Tumefaciens LBA4404 Research Articles

RNA-mediated silencing of PKS1 gene in Colletotrichum falcatum causing red rot in sugarcane

Red rot is one of the major threatening diseases of sugarcane. Due to frequent breakdown of resistance by simultaneous evolution of pathogen variants with the introduction of newer varieties red rot remains as a challenge for successful breeding programme in India. Earlier studies established that the pathogen has limited genotypic variation with enormous phenotypic variation, which could be mainly due to adaptability of pathogen to the newly released varieties and expression of pathogenicity factors including secondary metabolites viz., toxins, enzymes and pigments like melanin. It is also proved that a positive correlation remains between the production of melanin and disease expression and hence the melanin biosynthesizing genes viz., Polyketide synthase1 (PKS1), Scytalone dehydratase1 (SCD1) and Trihydroxynapthalene reductase1 (THR1) were characterized. In this study, we have utilized RNA interference (RNAi) approach using pSilent-1 vector which facilitates the generation of hair pin constructs that suppress the expression of target gene through Agrobacterium mediated transformation (ATMT) to functionally analyze the major gene PKS1 involved in the production of dihydroxy naphthalene (DHN) melanin to determine its role in virulence in C. falcatum. Functionally active sites of the PKS1 gene i.e., acyl transferase (AT) domain and the region responsible for conidial pigment (CP) polyketide synthase were chosen for knockdown approach. The above said regions were amplified in the virulent C. falcatum isolate (Cf671) and both the sense and antisense fragments were separately cloned into pSilent-1 vector. The expression cassette was then cloned into the binary vector and introduced into Agrobacterium tumefaciens LBA4404 which was later co-cultivated with the fungal spore suspension. Results indicated that the knockdown AT mutant failed to produce spores whereas the CP mutants produced spores as that of the parental strains. Further pathogenicity testing revealed that the disease symptoms of both AT and CP mutants were restricted within the inoculated internode until 15th and 5th dpi respectively while the symptoms of wild type were prominent and crossed beyond 3rd internode at 15 dpi during the host-pathogen interaction. Molecular confirmation of the knockdown mutants with the expression of hygromycin gene and absence of functional domains under RT-PCR clearly indicated that the PKS1 gene has a possible role in C. falcatum pathogenesis. However expression analysis by qRT-PCR indicated regaining of virulence in mutants during later part of host pathogen interaction. The results confirm the applicability of RNAi mediated silencing for functional analysis of pathogenicity gene homologs in C. falcatum.

TRANSFER OF GLUCANASE GENE TO RESIST LATE BLIGHT DISEASE IN POTATO (Solanum tuberosum L.)

Potato late blight is a pandemic disease caused by the highly virulent (Phytophthora infestans) fungus. The regeneration capacity was done among three tested potato cultivars (Spunta, Diamont and Desiree). Two different types of explants (inter-node and leaf) were cultured for calli induction and plant regeneration .The highest value of leaf explants was for Desiree cultivar (80%), which induced from developed callus in the 2,4-D medium. The best value of maximum shoot regeneration was also for Desiree cultivar in the BA, IAA and GA3 media from leaf which proved to be more effective. Then cloning of glucanase gene in pRI plasmid which carrying kanamycin resistance (nptII) gene was perfomed and followed by transformation in Agrobacterium tumefaciens LBA4404 strain which used for plant transfection. Nucleotide and amino acid sequences of transformed Agrobacterium were analyzed. The putative transgenic plantlets genomes and cDNA of the isolated RNA for glucanase gene.

Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Agrobacterium tumefaciens-mediated transformation system for the important medicinal plant Dendrobium catenatum Lindl

The orchid Dendrobium catenatum Lindl. has both ornamental and medicinal value. Studies on genetic transformation of other Dendrobium species have been reported; however, due to the heterogeneity among the Dendrobium species, an independent transformation system is required for optimum D. catenatum transformation. Agrobacterium tumefaciens-mediated transformation system for D. catenatum is reported herein. Three A. tumefaciens strains, GV3101, LBA4404, and EHA105, were tested for their ability to transform D. catenatum protocorms. It was found that infection with strain GV3101 at an OD600 of 0.6 yielded the highest transformation frequency of 56.5%. Addition of 0.001% (v/v) Triton™ X-100 or pretreatment with ultrasound (300 W, 40 kHz, 3 min) increased the transformation frequency by up to 2-fold. After 1 year, both green fluorescent protein (GFP) imaging and glucuronidase (GUS) histochemical analysis of transgenic plants demonstrated that DcObg-GFP and pDcObg-GUS construct DNA was transferred into the D. catenatum and expressed successfully. Functional and gene expression level analyses of transgenic plants containing a D. catenatum chloroplast-targeted homolog of Spo0B-associated GTP-binding protein (DcObgC) RNA interference construct (DcObgC-RNAi) further demonstrated that this transformation system was reliable and efficient for D. catenatum. The A. tumefaciens-based transformation protocol established and optimized in this current report could provide a powerful and efficient tool for academic research and genetic engineering-based breeding of D. catenatum.

Sprouted Sorghum Extract Elicits Coleoptile Emergence, Enhances Shoot and Root Acclimatization, and Maintains Genetic Fidelity in indica Rice

The high growth-stimulating effect of plant extract has urged the plant biotechnologists to use natural supplements in the culture media instead of synthetic phytohormones. We advocated the effect of sprouted sorghum extract (SSE) on emergence, in vitro acclimatization, and genetic fidelity in coleoptile derived callus of indica rice variety ADT36. The use of SSE with Murashige Skoog medium efficiently acclimatized the root and shoot apical systems. A higher mat and seminal roots (3.4 g biomass) with an efficient shoot primordium elongation were observed with an increase in the concentration of SSE. Seeds treated with SSE medium showed higher germination and earlier coleoptile maturation about 48 h compared to untreated seeds, and there was a higher expression of eEF-1α with an increase in coleoptile length. B5 medium was effective on inducing embryogenic and nodular callus from 3-day-old coleoptile with 3.0 mg/L 2,4-dichlorophenoxyacetic acid and further proliferated effectively with 0.8 mg/L kinetin with a fresh weight of 180 mg. Highly significant regeneration was observed with combination of 2.5 mg/L 6-benzylamino purine and 3.0 mg/L α-naphthaleneacetic acid. The metabolic and genetic profiles of in vitro and directly cultivated plants were the same, examined through Fourier-transform infrared spectroscopy, random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and R-ISSR (combination of RAPD and ISSR) markers, respectively, and thus confirming the significant efficacy of the SSE incorporated medium. Disarmed T-DNA was transformed to coleoptile derived callus through Agrobacterium tumefaciens LBA4404 and confirmed by GUS assay. The T-DNA integration was confirmed by DNA blot analysis using DNA from transient GUS-expressed explants. Thus, SSE can be used as a natural and organic supplement for organogenesis and efficient acclimatizations of shoot and root apical meristems in regenerated plants.

Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria.

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.

PREPARATION OF PLANT VECTOR CONSTRUCT CONTAINING DEHYDRIN GENE At2g21490

This work is focused on preparation of a plant transformation vector pDH2 containing the expression unit of the dehydrin gene At2g21490 under a control of the constitutive dCaMV 35S promoter and the selectable marker neomycin phosphotransferase gene controlled by the nos promoter. The gene At2g21490 is classified as a dehydrin of the type K2SY2. The gene was isolated from Arabidopsis thaliana by PCR approach. The plasmid pDH2 was transformed into the strain Agrobacterium tumefaciens LBA 4404. The stability of the plasmid pDH2 in Agrobacterium has been verified by restriction analysis after re-transformation of the pDH2 into Escherichia coli.

Genetic chimerism of CRISPR/Cas9-mediated rice mutants

The CRISPR/Cas9 technology is useful for genome editing to generate targeted mutants. Based on this genome editing technology, it was attempted to generate the rice mutant which lacks JAZ9 activity to understand its function in stress response. The sequence of guide RNA for the recognition of target site was obtained from CRISPR-PLANT website ( http://www.genome.arizona.edu/crispr ) to minimize off-target effect and was recombined into the CRISPR/Cas9 binary vector pRGEB31. Embryonic calli regenerated from the mature seeds (O. sativa L. cv. Nakdong) were co-cultivated with transformed Agrobacterium tumefaciens LBA4404, and 26 individual transgenic plants were obtained through the hygromycin selection process. Nucleotide sequence analysis showed that most of T0 plants carried both edited and unedited wt sequence of JAZ9, suggesting genetic chimerism of T0 plants. Even though 2 individual lines carried homozygous mutation on JAZ9, they were also chimeric due to biallelic mutation. The relative ratio between edited and unedited wt sequence was variable among individual lines. Expression level of Cas9 is correlated with the frequency of genome editing frequency. In some plants, the enrichment ratio changed along with developmental stage. The nucleotide sequence analysis revealed that Cas9-mediated A/T addition occurred at -3 nucleotide position from protospacer adjacent motif (PAM), whereas G addition at -5 nucleotide position from the PAM. Further analysis of T1 transgenic plants showed that the genome editing patterns were similar between T0 plants and their T1 sibling plants. These suggested that earlier selection of T0 plants with homozygous mutation is an efficient way to obtain homozygous mutants in T1 generation.

Transformasi Genetik Tembakau dengan Gen Cold Shock Protein melalui Perantara Agrobacterium tumefaciens

<p>Cold<br />shock protein (Csp) essential for organisms to survive in<br />abiotic stress condition. CspB gene has been fused to<br />ubiquitin promoter in the T-DNA region of pCambia 1300int,<br />and introduced into Agrobacterium tumefaciens LBA4404.<br />This research had an objective to transform genetically<br />Nicotiana tabacum cv. Samsun by CspB gene under the<br />control of Ubiquitin promoter and NOS terminator mediated<br />by A. tumefaciens. Leaf discs were co-cultivated with A.<br />tumefaciens LBA 4404. Based on the number of hygromycinresistant<br />calli, the efficiency of transformation was 57.5%. In<br />the selective medium containing 50 μg/l hygromycin, the<br />efficiency of regeneration of transgenic shoots was 82.6%.<br />Based on PCR analysis using primers corresponding to<br />ubiquitin promoter and CspB gene, 18 putative tobacco<br />transgenic containing CspB gene under the control of<br />ubiquitin promoter.</p>

Penyisipan Gen Inhibitor α-amilase pada Plasmid Biner pCambia 1301

<p class="p1">The experiment was conducted at the Molecular Biology Laboratory of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor. The objective was to construct <em>-ai </em>gene on a binary plasmid <em>p</em>Cambia 1301. This experiment was carried out using construction method by ligation process between fragments of <em>α-ai </em>gene from <em>p</em>TA<span class="s1">3 </span>plasmid and <em>p</em>Cambia 1301 on <em>Hind</em>III site. The result of ligant transformation into <em>E. coli </em>DH5<em>α </em>was 182 surviving colonies on YEP medium containing kanamycin. DNA samples were obtained from 60 randomly selected colonies. The restriction pattern was tested by digesting each DNA sample using <em>Hind</em>III showed colonies containing two fragments expected of sizes wich are 11.837 and 4.887 kb. Two colonies are predicted containing of <em>α-ai </em>gene on its the binary plasmid. Advanced tests using restriction enzymes <em>Bam</em>HI and <em>Xba</em>I showed two directions (right and left) of <em>α-ai </em>gene. The right direction was shown by <em>p</em>Cambia-<em>α-ai</em>1 from colony number 43. This plasmid showed expected fragments of sizes 13.485 and 3.219 kb when digested with <em>Bam</em>HI and two fragments of sizes 15.421 and 1.303 kb when digested with <em>Xba</em>I. The left direction was shown <em>p</em>Cambia-<em>α-ai</em>2 from colony number 58. This plasmid also demon-strated expected fragments of sizes 15.026 and 1.698 kb when digested with <em>Bam</em>HI and two fragments of sizes 13.082 and 3.642 kb when digested with <em>Xba</em>I. Both <em>p</em>Cambia-<em>α-ai</em>1 and <em>p</em>Cambia-<em>α-ai</em>2 were transformed into <em>A. tumefaciens </em>LBA4404.</p>

A potent bidirectional promoter from the monocot cereal Eleusine coracana

Ragi bifunctional α-amylase-trypsin inhibitor (RBI) of Eleusine coracana (L.) Gaertn. (finger millet) simultaneously inhibits α-amylase and trypsin. In continuation of previous work on the cloning, expression and characterization of RBI, a bidirectional promoter from finger millet was explored on the basis of experimental observations. Two trypsin inhibitors were identified while purifying RBI from a trypsin-Sepharose column eluent. Using an FPLC gel filtration column, these three inhibitors were purified to homogeneity and subjected to MALDI-TOF-TOF-MS/MS analysis and N-terminal sequencing. Both ragi trypsin inhibitors (RTIs) showed the same N-terminal sequence and considerable sequence similarity to RBI, indicating the presence of a multigene protease inhibitor family in finger millet. To gain insight into the evolution of these genes, the upstream region of RBI was explored by Genome Walking. Interestingly, on sequencing, a genome walking product of ∼1 Kb showed presence of an N-terminal RBI specific primer sequence twice but in opposite directions and leaving an intervening region of ∼0.9 Kb. The intervening region was presumed to represent an E. coracana bidirectional promoter (EcBDP), intuitively having a divergent RBI-RTI gene pair at two sides. For assaying the bidirectionality of promoter activity, a dual reporter GUS-GFP vector construct was made for plant expression containing the reporter genes at two ends of EcBDP, which was used to transform Agrobacterium tumefaciens LBA 4404. Transient plant transformation by recombinant Agrobacterium cells was carried out in onion scale epidermal cells and finger millet seedling leaves. Simultaneous expression of GUS and GFP under EcBDP established it as a potent natural bidirectional promoter from monocot origin, thereby potentially having vast application in cereal gene manipulations. In addition, inducibility of the EcBDP by either abscisic acid or cold treatment, as determined by transient transformation in onion, would substantiate more precise control of gene expression to mitigate the effects of adverse environmental conditions.

Efficient Agrobacterium-mediated transformation of Artemisia annua L. using young inflorescence

Artemisia annua L. is currently the only practical source of artemisinin, an effective antimalarial agent. Development of an efficient gene delivery system is crucial for genetic manipulation of this medicinal plant to enhance artemisinin production. Here, we report a protocol for efficient Agrobacterium-mediated transformation and plant regeneration of A. annua using young inflorescence as explants. We evaluated the influence of explant types and Agrobacterium strains on transformation and regeneration efficiency. Young inflorescence and leaf explants were co-cultivated with Agrobacterium tumefaciens strains, EHA105 or LBA4404, carrying the pCAMBIA2301 binary vector containing a s-glucuronidase reporter. Shoot regeneration was observed when explants were incubated on modified Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 6-benzylaminopurine and 0.3 mg L−1 α-naphthalene acetic acid. Shoot bud development from young inflorescence explants was observed within 7 wk of culture whereas those from the leaf explants regenerated after 9 wk. PCR and Southern blot analyses showed that 95% of the putative transgenic lines were positive for the GUS reporter gene. The maximum transformation frequency of 17% was achieved by co-cultivation of A. tumefaciens strain EHA105 with young inflorescence, representing a twofold improvement compared to using leaf explants. Molecular and genetic analysis of T1 progeny further confirmed the inheritance of the introduced gene. The transformation protocol using young inflorescence of A. annua reduces the transformation time and increases the efficiency; hence, this represents a practical platform for metabolic engineering of this important medicinal plant.

Highly efficient plant regeneration and Agrobacterium-mediated transformation of Helianthus tuberosus L.

In our current study, to develop an efficient regeneration and transformation system of Helianthus tuberosus, we verified effects of plant growth hormones, growth conditions, explant type, and transformation conditions. Leaf segments from regenerated shoots showed higher regeneration efficiency on MS basal medium containing 1.0mgL−1 zeatin under darkness than those from maintained in vitro plant. To carry out transformation, various parameters including plant materials, Agrobacterium cell density, immersion time, and Agrobacterium strains (leaf segment, OD600-0.6, 60min, and Agrobacterium tumefaciens LBA4404 harboring binary vector pCAMBIA1301) have been determined. The putatively transformed H. tuberosus were screened by survival rate and beta-glucuronidase (GUS) histochemical assay following two cycles of 3.0mgL−1 hygromycin selection at callus stage. The presence of the selectable marker gene hygromycin phosphotransferase and the GUS reporter gene with intron was then confirmed by genomic PCR, Southern blot, reverse transcriptase PCR (RT-PCR) and GUS histochemical assay. The method presented here could be helpful in genetic improvement of H. tuberosus through efficient shoot regeneration and stable Agrobacterium-mediated transformation.

Agrobacterium-mediated genetic transformation of Populus deltoides marsh clone G48 with gus and npt-II genes

Agrobacterium-Mediated Genetic Transformation of Populus deltoides Marsh Clone G48 with gus and npt-II Genes The present investigation had been carried out to standardize a protocol for Agrobacterium- mediated gene transfer in Populus deltoides Marsh. Clone G48 using petiole explants. Reproducibility of already standardized regeneration protocol for clone G48 had been evaluated. High frequency shoot regeneration (72%) from petiole explants was obtained on MS medium supplemented with 0.5 mg l-1 BAP, 0.2 mg l-1 IAA and 15 mg l-1 adenine sulphate. Root regeneration (100%) in in vitro developed shoots was obtained on MS medium supplemented with 0.1 mg l-1 IAA. Increasing concentrations of kanamycin (10-50 mg l-1) were given to find out the minimum dose of kanamycin required for the selection of putative transformed cells during genetic transformation. It was observed that a doze of 50 mg l-1 kanamycin inhibited callus formation and shoot regeneration and the explants turned brown and started dieing. This concentration of kanamycin would be the most useful for selection of npt-II gene transformed petiole cells/ tissues. Disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β-glucuronidase (gus) gene in binary vector pBI 121 along with kanamycin resistance gene (npt-II) was used for genetic transformation experiments. Only the transformed cells/ shoots were able to grow on selective shoot regeneration medium containing 50 mg l-1 kanamycin, whereas control explants did not survive. Regenerated putative transgenic shoots were subjected to transient gene expression analysis using spectrophotometric GUS assay and were found positive. This genetic transformation protocol will provide a platform for genetic manipulation of P. deltoides clone G48 for incorporation of genes governing various silviculturally important traits in future....

English

In the present investigation, explants from Kalanchoë blossfeldiana were used for gene transformation. The young leaves were inoculated with Agrobacterium tumefaciens LBA4404 strain with a binary vector plasmid pArtblue containing F3'5'H gene under control of CaMV35S promoter and nptII selectable marker gene. After inoculation, the explants were transferred to the co-cultivation medium. They were then transferred to the selection medium containing kanamycin and were sub-cultured every two weeks. Leaves of the putative transgenic shoots that survived in the selection medium were used in reverse transcription polymerase chain reaction (RT-PCR) analysis to detect gene expression. The RT-PCR analysis showed the presence of 550 bp F3'5'H amplification products and had an expression of F3'5'H gene. Plants with the introduced F3'5'H gene produced totally pale red flowers. Key words: Kalanchoë blossfeldiana, Agrobacterium-mediated transformation, young leaf, F3'5'H gene, reverse transcription-polymerase chain reaction (RT-PCR).

Conserved sequence of replicase gene mediated resistance in Nicotiana tabacum L. cv Abirami through RNA silencing

Tobacco streak virus (TSV) is economically the most important virus infecting several crop plants in India. Symptomatic samples collected from sunflower and okra fields were subjected to RT-PCR with TSV replicase gene specific primers. The replicase (Rep) genes of these isolates were sequenced in order to develop transgenic resistance to Tobacco streak virus (TSV) in tobacco (Nicotiana tabacum L.) cv Abirami by expressing hairpin RNA transcript (hpRNA). The sequence analysis of replicase gene of these isolates showed a sequence identity of around 99% at nucleotide level with Tamil Nadu okra isolate. The hairpin construct was generated using pHANNIBAL vector with the conserved nucleotide sequence corresponding to position 3065 to 3405 of the TSV replicase gene. The Rep hairpin construct was cloned into pART27 and mobilized into Agrobacterium tumefaciens LBA4404 and introduced into tobacco by Agrobacterium mediated transformation. The T0 plants obtained were screened by PCR and Southern blot analysis using the genomic DNA from transformed tobacco plants. The transformants produced ~299 bp and 340 bp amplicons corresponding to nptII gene and Rep gene respectively. The Southern blot analysis showed single and multiple-copy integration of the transgenes. The transgenic T0 tobacco plants showed resistance against TSV upon mechanical inoculation of TSV without producing any visible symptoms; resistance was also confirmed by DAC-ELISA.

Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

Ocimum gratissimum is renowned for its immense role in traditional, ayurvedic and unani system of medicines. The essential oil has a plethora of active ingredient like triterpenes, polyphenols and phenylpropanoids. The active ingredients claim most of the properties like anticancerous, anti HIV, antipyretic, antinociceptive, antiurolithiatic etc. Screening of biologically active ingredients from this valuable plant may provide us the basis to produce novel pharmaceutically important drugs and medicines. Considering the importance of this plant, it is the need to have a well developed regeneration and transformation protocol.The present report focuses on the development of an efficient protocol for direct regeneration and Agrobacterium mediated genetic transformation of O. gratissimum. CN; when inoculated on MS+1.5mg/L+0.5mg/L+0.5mg/L KN medium has shown the best response with a regeneration frequency of 98%±0.4. A. tumefaciens LBA4404 containing binary vector pBI121, harboring gus-A reporter gene with intron under the transcriptional control of Cauliflower Mosaic Virus (CaMV) 35S promoter and neomycin phosphotransferase (npt-II) as selection marker genes, to achieve maximum transformation frequency (20%±0.7). To carry out transformation, parameters like O.D., infection duration, concentration of AS, surfactant, (O.D600-0.6, 15min, 300μM, 0.01%) were standardized. The putative transformants were screened on SMM having 50mg/L kanamycin and subsequently rooted on ½ MS. The confirmation was done through PCR. The established regeneration and transformation protocol could be used as a tool to elucidate the functional genomics of important/novel compound which can be used in the preparation of therapeutic formulations for future.

Using leaf explants for transformation of Chrysanthemum morifolium ramat. mediated by Agrobacterium tumefaciens

An Agrobacterium-mediated transformation for chrysanthemum using leaf explants of two different cultivars was conducted. Leaves were first infected with Agrobacterium tumefaciens LBA4404 strain, harbouring a binary vector pBI121 carrying the GUS reporter gene and the nptII as the selectable marker gene. 30 seconds for infection of leaves proved to be the optimum duration. Best results were achieved when explants were cocultivated with A. tumefaciens for two days. Shoot induction medium was supplemented with 7 mgl -1 kanamycin and 300 mgl -1 cefotaxime. Putative transgenic plants were obtained after rooting on 1/2 MS medium containing 7mgl-1 kanamycin in the absence of cefotaxime. Transformation efficiency of ‘Reagan Elite Salmon’ (29.1%) was higher than ‘Resomee Splendid’ (9.1%). Presence of the gus gene was verified with histochemical GUS assay and polymerase chain reaction (PCR) of transformed plants.

Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum.

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable.

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry.