Highest Transient GUS Expression Research Articles

Aztreonam is a novel chemical inducer that promotes Agrobacteium transformation and lateral root development in soybean.

Agrobacterium-mediated soybean transformation is the simplest method of gene transfer. However, the low transformation due to the intractable nature of soybean genotypes hinders this process. The use of biochemicals (acetosyringone, cinnamic acid, flavonoids, etc.) plays an important role in increasing soybean transformation. These biochemicals induce chemotaxis and virulence gene activation during the infection process. Here we identified a biochemical, aztreonam (a monobactam), for high agrobacterium-mediated transformation in soybean. The soybean explants from three genotypes were inoculated with A. tumefaciens (GV3101) harboring the pMDC32 vector containing hpt or the GmUbi-35S-GUS vector containing the GUS gene during two separate events. High transient GUS expression was obtained during cotyledon explant culture on MS media supplemented with 2.5 mg/L aztreonam. The aztreonam-treated explants showed high efficiency in transient and stable transformation as compared to the untreated control. The transformation of aztreonam-treated explants during seed imbibition resulted in an average of 21.1% as compared to 13.2% in control by using the pMDC32 vector and 28.5 and 20.7% while using the GUS gene cassette, respectively. Based on these findings, the metabolic analysis of the explant after aztreonam treatment was assessed. The high accumulation of flavonoids was identified during an untargeted metabolic analysis. The quantification results showed a significantly high accumulation of the four compounds, i.e., genistein, apigenin, naringenin, and genistin, in cotyledon explants after 18 hours of aztreonam treatment. Alongside this, aztreonam also had some surprising effects on root elongation and lateral root formation when compared to indole-3-butyric acid (IBA). Our findings were limited to soybeans. However, the discovery of aztreonam and its effect on triggering flavonoids could lead to the potential role of aztreonam in the agrobacterium-mediated transformation of different crops.

Genotypic Variability in Soybean [Glycine max (L.) Merrill] through Agrobacterium-Mediated Transformation

Embryonic tip explants of 92 Indian soyabean and 7 advanced breeding lines derived from soaked mature seeds were inoculated and co-cultivated for 5-day with Agrobacterium strain EHA105 carrying the binary vector pCambia1305.1 containing a hygromycin and kanamycin resistance gene as plant and bacterial selectable markers, respectively. Transient expression of transgene was monitored by histochemical localization of β-glucouronidase (GUSPlus) reporter activity in transformed ET tissues. A high genetic variability for Agrobacterium-infection ranging from 3.8 to 100% was observed in the form of transient GUS expression. Five highly efficient genotypes, namely DS-228, JS 335, JS 72-44, KHSb2, and JS 72-280 with transient GUS expression of 100, 98.1, 96.5, 96 and 92%, respectively were identified. In addition, various infectivity patterns in these genotypes were observed. Genotypes with very high transient GUS expression identified in this study may improve success rate of development of transgenic soybean.
 Plant Tissue Cult. & Biotech. 30(2): 231-242, 2020 (December)

A high-efficiency Agrobacterium tumefaciens mediated transformation system using cotyledonary node as explants in soybean (Glycine max L.)

A high-efficiency Agrobacterium tumefaciens mediated transformation system was established through testing and optimising a range of variables using soybean cotyledonary as explants and glufosinate as selective agent. Seven cultivars (Heihe 19, Heihe 25, Heinong 37, Ha 03-3, YC-1, YC-2, and Zhonghuang 39) were used to screen the optimal cultivars. As a result, cultivars YC-1 and YC-2 were screened, which had the highest transient GUS expression percentage and shoot induction frequency. The cell density of Agrobacterium was found to be related with the transient GUS expression percentage; its optimal cell density was OD 0.8–1.0. It was beneficial to A. tumefaciens infection at 23 °C and dim light co-cultivation condition. Interestingly, the infection time showed no relation to the GUS transient percentage. Three types of antioxidant solutions were tested on their effects of alleviating cotyledonary explant browning and necrosis. The results showed that addition of 20 μM lipoic acid on solid CCM had the lowest cotyledonary explant necrosis and a high transient GUS expression rate. Following the optimized A. tumefaciens mediated transformation system, the bar gene was successfully transferred into soybean with the transformation efficiency reaching 14.71 %, more than 50 times increase from the un-optimized procedure. Southern and T 1 progeny analysis showed the exogenous gene had been integrated into the soybean genome and could be inherited next progeny.

Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg l−1 hygromycin. After two more selection steps, green shoots were rooted on MSB5 medium with 50 mg l−1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T1 generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.

Comparison of procedures for DNA coating of micro-carriers in the transient and stable biolistic transformation of sugarcane

Biolistic gene transfer is one of the preferred methods for both transient gene expression studies and stable genetic transformation for improvement of crops including sugarcane. In preparation for bombardment, DNA is precipitated onto micro-particles in the presence of a cationic polyamine and buffer. Three different DNA coating procedures were compared for biolistic transformation of sugarcane using linear expression cassettes: spermidine (free base), protamine sulfate, and the Seashell DNAdel™ Gold Carrier with proprietary precipitation buffer from Seashell Technology. All three DNA coating procedures produced transient and stable sugarcane transformation events in multiple experiments. Spermidine resulted in three-fold and 2.65-fold higher transient GUS expression than protamine and Seashell DNAdel™, respectively when averaged over 10 coating reactions per treatment. However, no significant difference in stable genetic transformation was found between the three DNA coating procedures. This study determined that spermidine-, protamine-, and Seashell DNAdel™ Gold Carrier-mediated DNA coating can be equally effective for biolistic transformation of sugarcane.

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 tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis

Procedure for the Agrobacterium tumefaciens mediated T-DNA delivery into the elite clone(s) of Eucalyptus tereticornis using leaf explants from microshoots has been developed. Amongst two strains of A. tumefaciens namely, EHA105 and LBA4404 (harbouring pBI121 plasmid), strain EHA105 was found to be more efficient. Pre-culturing of tissue (2 days) on medium supplemented with 100 μM acetosyringone, before bacterial infection significantly increased transient expression of reporter gene (GUS). Co-cultivation period of 2 days and a bacterial density of 0.8 OD600 resulted in higher transient GUS expression. Method of injury to tissue, presence of acetosyringone in co-cultivation medium and photoperiod during co-cultivation also influenced the expression of transient GUS activity. Amongst the three clones tested, maximum transient GUS activity was recorded in clone ‘CE2’ followed by clone ‘T1’. Regeneration of transformed shoots was achieved on modified Murashige and Skoog medium (potassium nitrate was replaced with 990 mg/l potassium sulphate and ammonium nitrate with 392 mg/l ammonium sulphate, and mesoinositol concentration was increased to 200 mg/l). Stable transformation was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to uidA and nptII genes. The absence of bacteria in the stable transformed tissues was confirmed by PCR amplification of fragment specific to 16S rRNA of bacteria.

Factors Affecting Transient Gene Expression in Mango Tissue Using Particle Bombardment

Particle bombardment seems to be a promising method for genetic transformation of mango and the information on various factors affecting transient gene expression is inconclusive. Pro-embryonic masses (PEMs) of mango (Mangifera indica L. cv. Kensington Pride) used for particle bombardment were established and maintained in vitro using various liquid and semisolid media containing 2,4-D. Various factors affecting the biolistic transformation efficiency in mango and subsequent transient gene expression were optimized using β-glucuronidase (GUS) as a reporter gene driven by CaMV 35S promoter. Our results show that bombardment pressure significantly affects transient gene expression with the best results obtained from PEMs bombarded at 1200 kPa of helium pressure under vacuum. The application of osmoticum pre and post-bombardment also significantly increased the transient gene expression in the PEMs as compared to the controls. Mannitol (0.2 m) proved to be the best osmoticum in improving transient GUS expression as compared to sorbitol. The duration of exposure of PEMs to mannitol (0.2 m) both pre and post-bombardment also played a significant role in improving transient gene expression. The transient GUS expression was significantly highest with a pre-treatment of 0.2 m mannitol for 4 hours as compared to 0, 8 and 12 hours. The post-bombardment treatment of 0.2 m mannitol for 16 hours resulted in significantly highest transient gene expression as compared to 0, 4, 8, 12, 20 and 24 hours. In conclusion, PEMs of `Kensington Pride' mango bombarded at 1200 kPa, which were exposed to mannitol (0.2 m) for 4 and 16 hours pre and post bombardment respectively, resulted in the highest transient GUS expression (25.1 GUS foci/mg PEMs).

Optimised DNA delivery into Coffea arabica suspension culture cells by particle bombardment

An optimised bombardment protocol to introduce DNA into Coffea arabica suspension culture cells was developed. Osmotic preconditioning of cells and physical bombardment parameters including Helium pressure, gap and target distances affecting DNA delivery were evaluated by monitoring transient expression of the uidA gene driven by the CaMV35S promoter. The highest transient GUS expression was obtained when cells were subjected to a 0.5 M mannitol–sorbitol pre-treatment 4 h prior to bombardment and a Helium pressure of 1550 psi, a 9-mm gap distance and 12 cm target distance as physical bombardment parameters. The optimised protocol was tested with two coffee promoters: α-tubulin and arabicin, which presented similar activity to the CaMV35S promoter in suspension culture cells by fluorometric GUS assays. GUS expression was reduced in bombarded tissue culture leaves, and only the CaMV35S and arabicin promoters showed histochemical activity in coffee endosperms. This is the first report of optimization of particle bombardment on coffee suspension culture cells, equivalent CaMV35S activity for a coffee promoter and transient β-glucoronidase expression in coffee endo-sperms.

High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus)

Five different varieties of Brassica napus (Cyclone, Dunkled, Oscar, Rainbow and KS75) were tested for their regeneration response. Cyclone showed a very high frequency of regeneration (92%). The use of silver nitrate was a pre-requisite for efficient shoot regeneration. Hypocotyls were selected as the starting material for transformation experiments on the basis of high transient GUS expression. Explants were co-cultivated with Agrobacterium strain EHA101 harboring a binary vector pIG121Hm containing neomycin phosphotransferase II (NPTII) gene, conferring resistance to kanamycin, hygromycin phosphotransferase (HPT) gene, conferring resistance to hygromycin as selectable markers and β-glucuronidase (GUS) gene as a reporter. Acetosyringone promoted the transformation but was not an absolute requirement. A pre-selection period of 7 days after co-cultivation was essential for successful transformation. Kanamycin was efficient selective agent for selection and maximum transformation efficiency was 24%. GUS activity was evident in leaf tissues. All the transgenic plants have an expected band of 0.43 kb fragment by PCR analysis confirming the presence of foreign DNA into plant genome.

Transformation of lentil (Lens culinaris M.) cotyledonary nodes by vacuum infiltration ofAgrobacterium tumefaciens

Lentil cotyledonary nodes are some of the most regenerative tissues in legumes. Attempts to transform them by vacuum filtration have been limitedly successful. This report describes a rapid and convenient transient expression protocol based on vacuum infiltration ofAgrobacterium cells into lentil cotyledonary nodes. Vacuum-infiltrated tissues had significantly (P<.05) higher transient GUS expression than did noninfiltrated tissues. Under optimal conditions (infiltration at 200 mmHg for 20 min), 95% of theAgrobacterium-infiltrated explants exhibited an average of 16 blue foci. We believe this to be the first report of this technique for transient gene expression in lentil cotyledonary nodes.

Transformation of Statice (Limonium sinuatum Mill.) by Agrobacterium tumefaciens-Mediated Gene Transfer.

A successful transformation procedure using Agrobacterium has been established for the most important ornamental statice, Limonium sinuatum Mill. Cell suspension cultures derived from friable calli, which were originated from leaf segments, were infected with Agrobacterium tumefaciens strain EHA105 (pIG121Hm) and co-cultivated for 2 days. The concentration of acetosyringone in co-cultivation medium and the growth stage of host plant cell used for infection were important factors for obtaining high transient GUS expression. After selection on medium with 75mgl-1 kanamycin, approximately 20 putative transgenic callus lines with kanamycin-resistance and GUS expression were obtained from 1g F. W. of cell cultures infected. Adventitious shoots were induced from 5 different putative transgenic callus lines on the regeneration medium containing 75mgl-1 kanamycin, and finally 16 transgenic plants which showed GUS expression in leaves were obtained. Presence of NPT II and GUS genes was confirmed by PCR analysis in the genomic DNAs of transgenic callus. Integration of GUS gene into the genome of transgenic plantlets derived from different transgenic callus lines was detected by Southern blot analysis.

Biological parameters affecting transient GUS gene expression in oil palm ( Elaeis guineensis Jacq.) embryogenic calli via microprojectile bombardment

Biological parameters affecting microprojectile bombardment delivery of DNA into oil palm embryogenic calli were optimized by monitoring transient GUS gene expression. The parameters optimized were the following: explant type using gold and tungsten microcarrier, bombardment preculture, time between bombardment and GUS staining, genotype, immature embryo preculture, DNA concentration, osmoticum type and concentration, and osmoticum treatment duration before and after bombardment. An independent experiment was carried out to study the effect of each parameter and its variables on transient GUS expression. ANOVA was carried out for each experiment using completely randomized design and the treatment means were compared using Duncan's Multiple Range Test. The highest transient GUS expression was observed 2 days post-bombardment using embryogenic calli derived from immature embryos. Bombardment was carried out using 300 μg of gold microcarriers coated with 1.5 μg of DNA 24 h after transfer to fresh medium. GUS expression could be further enhanced when calli were transferred to medium containing osmoticum (0.4 M mannitol) 2 h prior to bombardment. Highly significant differences between the variables were observed for all the parameters studied except genotype.