of highly efficient tissue culture, transformation and genome editing systems can provide a powerful platform for supporting fundamental research on the molecular mechanisms of secondary metabolism, fruit development, and production of important compounds by biotechnology.

An efficient and rapid kanamycin screening assay for identifying true transgenics in cotton

Genetic engineering is the best suitable approach for the generation of insect resistant cotton in private and public funded laboratories. Neomycin Phosphotransferase-II is the most commonly used selectable marker gene that can catabolize to kanamycin resistant protein which helps in screening and differentiation of resistant and sensitive plants. In current paper, the optimal concentration of kanamycin was determined as 200 mg/L in control plants. Based on this standardized a procedure for screening transformed plants by spraying 200 mg/L at seedling stage. The resistant plants were reconfirmed by PCR using Npt II specific primers. About 51% and 45% of kanamycin resistant plants were showed amplification of Npt II gene at T1 and T2 generation respectively. This method is simple and fast which can be exploited for screening large number of seedlings with low cost in effective manner.

An improved method for transformation of Actinidia arguta utilized to demonstrate a central role for MYB110 in regulating anthocyanin accumulation in kiwiberry

Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes. Using a newly developed transformation method we were able to generate transgenic lines of three Actinidia arguta genotypes and demonstrate an important role for MYB110 in anthocyanin accumulation in kiwiberry.

Improving peanut protein quality: Expression of a synthetic storage protein

Peanut is an affordable legume used in most households. It represents one of the most important protein supplies worldwide. However, peanut proteins are deficient in several essential amino acids (EAA), like most plant proteins; whereas plants are the main source of dietary proteins consumed by humans and livestock. This could lead to protein malnutrition in areas where people diet relies on one or two staple foods. Based on its high nutritional value, peanut is a good candidate for genetic biofortification. This study is aimed at expressing an EAA-rich artificial storage protein (ASPx) into peanut seeds for increased nutritive value. The ASPx derived gene was introduced into peanut via Agrobacterium-mediated transformation. Molecular analysis of regenerated kanamycin resistant plants using polymerase chain reaction (PCR) and Southern hybridization indicate the stable integration of one copy ASPx gene in transgenic plants. The expression of the ASPx in transgenic peanuts seeds was detected by mass spectrometry (multiple reaction monitoring). Amino acids analysis showed an increase of 12 to 19% of most EAA (Val, Tyr, Phe, Iso, Leu, Met) in a transgenic line. The results show that the nutritional quality of peanut could be improved. Key words: Genetic biofortification, essential amino acid, storage protein, peanut.

Effect of ‘Chloroxynil’ on Agrobacterium-mediated transformation efficiency of Lilium cv ‘Manissa’

As a powerful technique, gene transformation can produce new lily cultivars with specific characteristics such as new flower colors and resistance to pathogens. However, it is difficult to use this technique in monocot plants like lilies. A new phenolic compound called ‘chloroxynil’ has been introduced to replace ‘acetosyringone’ and is considered to improve transformation efficiency of Lilium. Our results showed that the addition of chloroxynil into the inoculation and co-cultivation media significantly improved the transformation efficiency. At 4 μM, chloroxynil-mediated callus regeneration increased 7 days after it was transferred to the regeneration medium. Transformation efficiency was 6.6 % at 100 μM acetosyringone, while 11.1 % at 4 μM CX. Finally, 11 kanamycin-resistant plants were successfully produced at 4 μM CX, and gene transformation in the plantlets was validated by PCR and RT-PCR techniques.

Development of transgenic cotton (Narasimha) using triple gene Cry2Ab-Cry1F-Cry1Ac construct conferring resistance to lepidopteran pest

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they are recalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing three Cry genes driven by three different promoters conferring insect resistance. The meristematic region of embryo axis explants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100 vector with three Cry gene cassettes (alpha-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt II as a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds of kanamycin selectionmedium.About 2551T1 seedswere collected from111T0 plants and these seeds screened again with kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR, lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showed amplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as 395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteins accumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry genes were accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effective resistance againstHelicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentage of insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgenic cotton plants carrying triple Cry genes could be an excellent germplasmresource for the breeders for introgressions.

Overexpression of the OvBAN gene enhances the proanthocyanidin content in transgenic alfalfa (Medicago sativa L.)

Sainfoin (Onobrychis viciaefolia Scop) is a forage crop in the Leguminosae family. The leaves of this plant and other tissues have a high level of proanthocyanidins (PAs). The BAN gene encodes anthocyanidin reductase (ANR), which is a key enzyme in PA biosynthesis. This report describes cloning of a BAN homolog gene (viz., OvBAN) from local sainfoin and overexpression of this gene in alfalfa (Medicago sativa L.), a globally important forage crop. The open reading frame (ORF) of OvBAN contains 1020 bp, which is deduced to encode 339 amino acids. Alignment with amino acid sequences showed that OvBAN had 99%, 87%, and 85% identity to homologs from another Onobrychis viciaefolia variety, known as Lotus corniculatus, and Medicago sativa, respectively. The ORF of OvBAN was cloned into a binary vector under the control of the CaMV35S promoter, in which the nptII gene was used as a selectable marker. The resulting binary vector was introduced to alfalfa using Agrobacterium tumefaciens-mediated transformation. Kanamycin-resistant plants were obtained and grown in potting soil. PCR and Southern blot analyses demonstrated the integration of the OvBAN transgene in transgenic plants. Quantitative real-time PCR analysis revealed that OvBAN was highly expressed in transgenic plants. Crude enzyme was extracted from transgenic and wild-type plants. Enzyme tests showed higher ANR activity in the transgenic samples than in the wild-type. Furthermore, butanol-HCl-based cleavage analysis showed that the PA content was higher in transgenic plants than in wild-type plants. These results demonstrate that OvBAN is an appropriate gene for the enhancement of PAs in alfalfa.

Gene Expression of the Antimicrobial Peptide Bombinin Increases the Resistance of Transgenic Tobacco Plants to Phytopathogens

Transgenic tobacco (Nicotiana tabacum L.) plants with an artificial gene from the antimicrobial peptide bombinin (bom) have been obtained and studied. The presence of the bom gene in the genome of kanamycin-resistant plants was shown by PCR. Expression of the bom gene was confirmed by antimicrobial activity measurements in leaf extracts. The obtained plants were morphogenetically resistant to Erwinia carotovora bacteria and Rhizoctonia solani fungi phytopathogens. In addition, the protective oxidative reaction to the infection, i.e., the SOD activity and proline content, were lower in transgenic plants than in the infected nontransgenic plants. Plants with the expression of the antimicrobial bombinin peptide gene are promising for use in agricultural biotechnology as plant protectors.

Potent insect gut binding lectin from Sclerotium rolfsii impart resistance to sucking and chewing type insects in cotton

Cotton is an important crop that is continuously cultivated around the world. However, its production has decreased in recent times due to wide ranging insects and also current practices of using synthetic insecticides that are not precise and their residues impairing the biodiversity. Hence, the search for newer classes of efficient entomotoxic proteins continues. Genetically modified cotton crops with cry genes from Bacillus thuringiensis, have been cultivated across the world, which overcome the chewing type insect menace. In the present study, we assess the development of transgenic cotton plants by Agrobacterium, wherein the confirmed kanamycin resistant T0 plants were advanced to T1 generation and the gene integration was studied by molecular analysis. Western blot and ELISA assays demonstrated the expression of 0.46% lectin of the total soluble leaf proteins. In planta bioassay showed 69% of aphid, Aphis gossypii population reduction with T1 generation plants. Whereas 100% insect mortality is occurred in Spodoptera litura larvae by 96 h. Present findings shows the potent insecticidal effect of Sclerotium rolfsii lectin on sucking (homopteran) and chewing (lepidopteron) insects, underlining its significance and strengthening genetic resources in cotton breeding against different order insect pests.

Expressing a modified cowpea trypsin inhibitor gene to increase insect tolerance against Pieris rapae in Chinese cabbage

To increase the tolerance of Chinese cabbage (Brassica campestris L. ssp. pekinensis) to Pieris rapae, we introduced a modified cowpea trypsin inhibitor (CpTI) gene, SCK, into various cultivars. SCK was derived from CpTI, an insect-resistance gene. The protein accumulating capacity of CpTI can be improved by adding a signal peptide sequence at the 5′ end and an endoplasmic reticulum-detained signal sequence at the 3′ end. Using an optimized Agrobacterium tumefaciens-mediated transformation system in Chinese cabbage, we obtained a maximum transformation efficiency of ~6.83%. Insect resistance tests and CpTI enzymatic assays showed that most of the transgenic plants had significant resistance to cabbage worm (Pieris rapae) larvae and that the plants with the highest levels of insect resistance had the greatest CpTI-related capacity, indicating a high correlation between SCK expression and insect resistance. An evaluation of segregation patterns in the independent transgenic line with the highest insect resistance, ‘ZB-08-04’, showed that kanamycin resistant versus sensitive plants segregated in a 3:1 Mendelian fashion. This study provides a potential germplasm resource for Chinese cabbage breeding in the future.

<i>Agrobacterium tumefaciens</i>-Mediated Transformation of Wild Tobacco Species <i>Nicotiana debneyi</i>, <i>Nicotiana clevelandii</i>, and <i>Nicotiana glutinosa</i>

Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), 50 mg·L-1 kanamycin and 250 mg·L-1 timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg·L-1 kanamycin and 250 mg·L-1 timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T0 plants and T1 seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.

Effect of Explant, Genotype and Plant Growth Regulators on Regeneration and Agrobacterium-Mediated Transformation of Potato

A B S T R A C T The objective of the present study is to develop an efficient protocol for plant regeneration and Agrobacterium-mediated transformation of potato cultivars Desiree, Agria and Marfona grown under Iranian agricultural conditions. The regeneration efficiency from internode, leaf and petiole explants was examined. The results from all Shoot Induction Medium (SIM) combinations and all cultivars together indicate highly efficient shoot regeneration from internode (4.56 shoots per explant). The response of petiole and leaf explants was lower, 3.83 and 2.55 shoots per explant, respectively. The highest efficiency of shoot regeneration was achieved with internode explants of cultivar Desiree (6.64 shoots per explant) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) 1 mg LG 1 + 6-benzyladenine (BAP) 1 mg LG 1 . For plant transformation, internode explants were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing β-glucuronidase (GUS) and nptII genes. Reverse transcriptase-polymerase chain reaction analysis and histochemical assay for β-glucuronidase indicated that the gene coding for this enzyme was integrated in the potato genome and could be expressed in potato tissues. The presence of nptII gene in the kanamycin resistant plants was verified by polymerase chain reaction analysis. The transformation frequency ranged from 22-42%.

Comparison and evaluation of two diagnostic methods for detection of npt II and GUS genes in Nicotiana tabacum.

To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and also a visual detection protocol on the basis of npt II and GUS genes in transgenic tobacco plants were used. Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum leaf discs was performed with plant transformation vector of pBI 121. From kanamycin-resistant plants selected by their antibiotic resistance, four plants were selected for DNA isolation. Presence of the transgene was confirmed in the transformants by PCR and LAMP. In this regard, all LAMP and PCR primers were designed on the basis of the gene sequences of npt II and GUS. The LAMP assay was applied for direct detection of gene marker from plant samples without DNA extraction steps (direct LAMP assay). Also, a novel colorimetric LAMP assay for rapid and easy detection of npt II and GUS genes was developed here, its potential compared with PCR assay. The LAMP method, on the whole, had the following advantages over the PCR method: easy detection, high sensitivity, high efficiency, simple manipulation, safety, low cost, and user friendly.

Ectopic expression of the PttKN1 gene in Cardamine hirsuta mediated via the floral dip method

PttKN1 gene (Populus tremula × P. tremuloides KNOTTED1) was isolated from the vascular cambium of a hybrid aspen. Previous studies on transformed plants with the PttKN1 gene suggested that it plays roles in plant development (typically in meristem initiation), maintenance and organogenesis in simple-leaved species. To investigate the gene functions further, sequence analysis of the deduced amino acid was conducted. The results suggested that the gene belongs to the class I KNOX gene (KNOTTED1-like homeobox genes) family and might play important roles in plant development by coding a transcription factor. The gene was introduced into Cardamine hirsuta using the floral dip method mediated via Agrobacterium tumefaciens. The primary transformed plants were obtained via kanamycin selection. Compared to the wild type, the kanamycin resistant plants demonstrated several morphological alterations, such as abnormal cotyledons, abnormal shoot meristem, flattened stem, and lobed and cup-shaped leaves. RT-PCR results showed that the above five types of kanamycin resistant plants expressed the same specific PttKN1 gene band. This suggested that the morphological alterations were caused by the insertion and expression of the gene. However, these phenotypes were similar to other PttKN1 transformed plants, despite the fact that C. hirsuta is a species with compound leaves and the other species have simple leaves. Therefore, the functions of the PttKN1 gene on compound-leaf species have yet to be investigated via the comparison between related species such as Arabidopsis thaliana and C. hirsuta.

Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both β-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.

Plant Bioreactors for the Antigenic Hook-Associated flgK Protein Expression

Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seed-specific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

A new double right border binary vector for producing marker-free transgenic plants.

BackgroundOnce a transgenic plant is developed, the selectable marker gene (SMG) becomes unnecessary in the plant. In fact, the continued presence of the SMG in the transgenic plant may cause unexpected pleiotropic effects as well as environmental or biosafety issues. Several methods for removal of SMGs that have been reported remain inaccessible due to protection by patents, while development of new ones is expensive and cost prohibitive. Here, we describe the development of a new vector for producing marker-free plants by simply adapting an ordinary binary vector to the double right border (DRB) vector design using conventional cloning procedures.FindingsWe developed the DRB vector pMarkfree5.0 by placing the bar gene (representing genes of interest) between two copies of T-DNA right border sequences. The β-glucuronidase (gus) and nptII genes (representing the selectable marker gene) were cloned next followed by one copy of the left border sequence. When tested in a model species (tobacco), this vector system enabled the generation of 55.6% kanamycin-resistant plants by Agrobacterium-mediated transformation. The frequency of cotransformation of the nptII and bar transgenes using the vector was 66.7%. Using the leaf bleach and Basta assays, we confirmed that the nptII and bar transgenes were coexpressed and segregated independently in the transgenic plants. This enable separation of the transgenes in plants cotransformed using pMarkfree5.0.ConclusionsThe results suggest that the DRB system developed here is a practical and effective approach for separation of gene(s) of interest from a SMG and production of SMG-free plants. Therefore this system could be instrumental in production of “clean” plants containing genes of agronomic importance.

Protein extract of tobacco expressing StoVe1 gene inhibits Verticillium dahliae proliferation

Verticillium dahliae is a principal pathogen causing verticillium wilt in Solanaceae crops. StoVe1 is a gene resisting to verticillium wilt isolated from Solanum torvum. In order to generate resistant tobacco plants, StoVe1 was inserted in the orientation behind CaMV 35S promoter of vector pGS and this construct was introduced into tobacco by Agrobacterium-mediated transformation. A total of 12 kanamycin-resistant plants were generated and 7 independent transgenic lines were identified by PCR analysis. Quantitative RT-PCR analysis showed that the levels of StoVe1 transcript in transgenic lines were up to 2–6 fold higher than in the control. Anti-fungal assay indicated that the protein extract of transgenic tobacco lines showed strong inhibition activity to V. dahliae, 2 fold or higher compared to control plants. This result reveals that StoVe1, as a V. dahliae resistance gene, has an application potential in plant breeding for verticillium wilt resistance.

Expression of the MSI-99m Gene in Transgenic Potato Plants Confers Resistance to Phytophthora infestans and Ralstonia solanacearum

Magainins are a class of antimicrobial peptides isolated from skin secretions of the African clawed frog Xenopus laevis. MSI-99 is a synthesized magainin II analogue with high inhibitory effects to a wide spectrum of microbial organisms, including bacteria, fungi and viruses. To verify the resistance conferred by the MSI-99m gene (a MSI-99 synthetic gene with codon usage adapted for expression in potato) to potato pathogens and to generate multi-resistant potato materials, we transferred the MSI-99m gene into potato plants using Agrobacterium-mediated transformation. PCR and Southern blot analyses of eight kanamycin-resistant plants showed that MSI-99m gene was present and expressed in five independent transgenic lines. These five transgenic plants exhibited enhanced resistance to Phytophthora infestans and Ralstonia solanacearum. The results demonstrate that the MSI-99m gene can be used to potentially improve potato disease resistance genetically.