Stable Expression Of GUS Gene Research Articles

Optimization of Agrobacterium Mediated Genetic Transformation in Paspalum scrobiculatum L. (Kodo Millet)

An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM-EHA 105, 250 µM-LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.

Optimization of Factors Influencing Agrobacterium mediated Genetic Transformation in Eclipta alba (L.) Hassk.

An efficient and reproducible protocol for in vitro plant regeneration and Agrobacterium-mediated genetic transformation was developed for Eclipta alba (L.) Hassk. using nodal explants. Several parameters affecting Agrobacteriummediated gene delivery were investigated and optimized. These include antibiotic concentration, preculture period, infection time, use of acetosyringone, co-cultivation period and temperature and increased level of copper in induction, co-cultivation and regeneration medium. Incorporation of elevated level of CuSO4 led to significant improvements in plant regeneration and recovery of transformed plants. The highest frequency of transformation was observed when explants were infected and co-cultivated at 23oC. Higher transformation frequency was attained by augmenting the medium with 1.0 ?M CuSO4 which was ten times the normal MS level. Addition of acetosyringone in the infection and co-cultivation media was very beneficial and resulted in greater transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected explants and leaves of regenerated transformants, respectively. Molecular analysis of transformed plants with nptII and gus A specific primers revealed the amplification of nptII and gus gene thereby demonstrated the efficacy of optimized protocol for A. tumefaciens-mediated genetic transformation in Eclipta alba for the first time.Plant Tissue Cult. & Biotech. 25(2): 155-164, 2015 (December)

Optimization of Key Bombardment Parameters in Biolistic Mediated Transformation in Wheat

Biolistic mediated method is an important approach in wheat genetic transformation. The objective of this study was to optimize the bombardment parameters and establish an efficient and stable biolistic transformation system using common wheat (Triticum aestivum L.) variety Kenong 199 with 12 treatments of parameter combinations composed of gold particle size, target distance, and acceleration pressure. The results indicated that different combinations of parameters had different effects on the transient expression of GUS gene encoding β-glucuronidase, regeneration of calli, and final transformation efficiency. The parameter combination of 0.6 μm gold, 5.5 cm target distance, and 650 psi acceleration pressure exhibited good regeneration and the highest level GUS transient expression. Furthermore, stable expression of GUS gene in transgenic lines was confirmed by histochemical staining assay. Of the 28 transgenic T0 plants obtained, 12 were from the treatment with the above optimized parameter combination, with an average transformation efficiency of 2.45%.

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 spinach plants produced by Agrobacterium-mediated method based on the low temperature-dependent high plant regeneration ability of leaf explants

Agrobacterium strain EHA101 harboring plasmid pIG121-Hm containing the genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII) was co-cultivated with leaf explants from in vitro grown spinach plants. Hygromycin-resistant calli were obtained 1 month after selection of transformed cells on 2.5 g l−1 gellan gum-solidified MS medium containing 30 g l−1 sucrose, 0.5 mg l−1 2,4-D, 2 mg l−1 kinetin, 20 mg l−1 hygromycin and 20 mg l−1 meropenem trihydrate. Regeneration of adventitious shoots was affected by culture temperature, and the highest frequency of shoot formation was 40% when the calli were cultured at 14°C during regeneration process on 2.5 g l−1 gellan gum-solidified MS medium containing 30 g l−1 sucrose, 0.01 mg l−1 2,4-D, 1 mg l−1 kinetin, 1 mg l−1 gibberellic acid (GA3), 20 mg l−1 hygromycin and 10 mg l−1 meropenem trihydrate. Stable expression of gus gene was indicated by histochemical GUS assay in the leaves and roots of putative transgenic plants. Southern blot analysis of genomic DNA isolated from T0 plants confirmed the successful integration of T-DNA into the plant genome. Segregation of the gus gene in transgenic T1 progeny was confirmed by PCR analysis. These results show that Agrobacterium-mediated transformation combined with plant regeneration at a low temperature, 14°C, can be efficiently used for producing transgenic spinach plants with useful genes.

In vitro Regeneration and Agrobacterium-mediated Genetic Transformation of Tomato (Lycopersicon esculentum Mill.)

Agrobacterium-mediated genetic transformation system has been developed for two tomato (Lycopersicon esculentum Mill.) varieties, namely Pusa Ruby (PR) and BARI Tomato-3 (BT-3). Prior to the establishment of transformation protocol cotyledonary leaf explants from the two varieties were cultured to obtain genotype independent in vitro regeneration. Healthy multiple shoot regeneration was obtained from the cut ends of cotyledonary leaf segments for both the varieties on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. The maximum root induction from the regenerated shoots was achieved on half the strength of MS medium supplemented with 0.2 mg/l IAA. The in vitro grown plantlets were successfully transplanted into soil where they flowered and produced fruits identical to those developed by control plants. Transformation ability of cotyledonary leaf explants was tested with Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121, containing GUS and npt II genes. Transformed cotyledonary leaf explants were found to produce multiple shoots on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 200 mg/l since kanamycin resistant gene was used for transformation experiments. Shoots that survived under selection pressure were subjected to rooting. Transformed rooted plantlets were transferred to soil. Stable expression of GUS gene was detected in the various tissues from putatively transformed plantlets using GUS histochemical assay. Key words: In vitro regeneration, transformation, tomato D.O.I. 10.3329/ptcb.v19i1.5004 Plant Tissue Cult. & Biotech. 19(1): 101-111, 2009 (June)

Agrobacterium-mediated Genetic Transformation for Local Cultivars of Potato (Solanum tuberosum L.) Using Marker Genes

Genetic transformation using nodal and internodal segments from three economically important potato (Solanum tuberosum L.) varieties namely, Diamant, Cardinal and Granola was conducted using an Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI12 containing the GUS and nptII genes. Node and internodal segments were used for direct regeneration as well as regeneration with the intervention of callus. best responses were obtained for direct regeneration of shoots when the explants were cultured on MS supplemented with 4.0 mg/l BAP +1.0 mg/l IAA, 1.5 mg/l BAP + 0.5 mg/l IAA and 5.0 mg/l BAP +1.0 mg/l IAA in Diamant, Cardinal and Granola, respectively. In Diamant spontaneous in vitro microtuberization was obtained from these proliferated shoots. Further culturing of these in vitro grown green microtubers regenerated a large number of shoots on MS containing 4.0 mg/l BAP +1.0 mg/l IAA. By combining the best treatments, this protocol yielded an average transformation rate of 87% of treared explants. Stable expression of GUS gene was visualized in the various parts of transformed shoots through histochemical assay. Genomic DNA was isolated from transformed shoots and stable integration of the GUS and nptII genes was confirmed by PCR analysis. Key words: Potato, in vitro regeneration, transformation D.O.I. 10.3329/ptcb.v20i2.6894 Plant Tissue Cult. & Biotech. 20(2): 145-155, 2010 (December)