Infection of Early and Young Callus Tissues of Indica Rice BPT 5204 Enhances Regeneration and Transformation Efficiency

Manimaran, P.; Kumar, Gaurav; Reddy, M. Raghurami; Jain, Sudha; Rao, Talluri Bhaskar; Mangrauthia, Satendra K.; Sundaram, R. M.; Ravichandran, S.; Balachandran, S. M.

doi:10.1016/s1672-6308(13)60153-5

Cited by 24 publications

(20 citation statements)

References 55 publications

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“…Based on high competency to regenerate via somatic embryogenesis only 6 weeks old calli were only used for further transformation studies. In contrast, 61.3 % regeneration was reported by 6 days old callus with indica rice cultivar BPT5204 (Manimaran et al 2013). The slight difference observed in callus regeneration frequency with respect to both cultivars might be due to the genotypic variation or culture media and/or growth conditions.…”

Section: Callus Age On Regeneration Efficiencymentioning

confidence: 86%

Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker

Chakraborty

Reddy

Narasu

et al. 2016

Physiol Mol Biol Plants

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Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l −1 ) and Paramomycin (70 mg l −1 ) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe.

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Section: Callus Age On Regeneration Efficiencymentioning

confidence: 86%

Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker

Chakraborty

Reddy

Narasu

et al. 2016

Physiol Mol Biol Plants

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“…Subsequently, the binary plasmids in the backbone of pCAMBIA3300 carrying different promoter deletion fragments were mobilized into Agrobacterium strain EHA105 by tri-parental method (Lichtenstein and Draper 1985). Embryogenic calli (21 days) of indica rice cultivar BPT 5204 were transformed using Agrobacterium tumefaciens followed by cocultivation and washing of transformed calli as described by Manimaran et al (2013). The washed calli were maintained in selection MS basal salts (Murashige and Skoog 1962) with 2 mg/L 2,4-D, 0.5 mg/L kinetin, 500 mg/L L-proline, 500 mg/L casein hydrolysate, 30 g/L maltose, solidified with 0.3 % phytagel and supplemented with 8 mg/L phosphinothricin (Duchefa, the Netherlands) for 15 days in dark.…”

Section: Gene Constructs and Rice Transformationmentioning

confidence: 99%

“…Total genomic DNA extraction, PCRs and DNA blot analyses of transgenic plants and non-transformed control plants were followed as described previously (Manimaran et al 2013). For confirmation of transgenic plants by PCR, primer pairs were designed from promoter and GUS region; OSIPP3 forward: 5 0 -AAAAGGCAACACCAAGTTTAGC C-3 0 and GUS reverse: 5 0 -ATCCACGCCGTATTCGG-3 0 .…”

Section: Molecular Confirmation and Inheritance Of Transgenementioning

confidence: 99%

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica

et al. 2015

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Pollen-specific expression. Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5' deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5' promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504 bp), OSIPP3-∆2 (968 bp), OSIPP3-∆3 (388 bp) and OSIPP3-∆4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated that they were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

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“…Different types of explants are used for transformation, such as mature seed derived calli, immature embryo-derived calli, leaf base derived calli and shoot apex. Among these explants, calli derived from mature seeds showed high transformation efficiency (Hiei and Komari 2008;Rance et al, 1994;Manimaran et al, 2013). In rice, many factors are influencing the efficiency of T-DNA delivery to plant cell such as type of explants, optical density of Agrobacterium, infection time, co-cultivation medium with acetosyringone for vir gene induction.…”

Section: Introductionmentioning

confidence: 99%

Standardization of Agrobacterium mediated genetic transformation in Indica rice cv BPT-5204

et al. 2018

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The BPT-5204 genotype from Indica rice cultivar (cv) is recalcitrant and showed low transformation frequency compare to japonica rice cv. Here we have optimized the efficient transformation protocol to minimize the time scale and enhance the transformation frequency by altering the key parameters like, acetosyringone (AS) concentration, optical density of bacterial culture and co-cultivation time. Highly proliferated 21 days old Scutellum derived embryogenic calli were infected with Agrobacterium tumefaciens harbouring pCAMBIA2300-Ds-En-Bar binary vector. T-DNA contains tetrameric CaMV35S enhancer elements along with bar gene which embedded between the transposable Ds element. At 0.5 OD600nm of Agrobacterium culture and co-cultivation for 2 days on MS co-cultivation medium containing 100 μM acetosyringone proved to be optimal and attained 6.2 % of transformation efficiency. The transformed calli and regenerated plantlets were proliferated on Murashige and Skoog (MS) medium containing phosphinothricin (PPT). Polymerase chain reaction (PCR) confirmed that intact T-DNA was successfully integrated in the rice genome. This protocol can be employed to develop transgenic rice plants with gain of functional mutagenesis.

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Infection of Early and Young Callus Tissues of Indica Rice BPT 5204 Enhances Regeneration and Transformation Efficiency

Cited by 24 publications

References 55 publications

Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker

Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica

Standardization of Agrobacterium mediated genetic transformation in Indica rice cv BPT-5204

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