Agrobacterium tumefaciens Mediated Transformation of Symbiodinium spp.

Symbiodinium spp conducts symbiosis mutualism within a wide phyletic range of marine invertebrate hosts including corals and anemones. The present study investigates the transformation of foreign genes into the free living cultured Symbiodinium spp by co-cultured Symbiodinium cells with A. tumefaciens. Ti-plasmidbased binary vector harboring the GUS and GFP genes were transformed into Symbiodinium cells by co-cultivation. GUS histochemical assay was monitored in Symbiodinium cells under light microscopy. Putative GFP in transformed Symbiodinium cells was detected by immunoassaying with antibodies against GFP protein. These results suggest that A. tumefaciens could provide efficient tools for gene transformation of Symbiodinium cells.

Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars

The developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Désirée and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with β-glucuronidase gusA gene under the control of 35S CaMV promoter. Kanamycin was used as plant selectable marker for screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10 % using the internodal explant, and 15, 12, 17, 8 and 6 % using leaf discs as explant in Lady Olympia, Granola, Agria, Désirée and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptII genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility make this protocol suitable for wider application of transgenic potato plants.

Tinkering Cis Motifs Jigsaw Puzzle Led to Root-Specific Drought-Inducible Novel Synthetic Promoters

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

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)

Transient Transformation of Artemisinic Aldehyde ∆ 11 (13) Double Bond Reductase (dbr2) Gene into Artemisia annua L.

Global demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering-based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens-mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303-dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S-408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co-cultivated on MS co-cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA-dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA-dbr2 construct and transformation into A. annua have been successfully performed.

Agrobacterium?mediated Genetic Transformation of Two Varieties of Brassica juncea (L.) Using Marker Genes

Protocol for Agrobacterium?mediated genetic transformation using hypocotyl and cotyledonary leaf with petiole from two local varieties of Brassica juncea was established by optimizing various factors influencing transformation. GUS histochemical assay revealed that the cotyledonary leaf with petiole and hypocotyl explants had positive interaction with the Agrobacterium strain LBA4404 containing the binary plasmid pBI121 which has marker genes like, GUS and nptII. Maximum transformation was obtained with bacterial suspension having an optical density of 0.8 at 600 nm, 30 min of incubation and 72 hours of co?cultivation. The transient and stable integration of the marker genes were confirmed through histochemical GUS assay, as well as PCR analysis.Plant Tissue Cult. & Biotech. 26(1): 55-65, 2016 (June)

Agrobacterium-Mediated Transformation of Three Freshwater Microalgal Strains

Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp.

In vitro Regeneration and Agrobacterium-mediated Transformation of Potato (Solanum tuberosum L.) Cultivars Grown in Mexico

Prior to Agrobacterium-mediated genetic transformation in vitro regeneration protocol was established for three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) grown in Mexico using leaf, node and internodal explants. Regeneration protocol was developed with or without the intervention of callus. Two potato cultivars, namely, Cambray Rosa Morelos and Alpha were transformed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing the GUS and nptII genes. GUS histochemical assay and PCR analysis were conducted on rooted shoots grown in media without hormones but supplemented with antibiotics. Transformed shoots tested positive through GUS histochemical assay and integration of nptII gene was confirmed by PCR analysis DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14193 Plant Tissue Cult. & Biotech. 22(2): 93-105, 2012 (December)

Agrobacterium-mediated Genetic Transformation of Local Cultivars of Chickpea (Cicer arietinum L.)

Agrobacterium-mediated genetic transformation protocol was established for two chickpea (Cicer arietinum L.) varieties, namely Barichhola-4 (Bch-4) and Barichhola-5 (Bch-5). Transformation ability of different explants such as decapitated embryo with single cotyledon disc (DEC), decapitated embryo (DE) and slice embryo decapitated at shoot end with single cotyledon disc (SEC) were tested using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing the GUS and nptII genes. Maximum transformation ability was exhibited by explants of decapitated embryo (DE) from Barichhola-5 (Bch-5). The optimum regeneration from the transformed tissue was achieved on MS supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn and 0.2 mg/l NAA along with double the amount of CaCl2 and KNO3. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 150 mg/l. Stable expression of the GUS gene was detected in various parts of the transformed shoots through GUS histochemical assay. Stable integration of nptII gene within the genomic DNA from these transformed shoots was confirmed through PCR analysis.DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11258 Plant Tissue Cult. & Biotech. 22(1): 41-50, 2012 (June)