Maximum Transformation Efficiency Research Articles

High-efficiency transformation of Yarrowia lipolytica using electroporation.

Yarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6×104 transformants per μg DNA and 2.8×104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.

An efficient mesophyll protoplast isolation, purification and PEG-mediated transient gene expression for subcellular localization in Chinese kale

Abstract Transient gene expression serves as a valuable tool for gene functional study in plants. Here we reported protoplast isolation and purification from the plantlet young leaves of Chinese kale (Brassica oleracae var. alboglabra Bailey), and their transient gene expression with polyethylene glycol (PEG)-mediated transformation and subcellular localization of phytoene desaturase (BaPDS1). The procedures of isolation andtransformation ofmesophyll protoplasts derived from Chinese kale were optimized, and the influencing factors were analyzed. The results showed that the optimal protocol of protoplast isolation and purification was initialized by digestion in enzyme solution (2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol) for 9 h. After filtered through 400 mesh and centrifuged at 179 ×g for purification, the total yield of protoplast reached as high as 6.04 × 107 protoplasts g−1 fresh weight (FW) and the viability of the protoplasts was up to 95%. A maximum transformation efficiency of approximately 30% measured by using green fluorescent protein (GFP) as a detecting gene was obtained when PEG4000 was at a final concentration of 40% and transformation time was set to 15 min. In addition, the subcellular localization of BaPDS1 in Chinese kale was targeted to the chloroplast, confirming the efficiency and reliability of this transient transformation system. Taken together, an efficient protoplast isolation, purification and transformation system in Chinese kale was established in this study, laying a foundation for future research in molecular biology and gene function in Chinese kale and other Brassica vegetables.

HTS YBCO Resonator Configuration With a Coplanar Optimized Flux Concentrator Strongly Coupled to rf SQUID

We developed a novel magnetic coupling module formed of a monolayer superconducting flux concentrator, which is integrated with a coplanar resonator strongly coupled to HTS rf-SQUID. Three types of resonators, including a long stripline resonator between input loop and pick-up loop of the flux concentrator, a complementary split ring resonator (CSRR), and also a spiral shape inside the input loop are explored. The resonance quality factors as well as the coupling to the SQUID of different patterns of these three types of the resonators is evaluated using Finite Element Method (FEM) simulations. Several readout methods to couple the electronic system to the resonators are tested, including inductive (coil) and capacitive (transmission line) couplings, and the optimum readout is reported for each of the resonators. Among the evaluated resonator types, a spiral shape resonator with optimal design showing the highest quality factor (5900) together with the strongest coupling to the SQUID (-0.5 dB) at resonance frequency of 836 MHz, is fabricated using 200 nm thick superconducting YBCO on a 1 mm thick crystalline LaAlO3 substrate. The flux concentrator of the module is optimized by the variation of its linewidths and also its input loop radius to obtain maximum flux transformation efficiency.

The competence system of Streptococcus anginosus and its use for genetic engineering.

Streptococcus anginosus is considered a human commensal but improvements in species identification in recent years have highlighted its role as an emerging pathogen. However, our knowledge about the pathogenicity mechanisms in this species is scarce. One reason for this is the lack of published genetic manipulation techniques in the S.anginosus group. To establish a novel mutation technique we investigated the competence system of S.anginosus and created a Cre-recombinase-based mutation method that allows the generation of markerless gene deletions in S.anginosus. Insilico analysis of the competence system demonstrated that S.anginosus encodes homologues for the vast majority of genes that are known to be essential for the transformation of S.pneumoniae. Analysis of transformation kinetics confirmed that S.anginosus SK52 possesses an S.pneumoniae-like competence development with a rapid increase of competence after treatment with Competence Stimulating Peptide (CSP), reaching a maximum transformation efficiency of 0.24%±0.08%. The combination of CSP-induced transformation and the Cre-lox system allows the efficient and fast creation of markerless gene deletions and will facilitate the investigation of the pathogenicity of S.anginosus on a genetic level.

Strong Polarization Transformation of Bloch Surface Waves.

Polarization is an intrinsic attribute of optical waves, so manipulating the polarization state of optical surface waves can be of a fundamental importance for the next-generation information and bio-photonics technology. Here, we show theoretically that the polarization of the Bloch surface wave (BSW) on a dielectric multilayer can be transformed between a transverse-electric (TE) state and a transverse-magnetic (TM) state by using the laterally continuous grooves inscribed on this multilayer. This polarization transformation can be enhanced or inhibited by the interference between the reflected BSW beams, which can be tuned by the periodicity and depth of the grooves. The maximum polarization transformation efficiency can be achieved as high as 43% when the number of grooves is increased to 10. A generalized Fresnel formula is proposed to describe the polarization transformation of the BSW beams. Due to this polarization transformation, an anomalous reflection of BSW beams can be realized, which is the inequality between the incident angle and the reflection angle.

Remediation of lead-contaminated sediment by biochar-supported nano-chlorapatite: Accompanied with the change of available phosphorus and organic matters

Some rivers in China have been seriously contaminated due to the discharge of lead (Pb) smelting wastewater. In this study, biochar-supported nano-chlorapatite (BC-nClAP) was synthesized to immobilize Pb in contaminated sediment. The remediation effect of BC-nClAP on Pb-contaminated sediment was evaluated through batch experiments and the materials were characterized by x-ray diffraction, scanning electron microscope, Brunner-Emmet-Teller and electronic differential system. It was found that BC-nClAP can transform Pb effectively from labile fraction into stable fraction with a maximum transformation efficiency increasing to 94.1% after 30 days of treatment, and the stabilization efficiency of toxicity characteristic leaching procedure reached 100% only after 16 days of treatment. The content of available phosphorus (AP) in the sediments treated by BC-nClAP was much less than that treated by nClAP, which indicated a lower risk of eutrophication and suggested the dissolution-precipitation mechanism involved in Pb immobilization. BC-nClAP presented the best immobilization efficiency of Pb and the content of organic matters in BC-nClAP treated samples increased the most, thus the OM might play an important role during the Pb immobilization.

Withania somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application.

Withania somnifera, commonly known as Ashwagandha an important medicinal plant largely used in Ayurvedic and indigenous medicine for over 3,000 years. Being a medicinal plant, dried powder, crude extract as well as purified metabolies of the plant has shown promising therapeutic properties. Withanolides are the principal metabolites, responsible for the medicinal properties of the plant. Availability and amount of particular withanolides differ with tissue type and chemotype and its importance leads to identification characterization of several genes/ enzymes related to withanolide biosynthetic pathway. The modulation in withanolides can be achieved by controlling the environmental conditions like, different tissue culture techniques, altered media compositions, use of elicitors, etc. Among all the in vitro techniques, hairy root culture proved its importance at industrial scale, which also gets benefits due to more accumulation (amount and number) of withanolides in roots tissues of W. somnifera. Use of media compostion and elicitors further enhances the amount of withanolides in hairy roots. Another important modern day technique used for accumulation of desired secondary metabolites is modulating the gene expression by altering environmental conditions (use of different media composition, elicitors, etc.) or through genetic enginnering. Knowing the significance of the gene and the key enzymatic step of the pathway, modulation in withanolide contents can be achieved upto required amount in therapeutic industry. To accomplish maximum productivity through genetic enginnering different means of Withania transformation methods have been developed to obtain maximum transformation efficiency. These standardized transformation procedues have been used to overexpress/silence desired gene in W. somnifera to understand the outcome and succeed with enhanced metabolic production for the ultimate benefit of human race.

Establishment of genetic transformation system for Sophra alopecuroides and deletion analysis of SaLDC promoter

Establishing the genetic transformation system of medicinal plant is important to study their functional genes. Based on the established regeneration system of Sophra alopecuroides, 6 factors of genetic transformation were optimized, that was the concentration of Agrobacterium tumefaciens, the infection time, the co-cultivation time of agrobacterium tumefaciensand S.alopecuroides callus, the preculture time of S.alopecuroides callus, the adding method ofacetosyringone (AS) and the concentration of AS, respectively. The results showed that a maximum genetic transformation efficiency of 83.33% was achieved with 15d-precultured of S.alopecuroides callus, which was infected by A600=0.9 A. tumefaciens for 15 minutes and then co-cultivated for 48 hours with 200 μmol•L-1AS. The promoter sequence (1 260 bp) of upstream SaLDC was cloned from S.alopecuroides genomic DNA (gene bank accession number: KY038928). The deletion fragment of SaLDC promoter with different length (310,594,765,924,1 260 bp) were ligated with the GUS reporter gene to form five plant expression vectors named P310,P594,P765,P924,P1260, which were then transferred into S.alopecuroides callus. The GUS transient expression showed that all 5 different deletion fragment of SaLDC promoter can drive the GUS gene expression in S. alopecuroides callus. The SaLDC promoter we cloned has high promoter activity, and they may facilitate its function analysis in the future.

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

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

Enhanced production of an anti-malarial compound artesunate by hairy root cultures and phytochemical analysis of Artemisia pallens Wall.

Artemisinin and its derivatives are still one of the most effective drugs for the treatment of malaria. Artemisia pallens commonly known as Dhavanam, is an aromatic herb belonging to the family Asteraceae. Increasing the artemisinin content of A. pallens by genetic engineering would improve the availability of this much needed drug. In the present study, Agrobacterium rhizogenes (strain NCIM 5140) mediated genetic transformation of Artemisia pallens were carried out for hairy root induction. The effect of different media (Half MS, MS, MS along with BAP 0.5 mg/l and MS along with Kinetin 0.5 mg/l) and type of explants (leaf and stem) on hairy root induction and culture were also studied. Maximum transformation efficiency (70.0 %) was observed in case of stem explants when it was co-cultivated with Agrobacterium rhizogenes and kept on half strength MS media. Artesunate is a derivative of artemisinin, was quantified using HPLC from dried aerial extract and hairy roots. The content of artesunate in hairy roots was increased up to twofold as compared to aerial part of Artemisia pallens. The maximum amount of artesunate found in hairy roots was 5.62 ± 0.16 μg/g of dry weight. Apart from artesunate the other phytochemicals like alkaloids, polyphenols, and flavonoids are important because they impart the medicinal properties in this plant. Therefore, we have also quantified total alkaloids, flavonoids and polyphenolic content in the aerial part of the plants. The total alkaloids and flavonoids content were found 1.72 ± 0.00 mg/g dry weight in aqueous extract and 3.8 ± 0.00 mg/g in methanolic extract in terms of colchicine and rutin equivalents, respectively. Similarly, total phenolic content is 3.70 ± 0.01 mg/g in ethanolic extract in terms of tannic acid equivalent.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0496-5) contains supplementary material, which is available to authorized users.

High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA® (4 mg l−1) containing MS medium followed by BASTA® foliar spray on 15-day-old black gram plants (35 mg l−1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.

INTER-COUNTRY EFFICIENCY EVALUATION IN INNOVATION ACTIVITY ON THE BASIS OF METHOD FOR DATA ENVELOPMENT ANALYSIS AMONG COUNTRIES WITH DEVELOPED AND DEVELOPING ECONOMY, INCLUDING THE REPUBLIC OF BELARUS

The paper considers a problem on efficiency evaluation of innovation activity in 63 countries with developed and developing economies while using a method for data envelopment analysis. The following results of innovation activity have been used for calculation of an efficiency factor: export of high-technology products as percentage of industrial product export, export of ICT services as percentage of services export and payments obtained due to realization of intellectual property rights (in US dollars). A model of the data envelopment analysis with a changeable scale-dependent effect and which is directed on maximization of the obtained results (output-oriented VRS model) has been used for the analysis. The evaluation has shown that such countries as the USA, Israel, Sweden and some others have maximum efficiency of resource transformation into innovative activity output. The executed analysis has revealed that the Republic of Belarus has a potential for improvement of indices on innovation results.

Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD600, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.

Inducing osmotic stress leads to better genetic transformation efficiency in cotton (Gossypium hirsutum L.)

The present study investigated the effect of different salts on cotton shoot regeneration and transformation efficiency. Two-day-old germinating embryos of a local cotton cultivar (SG-125) were pretreated with 50 mM each of NaCl, CaCl2, and KCl for 60 min. The embryo explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid pTF101.1 that carried the insecticidal gene (cry1Ac) under control of wound-inducible promoter (AoPR1) and bilanafos acetyl reductase (bar) gene for plant selection. The salt-pretreated embryos showed maximum response on regeneration MS medium containing 0.50 mg/L 6-benzylaminopurine (BAP) and 0.10 mg/L indole-3-butyric acid (IBA), also supplemented with 5 mg/L bialaphos for in vitro screening of the transformed plantlets. The primary transformants were further screened by molecular techniques for integration and expression of the introduced gene. Maximum transformation efficiency (1.10%) was noted on KCl-treated explants compared to nontreated (control) explants. In conclusion, pretreatment of explants with 50 mM KCl for 60 min induced positive effects and triggered shoot regeneration in primary cotton transformants.

Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301–bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 µM acetosyringone and co-cultivated for 3 days on a medium containing 100 µM acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days.

English

Agrobacterium-mediated genetic transformation of rice (Oryza sativa L.) cultivar BRRI dhan56 was carried out in this study. Agrobacterium tumefaciens strain LBA 4404, which harbors the plasmid pIG121 that carries the genes for ß-glucuronidase gene, served as a reporter gene in the histochemical assay and the neomycin phosphotransferase ΙΙ (NPT ΙΙ) gene for the identification of resistance to kanamycin was used for genetic transformation. Twenty days old embryogenic calli from mature embryos of highly regenerating rice cultivar BRRI dhan56 were used to co-cultivate with 0.8 to 0.9 OD600 Agrobacterium for 25 min and the cultured was continued on agar medium for this study. The transformed colonies were selected by using 50 mg/L kanamycin and 50 mg/L rifampicin and confirmed by colony PCR. The PCR positive colonies were isolated to transform by using calli of indica rice cultivar BRRI dhan56. Putative leaf and root segments from plantlets obtained from transformation experiment with the plasmid pIG121 were GUS positive. Integration of the introduced gene into the genome was demonstrated by PCR. The maximum transformation efficiency of 32% was obtained by using 500 mg/L cefotaxime as a bacteriostatic agent to inhibit growth of Agrobacterium. In this study, 100 µM acetosyringone in co-cultivation medium and co-cultivation for 3 days were the optimum conditions for maximum transformation. The expression of GUS gene revealed that the calli were successfully transformed. The results of this study would be an effective tool for crop improvement and gene-function studies on the model monocot plant rice.   Key words: Agrobacterium, Oryza sativa L., acetosyringone, β-glucuronidase, cefotaxime, plasmid, phosphotransferase, rice, transformation.

Highly efficient Agrobacterium-mediated in planta genetic transformation of snake gourd (Tricosanthes cucumerina L.)

Regeneration of plants from Agrobacterium tumefaciens infected cell or tissue is a time-consuming process and somaclonal variations are the major bottleneck. Hence, in the present study, a tissue culture-independent genetic transformation system for snake gourd was developed using seed as an explant. Snake gourd seeds were infected with three A. tumefaciens strains harbouring pGA 492 plasmid, and the transformants were selected against BASTA®. Several factors influencing the in planta genetic transformation such as Agrobacterium strains, acetosyringone, sonication, and vacuum infiltration, have been evaluated. The maximum transformation efficiency (19.6 %) was recorded when the pre-cultured snake gourd seeds were sonicated for 30-min and vacuum infiltrated for 3-min at 750 mm of Hg in A. tumefaciens EHA 105 suspension containing 100 µM of acetosyringone. The bar gene integration into the snake gourd genome was confirmed by polymerase chain reaction and Southern blot hybridization. The transgene was successfully inherited into the T1 progeny plants. This transformation method can produce transgenic snake gourd plants in a relatively short time (35-days).

Agrobacterium-mediated in planta genetic transformation of sugarcane setts.

An efficient, reproducible, and genotype-independent in planta transformation has been developed for sugarcane using setts as explant. Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA(®). Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure, have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6min and vacuum infiltered for 2min at 500mmHg in A. tumefaciens C58C1 suspension containing 100µM acetosyringone, 0.1% Silwett L-77 showed the highest transformation efficiency of 29.6% (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6%). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time.

Effect of Various Factors on the Efficiency of Agrobacterium-Mediated Transformation of Grape (Vitis viniferaL.)

The present investigation was conducted to study the effect of inoculation time, cocultivation period and various concentrations of hygromycin and kanamycin on genetic transformation of grape (Vitis vinifera L.) cv. King's Ruby through Agrobacterium tumefaciens. The Chitinase gene (for fungal resistance) and GUS gene (for phenotypic expression of transgenic) were introduced in the embryogenic calli developed from leaf discs, through Agrobacterium strain LBA4404 harboring plasmid pBI121 nptII as selectable marker for GUS gene and hptII for Chitinase gene. Regarding transformation efficiency rate, 10 minutes inoculation period with bacterial suspension showed the highest transformation efficiency rate i.e. 2.83% with Chitinase gene and 2.5% with GUS gene. Infected calli with Chitinase gene and GUS gene co-cultivated for 2 days showed the maximum transformation efficiency of 2.75% and 3.25%, respectively. For elimination of excess bacteria, cefotaxime treatment (300 mg L−l) showed the highest survival rate of 3.16% for Chitinase gene and 2.5% for GUS gene. The maximum i.e.2.83% transformation efficiency rate was achieved at 10 mg L−l of hygromycin for selection of transformed calli. The highest 2.25% transformation efficiency was yielded when kanamycin was used at the rate of 100 mg L−l. Five out of 8 calli showed positive expression with 62.5% transformation efficiency through histochemical GUS assay. Present results may be helpful in improving genetic transformation efficiency of grape through Agrobacterium tumefaciens against fungal diseases and reduce the use of fungicides.

Application of sonication in combination with vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars.

Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.