nptII Gene Research Articles

Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.

An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

Genetic Transformation Via Biolistic Gene Gun Method and Regeneration of Common Bean

The objective of the present research was to utilize biotechnology to improve agronomical traits such as drought stress tolerance in common bean (Phaseolus vulgaris L.) as important recalcitrant crop species via a particle gun “Genebooster” for the bombardment of plant tissues with DNA. The process involves the high velocity acceleration of microprojectiles carrying foreign DNA, penetration of the cell wall and membrane by microprojectiles, and delivery of the DNA into plant cells. The initial phase of the two common bean varieties “Fonix” and “Maxidor” focused on the development of a highly regenerable tissue culture procedure amenable to the particle gun physical method. The results showed that MS medium supplemented with 1 mg/l BA and 0.1 mg/l NAA were the optimal for shoot induction from meristematic ring of cotyledonary node explants in the both bean varieties. The author has developed a tissue culture protocol that enables the “meristematic ring” that develops below axillary shoots on nodal bean explants to produce shoot buds, and developed an electric-discharge particle acceleration procedure to produce transgenic plants in the two common bean varieties. The plasmid pFF19K, harboring the kanamycin resistance gene nptII as a selectable marker and pFF19-mtlD harboring the mtlD gene which confers mannitol (drought stress) tolerance were used for adapting transformation in the two common bean varieties. The results indicated that the successful delivery of plasmids DNA and efficient gene transfer into common bean meristematic tissues included 20 bar nitrogen pressure, 135 mm shooting distance, 1.1 μm diameter size of microprojectiles, and twice bombarding of the target tissues. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 7.6% and 8.4% in “Fonix” and “Maxidor” bean varieties, respectively. Transgenic plants were recovered using both kanamycin (100 mg/l) and mannitol (1.2 M/l) screening to introduce the neomycin phosphotransferase II (nptII) and the mannitol-1-phosphate dehydrogenase (mtlD) genes into the two common bean varieties. The introduced nptII and mtlD genes have been shown to confer strong resistance and tolerance in transgenic beans to kanamycin and mannitol stress applications. The transgenic bean plants were healthy and the method has proven to be applicable to common bean crop. To my knowledge, this is the first report on the production of common bean transgenic plants with mtlD gene using biolistic gun-mediated gene transfer system.

Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.

Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for <i>Agrobacterium tumefaciens</i>-Mediated Transformation of Higher Plants

We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU14 and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline-based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.

An Agrobacterium-mediated transformation system of pyrethrum (Tanacetum cinerariifolium) based on leaf explants

Pyrethrum (Tanacetum cinerariifolium) is well-known for the production of natural pyrethrin insecticides, which are isolated from the dried flower heads. To allow genetic analysis and improvement of pyrethrum in terms of yield and quality by molecular breeding a transformation protocol would be desirable. To achieve this, first an efficient direct shoot regeneration system was set up. Leaf explants formed shoots within 4 weeks on MS medium supplemented with 2.0mgl−1 6-BA and 0.2mgl−1 IBA. The best regeneration frequency obtained was 87% with an average number of 2 shoots per explant. Subsequently, a genetic transformation protocol was developed. Kanamycin added to the medium significantly reduced explant regeneration, and no green shoots were formed after 4 weeks incubation at 10mgl−1 kanamycin. Severe phytotoxic effects of cefotaxime were observed at concentrations >300mgl−1 to control Agrobacterium. Thus, 10mgl−1 kanamycin and 200mgl−1 cefotaxime were selected as optimal for genetic transformation. Agrobacterium-mediated genetic transformation was carried out with a GUS gene under the control of the chrysanthemum RbcS promoter. Two days of pre-culture and 5min of infection were found to result in the most efficient transformation rates. Ninety-nine percent of leaf explants infected with Agrobacterium showed GUS expression 3–5 days after transfection and 12% of the regenerated shoots had a stable GUS-positive phenotype. The other plants had a transient kanamycin-resistant phenotype and eventually lost the NPT-II and GUS gene expression.

Influence of Marker Genes on Physicochemical Properties of Starch Produced by Transgenic Cassava (Manihot esculenta Crantz) Plants

The study monitored the influence of genetic modification of cassava plants with NPT-II and GUS genes on starch content and physicochemical properties of transgenic starch for three cycles of vegetative propagation from 2010-2012. There were no significant (p>0.05) differences in the starch content and physicochemical properties among the three cycles of vegetative propagation. The NPT-II and GUS genes were strongly expressed in the vascular bundle, starch synthesizing and storage cells. The type of TME 12 variety (transgenic vs non-transgenic) has significant (p<0.05) influence on starch content, crude fibre, pasting viscosity and breakdown value. Starch content and crude fibre of non-transgenic plant (83.9%, 3.6%) were significantly (p<0.05) higher than that of transgenic plant (77.5%, 2.9%). Similarly, pasting viscosity and breakdown value (783.2 mPa s, 284.2 mPa s) of non-transgenic plants were greater than that of transgenic plant (621.0 mPa s, 133.1 mPa s) significantly. The implications of the findings on future genetic modification of cassava with NPT-II and GUS genes for starch quality improvement are discussed.

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P SAG12 -ipt gene delays leaf senescence and enhances resistance to exogenous ethylene

KEY MESSAGE : The P ( SAG12 ) -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions. Transgenic plants of Rosa hybrida 'Linda' were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P( SAG12 )-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P(35S)-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10% and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1-6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5μll(-1)) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.

Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil

To introduce genetic resistance against alfalfa weevil (Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production.

Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker

BackgroundSpecies of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs) in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph) and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential.ResultsWe established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L) resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi.ConclusionThe neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic tractability of these important BCAs.

Analysis of mosaic expression of the nptII gene in transgenic tobacco plant lines contrasting in mosaicism

Differences in the frequency of occurrence of plants with the mosaic phenotype between the Nu5 and Nu6 lines of transgenic tobacco (Nicotiana tabacum) plants remained irrespective of the allelic state of the nptII marker gene. Transition of the nptII gene from the hemizygous state (T3) to the homozygous state (T4) was accompanied by an increase in the frequency of mosaics in both lines. Transition from the homozygous state (T4) into the hemizygous state (F1) resulted in a further increase in the frequency of mosaic plants in the Nu5 line, whereas in the Nu6 line this parameter remained at a high level. Hypermethylation of the pMAS promoter in plants of both lines, as well as differences in the degree of methylation of cytosines in the 5'-region of the coding part of a truncated nptII gene copy between the Nu5 and Nu6 lines, pointed to epigenetic regulation of the mosaic expression of the nptII gene.

Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst

Efficient Agrobacterium tumefaciens mediated T-DNA delivery and subsequent shoot organogenesis has been achieved from Bacopa monnieri. Various factors influenced T-DNA delivery as evident from transient GUS assay. The transient GUS expression was significantly higher (97.7 %) in explants that were pre-cultured before bacterial infection on medium supplemented with 100 μM acetosyringone. Incorporation of acetosyringone into the co-cultivation medium also enhanced transient GUS activity. Explant injury with carborundum paper, co-cultivation period of 2 days and a bacterial density of 0.4 OD600 showed higher transient GUS expression. Following co-cultivation, shoot organogenesis was achieved from leaf segments on basal Murashige and Skoog medium containing 58 mM sucrose. Supplementation of antibiotics (cefotaxime or carbenicillin) at > 250 μg/ml into the medium significantly promoted shoot organogenesis from leaf explants (71.5 % in control and > 83.0 % on medium containing 500 μg/ml of carbenicillin or cefotaxime). Stable transformation of regenerated shoots was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to reporter gene (uidA) and selection marker gene (nptII). The expression level of nptII gene in independent transgenic lines was studied using quantitative real time-PCR. Stable transformed shoots after rooting were successfully established in the pots.

Optimization of Agrobacterium-Mediated Genetic Transformation and Regeneration of Transgenic Plants in Indian Cultivar of Barley (Hordeum vulgare L. cv. BL 2)

An efficient Agrobacterium-mediated genetic transformation protocol has been developed for Indian winter barley. Several parameters affecting Agrobacterium-mediated gene delivery were investigated and optimized. These include use of acetosyringone, surfactants in the infection medium, antinecrotic treatment of explants and heat and centrifugation pre-treatment. Addition of acetosyringone in the infection and co-cultivation media was advantageous and resulted in increase in transformation efficiency from 3.7 to 11.1 %. Addition of surfactants (Tween-20/Pluronic acid F 68) at 0.1 % (v/v) had also resulted in improved gene delivery. Pre-treatment of immature embryos with antinecrotic mixture had significant positive effect on percent callus survival and transformation efficiency. Temperature pre-treatment of immature embryos prior to infection resulted in an increase in the number of GUS positive calli with optimum temperature of 40 °C for 3 min. However, centrifugation of immature embryos had a negative effect on barley transformation. Regenerated transgenic plants obtained after 8 to 10 weeks of selection were further rooted for 2 weeks. The plantlets established in green house condition showed normal growth. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of regenerated transformants, respectively. Molecular analysis of transgenic plants containing II gene (nptII) demonstrated the efficacy of optimised protocol for Agrobacterium-mediated genetic transformation in Indian cultivar of barley for the first time.

Transgenic lines of melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens

The oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases. In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR-positive lines. RT-PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T1 generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.

Stable Agrobacterium-mediated transformation of the halophytic Leymus chinensis (Trin.)

In this study, an efficient procedure for stable Agrobacterium -mediated transformation of Leymus chinensis (Trin.) was established. Agrobacterium tumefaciens strain EHA105, harboring a binary vector pCAMBIA2300, was used for transformation, along with a sweet potato 2-cysteine peroxiredoxin ( 2-Cys Prx ) gene under the control of the stress-inducible sweet potato anionic peroxidase 2 ( SWPA2 ) promoter and the neomycin phosphotransferase ( nptII ) gene under the control of the cauliflower mosaic virus (CaMV) 35 S promoter. We found that a one-month-old callus derived from mature seeds could be efficiently transformed. Seven-day preculture followed by inoculation with the addition of 100 μmolL -1 acetosyringone (AS) and then a 3 day co-cultivation were performed before selection. Selection of transgenic shoots was done in the presence of 150 mgL -1 kanamycin (KM). An optical density at a wavelength of 600 nm (OD 600 ) of approximately 0.4 for A. tumefaciens infection solution and 20 min of infection time gave the highest transformation efficiency. Polymerase chain reaction (PCR) analysis of KM-resistant plants and newly regenerated rhizomes revealed stable transformation of the 2- Cys Prx gene and the nptII gene, with the highest transformation frequency of 4.93%. RT-RCR analysis was conducted using salt stressed transgenic plants, and the results suggested that 2-Cys Prx had low transcription levels under non-stressed conditions, and increased transcription after 6 h of 200 mM NaCl stress. This gene continued to demonstrate high levels of transcription until 6 h after withdrawal of stress, with a slow recovery. The method reported herein provides a direct opportunity for improvement of the quality traits of L. chinensis via genetic transformation. Keywords: Leymus chinensis , Agrobacterium -mediated transformation, 2-Cys peroxiredoxin, gene transformation

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08% among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Identification of cis‐elements conferring high levels of gene expression in non‐green plastids

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.

Standardization of Protocol for Agrobacteriummediated Transformation in Potato (Solanum tuberosum L.)

The experiment was conducted at the Laboratory of Biotechnology, Biotechnology Division, Bangladesh Agricultural Research Institute, Gazipur- 1701 during July 2007 to June 2008. An efficient and reproducible protocol for the production of transgenic potato plants was developed by inoculating internode explants of potato with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121 having one reporter gene (gus) and selectable marker gene (nptII) resistant to Kanamycin. The transformation experiment was done by optimizing two important parameters names infection time and cocultivation period. Most of the explants produced shoots within 21 days on 5 mg/l Zeatin riboside (ZR) and 50 mg/l Kanamycin supplemented MS medium without introducing callus. The infected explants produced 8.27 and 6.42 shoots in Asterix and Diamant varieties, respectively within 21 days. Transgenes were confirmed by molecular analysis. DNA from well established rooted plants confirmed nptII positive through PCR analysis. The transformation rates were 28.97 and 24.37% in Asterix and Diamant, respectively. Putative transformed plants of Diamant and Asterix varieties produced roots in ½MS medium supplemented with 50/mg Cefotaxim, 50 mg/l Kanamycin and 0.5 mg/l IBA. DOI: http://dx.doi.org/10.3329/bjar.v37i2.11220 Bangladesh J. Agril. Res. 37(2): 185-194, June 2012

Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture

An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing β-glucuronidase gene (gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20min to agrobacterial inoculum (O.D. 0.4) followed by 3days of co-cultivation on medium supplemented with 100μM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0μM 6-benzylaminopurine, 8.0μM indole 3-acetic acid, and 50.0mgl(-1) kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5μM indole 3-butyric acid for 1week and then transferred to hormone free one-half MS basal for 2weeks. We were successful in achieving 100% frequency of transient GUS expression with 5% stable transformation efficiency using optimized conditions. PCR analysis of T0 transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant.

Use of sonication and vacuum infiltration for Agrobacterium – mediated transformation of an Indian lentil (Lens culinaris Medik.) cultivar

The regeneration and transformation protocol for lentil employing both sonication and vacuum infiltration of whole seed explants is described. This protocol overcomes the non-competence of whole seed explants to Agrobacterium mediated transformation though the regeneration potential of this explant is high as compare to other agro-competent explants. The decoated seeds of four genotypes were cultured on regeneration media that consisted of Murashige and Skoog (MS) salts and Gamborg (B5) vitamins (MSB5V) supplemented with different concentrations of 6-benzylaminopurine (2.5–20μM) and thidiazuron (0.1–10μM) separately. Best regeneration response with an average of 9 shoots per explant was observed in 95% of the cultures on MSB5V medium fortified with 5.0μM thidiazuron. The protocol was found independent to the effect of genotype. Among the effects of physical treatments, it was found that sonication at 20KHz for 60s followed by vacuum infiltration at −660mm of Hg for 5min resulted in highest transient GUS expression (68%) with no significant effect on regeneration. Rooting was observed in 45% of shoots on rooting medium containing half strength MS salts and full strength MS vitamins supplemented with 2.5μM IBA. Regenerated plantlets were established in soil with 50% survival rate and grown to maturity. GUS activity in leaves and roots of primary transformants recovered on kanamycin containing selection media confirmed the expression of transgene. These plants were tested and found positive for nptII gene by PCR and Southern analysis with an overall transformation frequency of 0.66%. PCR analysis of T0 progeny revealed the inheritance of transgene.

Agrobacterium-mediated Genetic Transformation in Lentil (Lens culinaris Medik.) followed by In vitro Flowering and Seed Formation

Genetic transformation system was developed for two microsperma varieties of lentil (Lens culinaris Medik.), namely Bari Masur-4 (BM-4) and Bari Masur-5 (BM-5) using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing GUS and nptII genes. Three different types of embryo explants, namely cotyledonary node (CN), decapitated embryo (DE) and cotyledone attached decapitated embryo (CADE) were used. Highest GUS positive expression was found in DE followed by CADE as detected by transient assays. Following Agrobacterium infection CADE showed better response in developing multiple shoots on MS supplemented with 2.22 µM BAP, 2.32 µM Kn, 0.29 µM GA3 and 30.35 µM tyrosine. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin up to 200 mg/l. Transgenic lentil shoots were produced with an overall frequency of 1.009%. In vitro rooting appeared to have a limitation in obtaining complete plantlets in lentil, therefore in vitro flowering and seed formation were induced in transformed shoots of lentil with a view to recovering of the transgenic progenies. GUS positive shoots were found to produce in vitro flowers and pods on half-strength MS containing 98.4 µM IBA and 2.69 µM NAA. Expression of gene was detected in various tissues of the transformed shoots. Stable integration of GUS gene was also confirmed through PCR analysis. Plant Tissue Cult. &amp; Biotech. 22(1): 13-26, 2012 (June) DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11243