Plasmid pCAMBIA Research Articles

Melatonin strongly enhances the Agrobacterium- mediated transformation of carnation in nitrogen-depleted media

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

Localization of PQ-LRP protein in Microsporum canis

Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein (EGFP) as a marker. Methods The total RNA was extracted from Microsporum canis, and reversely transcribed into cDNA. The PQ-LRP gene was amplified by PCR using the above cDNA as the template. The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300. Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation, in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein. Results The expression vector pCAMBIA-LRP-EGFP was successfully constructed, and the fusion gene LRP-EGFP was expressed integratedly in Microsporum canis. Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis, giving a granular or cluster-like appearance. Conclusion The infusion gene LRP-EGFP can be successfully expressed in Microsporum canis, and PQ-LRP protein is located on the cell membrane of Microsporum canis. Key words: Microsporum; Fiducial markers; Green fluorescent proteins; Microsporum canis; PQ-LRP; Protein localization

Regeneration and Agrobacterium-mediated transformation of japonica rice varieties developed for a cold region

So far, a large number of transformation systems have been established for japonica rice, but only a few have been reported for cold-region varieties. In our study, we established highly efficient tissue culture systems for two cold-region rice cultivars, Dongnong 427 and Longdao 14. Plant growth regulator (PGR) levels were optimized by an orthogonal experimental design. The culture ability, constituted by induction and differentiation rate, served as the detection index of orthogonal experiments. The optimal combinations of PGRs for callus induction and regeneration of Dongnong 427 and Longdao 14 were 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) + 2 mg/l 6-benzyladenine (BA) + 4 mg/l kinetin (KIN) + 0.2 mg/l α-naphthaleneacetic acid (NAA) and 1 mg/l 2,4-D + 4 mg/l 6-BA + 4 mg/l KIN + 0.5 mg/l NAA, respectively. Agrobacterium strain EHA 105 containing the plasmid pCAMBIA1301 was used for transformation. The frequency of transient transformation was expressed as the ratio between the number of calli showing GUS expression and the total number of calli kept for staining. The highest transformation efficiency in Dongnong 427 was obtained when calli were immersed in 0.272 OD<sub>600</sub> (optical density determined at 600 nm) for 10 min. While it was best for Longdao 14 calli to be infected with 0.592 OD<sub>600</sub> for 20 min. Infected calli of the two varieties were co-cultivated on two pieces of sterile filter paper moistened with 1 ml liquid co-cultivation medium for three days. The expression of the GUS gene was confirmed by PCR analysis of plants of both varieties.

In vitro Regeneration and Over Expression of Pea DNA Helicase 45 (PDH45) Gene into the Local Cultivars of Chickpea (Cicer arietinum L.) through Agrobacteriummediated Genetic Transformation

In vitro regeneration studies compatible to Agrobacterium-mediated genetic transformation were carried out using two different types of zygotic embryo derived explants namely, decapitated embryo (DE) and decapitated embryo with single cotyledon disc (DEC) from three varieties of chickpea (Cicer arietinum L.) such as BARI chhola-4, -5 and -9 cultivated in Bangladesh. The best responses towards in vitro shoot regeneration was obtained from decapitated embryo with DEC on MS containing 0.5 mg/l BAP, 0.5 mg/l Kn and 0.2 mg/l NAA. Healthy and effective roots from the regenerated shoots were developed on MS supplemented with 0.2 mg/l IBA. Genetic transformation was carried out with Agrobacterium strain LBA4404 containing the binary plasmid pCAMBIA1301- PDH45 to integrate salt tolerant PDH45 gene in locally grown varieties of chickpea. The transformed plantlets were successfully established in soil following adequate hardening. Integration of salt tolerant PDH45 gene within the genomic DNA was confirmed through GUS histochemical assay and PCR analysis.Plant Tissue Cult. & Biotech. 28(1): 125-140, 2018 (June)

Optimization of various factors affecting Agrobacterium-mediated transformation and regeneration of putative transgenic Malaysian MR219 rice with Bacillus SP 289 endo-β-1,3-1,4-glucanase gene

Sheath blight disease caused by Rhizoctonia solani is the most destructive diseases in rice cultivation. Development of transgenic indica rice MR219 through Agrobacterium tumefaciens strain EHA 105 harbouring the plasmid pCAMBIA 1305.2 with endo-beta-1,3-1,4-glucanase gene from Bacillus SP 289 is one of the strategies to engineer disease resistance. Four optimisation parameters were examined such as Agrobacterium culture cell density (0.1 to 1.0 based on OD600nm), callus immersion time in the Agrobacterium culture (30 to 120 minutes), duration of the subsequent drying time (15 to 120 minutes) and co-cultivation period (1 to 6 days). Hygromycin-resistant embryogenic calli developed after 8 to 10 weeks of transformation. Improved transformation rates were achieved when calli were incubated with an Agrobacterium suspension with a culture density of OD 600nm 0.2 for 30 mins, followed by 90 mins of drying time and co-cultivation for 3 days. PCR analysis of the transformants confirmed the presence of Bacillus SP 289 endo-beta-1,3-1,4-glucanase and hpt genes in the rice genome. The transgenic rice plants obtained in this study will be tested against sheath blight disease.

Agrobacterium tumefaciens-mediated transformation of drumstick (Moringa oleifera Lam.)

ABSTRACTDrumstick (Moringa oleifera Lam.) is an industrially important tree species. Due to the availability of an efficient regeneration system, the present study was undertaken to develop a reliable and stable Agrobacterium tumefaciens-mediated transformation system for the genetic improvement of drumstick. A. tumefaciens strain EHA105, harbouring the plasmid pCAMBIA1301 containing the hygromycin phosphotransferase II and ß-glucuronidase genes driven by the CaMV 35S promoter, was used to establish a transformation system in drumstick. The use of vacuum infiltration-assisted Agrobacterium infection (excluding a pre-culture step) and co-cultivation for two days significantly increased the transformation frequency. Genomic integration and transgene expression were confirmed by β-glucuronidase (GUS) assays, polymerase chain reaction (PCR), Southern blot analysis and real-time quantitative PCR (RT-qPCR). This transformation protocol provides a basis for the future development of genetic engineering techniques to improve the performance of drumstick.

Yield improvement of the king oyster mushroom, Pleurotus eryngii, by transformation of its cellulase gene

AbstractA plasmid pCAMBIA1301 containing

Optimasi Sistem Regenerasi dan Transformasi Padi Varietas Elit Indonesia

<p>New rice<br />variety can be generated by means of transgenic approach.<br />Transgenic rice researches have been conducted in many<br />institutions worldwide using Japonica, Indica, and Javanica<br />varieties. The most cultivated rice in Indonesia is Indica.<br />Indica type is known to have low responsive tissues in<br />culture and transformation media when compared to<br />Japonica rice. This research activity is aimed to optimize<br />regeneration and transformation systems of the Indonesian<br />elite rice varieties, so that good method can be achieved to<br />be used in the generation of transgenic elite rice varieties of<br />Indica type. The research consisted of two activities:<br />regeneration and transformation optimizations in varieties of<br />Dodokan (upland rice) and Inpari 6 (irrigated rice).<br />Immature embryo was used as the explant in this research.<br />The optimization studies used 2 types of media, NBH (N6<br />salts and vitamins, cassamino acid 0.5 g/l, L-proline 0.5 g/l,<br />sucrose 20 g/l, D-glucose 10 g/l, 2.4-D 2 mg/l, NAA 1 mg/l, BA<br />1 mg/l, agarose Type I 5.5 g/l) and NBH-M (N6 macro salts,<br />B5 micro salts, and vitamins, 0.3 g/l cassamino acid, 3 g/l Lproline,<br />20 g/l sucrose, 3 mg/l 2.4-D, 1 mg/l NAA, 1 mg/l BAP,<br />5.5 agarose Type I), 2 types of regeneration media, R1 (MS<br />base media and vitamis, 0.3 g/l glutamine, 30 g/l sucrose, 2<br />mg/l kinetin, 1 mg/l NAA, 3 g/l phytagel) and R2 (MS base<br />media and vitamins, 2 g/l cassamino acid, 20 g/l sucrose, 30<br />mg/l sorbitol, 2.5 mg/l kinetin, 0.25 mg/l NAA, 3 g/l phytagel).<br />Optimization transformation of Indonesian elite rice varieties<br />used developed an empty plasmid pCAMBIA 1301 containing<br />hpt gene. The transformation was conducted using two<br />types of co-cultivation media, K1 (N6 macro salts, B5 micro<br />salts, and vitamins, 0.5 g/l cassamino acid, 0.5 g/l L-proline,<br />20 g/l sucrose, 10 g/l glucose, 2 mg/l 2.4-D, 1 mg/l NAA, 1<br />mg/l BAP, 0.1 mM acetosyringone) and K2 (N6 macro salts,<br />B5 micro salts, and vitamins, 0.5 g/l cassamino acid, 0.5 g/l Lproline,<br />20 g/l sucrose, 10 g/l glucose, 2 mg/l 2.4-D, 1 mg/l<br />NAA, 1 mg/l BAP, 0.2 mM acetosyringone). The results<br />showed that Inpari 6 could form embryonic calli in NBH<br />media and further regenerated well in R1 media (13.8%).<br />The co-cultivation media K1 generated more selected calli<br />which then generated green plant of young embryo<br />compared to K2. Inpari 6 showed higher regeneration rates<br />after transformation (3.6%) compared to Dodokan (0%).<br />Molecular analysis showed that all 11 transformants (Inpari</p><p>6) tested contained the hpt gene. These results are expected<br />to support the development of transgenic Indica rice<br />generation in Indonesia.</p>

English

Cowpeas (Vigna unguiculata) are important grain legumes grown by resource poor farmers across Sub-Saharan Africa. In Kenya, cowpeas continue to produce low yields due to erratic rainfall caused by the current climate change. This study sought to enhance drought tolerance in Kenyan cowpeas through transformation by Agrobacterium tumefaciens using L-Pyrroline-5-carboxylate synthase (P5CS) gene. Cowpea variety K80 was inoculated with Agrobacterium tumefaciens strains EHA105 and LBA4404 harbouring recombinant plasmids pCAMBIA1301:VuP5CS or pCIP100:VuP5CS through vacuum infiltration of pre-germinated seeds at 60 kpa for 25 min. The presence of the transgene in the transformed cowpea plants and its transfer to progeny was confirmed by PCR analysis of T0 and T1 plants. The highest transformation efficiency of 20.5% was achieved with strain EHA105 harbouring plasmid pCAMBIA1301:VuP5CS. A segregation analysis of the transgenes gave a 1:4 ratio for the VuP5CS transformed: non-transformed plants and did not follow the 3:1 Mendelian inheritance pattern for dominant genes. There was no difference in proline content in the transformed and non-transformed T1 plants. However, the roots of the transformed plants were significantly longer than those of the non-transformed plants. The numbers of harvested seeds were also significantly higher in the transformed plants with 10 to 11 seeds per pod in comparison to the non-transformed plants with an average of 5.58 seeds per pod, indicating drought tolerance potential of the transformed plants. The T2 and T3 plants need to be screened further to evaluate the stable integration of the transgene and physiological characterization under water stress. Key words: Vigna unguiculata, in planta transformation, Agrobacterium tumefaciens, VuP5CS (Vigna unguiculata pyrroline-5-carboxylate synthase).

Agrobacterium tumefaciens - Mediated transformation of Woodfordia fruticosa (L.) Kurz.

In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

Improvement of adventitious organogenesis for regeneration of transgenic plants in blackberry

The introduction of foreign DNA into the plant genome by Agrobacterium tumefaciens is a promising technique of targeted gene transfer which depends on good working regeneration system. The aim of the work was to elaborate the system for efficient adventitious organogenesis and transgenic plant regeneration in Rubus fruticosus L. using explants from mature plants. Regeneration of putative transgenic shoots took place from flag explants cultivated vertically on MS medium with 1 mg l-1 TDZ and 0.02 mg l-1 IBA followed by transfer on MS medium with 1 mg l-1 BAP, 0.02 mg l-1 IBA and 0.1 mg l-1 GA3 supplemented with 10-15 mg l-1 hygromycin after transformation by A. tumefaciens strain LBA 4404 carrying plasmid pCambia 1304. Four putative transgenic plants of cv. 'Cacanska Bestrna' were rooted and acclimatized.

Finger millet [Eleusine coracana (L.) Gaertn

Millets are the primary food source for millions of people in tropical regions of the world supplying mineral nutrition and protein. In this chapter, we describe an optimized protocol for the Agrobacterium-mediated transformation of finger millet variety GPU 45. Agrobacterium strain LBA4404 harboring plasmid pCAMBIA1301 which contains hygromycin phosphotransferase (hph) as selectable marker gene and β-glucuronidase (GUS) as reporter gene has been used. This protocol utilizes the shoot apex explants for the somatic embryogenesis and regeneration of finger millet after the transformation by Agrobacterium. Desiccation of explants during cocultivation helps for the better recovery of transgenic plants. This protocol is very useful for the efficient production of transgenic plants in finger millet through Agrobacterium-mediated transformation.

English

Rubber tree belonging to the genus Hevea is an economically important crop of Thailand and South-east Asia . To optimize its agronomical trait for glyphosate-resistant, in vitro gene transformation through Agrobacterium tumefaciens was investigated. The bacteria carrying plasmid pCAMBIA 1304, harboring gus as screenable marker genes and EPSPs gene was used. The shoot tips were immersed in A. tumefaciens suspension at optical densities (OD 600 ) at 0.3, 0.6 and 0.9 for various times (15, 30 and 60 min). The results revealed that shoot explants immersed in A. tumefaciens suspension at OD 600 of 0.6 for 30 min gave the higher survival rate after being cultured on glyphosate containing MS medium for one and half months. Assessment of transformed shoots revealed positive results in GUS histochemical assay. The presence of the gus and EPSPs genes in transformed rubber tree were confirmed by polymerase chain reaction (PCR) technique, dot blot hybridization and Southern PCR hybridization. Specific primers for the gus and EPSPs genes were designed to amplify a 919 and 1,600 bps DNA fragment, respectively. Keywords: Transgenesis, glyphosate, inoculation time, Agrobacterium density, Hevea African Journal of Biotechnology , Vol 13(23) 2321-2329

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.

Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn

Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival of putative transformants were taken into consideration for the assessment of parameters. Optimum conditions for the microprojectile bombardment mediated genetic transformation of finger millet were 1,100 psi rupture disk pressure with 3 cm distance from rupture disk to macrocarrier and 12 cm microprojectile travel distance. Double bombardment with gold particles of 1.0 μm size provided maximum transient GUS expression and transformation efficiency. Osmotic treatment of callus with 0.4 M sorbitol enhanced efficiency of particle bombardment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634 bp sizes of the bands respectively from putative transformants and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of transgene in T1 generation plants. Thus a successful genetic transformation system was developed using particle bombardment in E. coracana with 45.3% transformation efficiency. The protocol will be helpful for the introgression of desired genes into E. coracana.

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato ( Ipomoea batatas [L.] Lam.)

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato ( Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l −1 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l −1 hygromycin and 200 mg l −1 cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.

A Rare Event of Agrobacterium rhizogenes-assisted Genetic Transformation of‘Silk’Banana (genotype-AAB)

Genetic transformations in monocots are achieved with low frequencies with Agrobacterium tumefaciens, often in conjunction with biolistic methods. However, we report here for the first time, the use of A. rhizogenes harbouring a binary vector and utilization of shoot bud explants, instead of embryogenic cultures, while also establishing that intervening hairy root stage can be bypassed. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene under the control of CaMV35S promoter was used for sonication-assisted transformation of shoot bud explants of an endemic banana cv. Nanjanagudu Rasabale (NR) grouped under genotype AAB. Transformation frequency up to 2.4% was obtained depending on the medium used. The use of hygromycin in the multiple shoot induction medium allowed the selection of transgenics having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of transformants and indicated single and multiple locus integrations.

Effect of over-expression of Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (LuPLR) gene in transgenic Phyllanthus amarus

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l−1, kan 50 mg l−1 and cephotaxime 62.5 mg l−1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l−1, kan 50 mg l−1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l−1 indole 3-butyric acid (IBA) and 5 mg l−1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.

Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recombinant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

Symbiotic reactions of sea-buckthorn roots transformed with the pea lectin gene

Sea-buckthorn (Hyppopha L.) transgenic roots transformed with the lectin gene were obtained using the wild-type strain of Agrobacterium rhizogenes 15834 preliminary transformed with the plasmid pCAMBIA 1305.1, which contained the full-size pea lectin gene. Effects of lectin gene expression on symbiotic responses of sea-buckthorn to inoculation with rhizobia (Rhizobium leguminosarum, pea symbiont) and actinomycetes of genus Frankia (sea-buckthorn symbiont) were studied. In sea-buckthorn seedlings, whose transgenic roots were inoculated with both microsymbionts simultaneously, atypical nodule-like structures were found along with typical actinorhizal nodules. Random amplified polymorphic DNA (RAPD) analysis of bacteria, isolated from these structures, revealed the presence of R. leguminosarum rhizobia and the absence of Frankia actinomycetes.