Biolistic transformation of Scoparia dulcis L.

Kota, Srinivas; Narra, Muralikrishna; Kumar, Kalva Bharath; Ellendula, Raghu; Mahender, Anumalla; Kasula, Kiranmayee; Yashodahara, Velivela; Abbagani, Sadanandam

doi:10.1007/s12298-016-0338-2

Cited by 13 publications

(7 citation statements)

References 28 publications

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“…Centrifugation was repeated three times, followed by washing in ethanol, and finally the pellet was suspended in 75 ll of absolute ethanol and kept on ice until bombardment. Following vortexing, 10 ll of the mixture was applied to macrocarriers for each bombardment event (Srinivas et al 2016).…”

Section: Preparation Of Microcarriersmentioning

confidence: 99%

Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment

et al. 2017

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Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6 µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene () and neomycin phosphotransferase II gene ( II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2 ± 1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of L. were determined as follows: 650 psi helium pressure and 6 cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50 mg l kanamycin) round. Finally, the transformants produced were subjected to histochemical assay, and integration of the transgenes ( and II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals.

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Section: Preparation Of Microcarriersmentioning

confidence: 99%

Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment

et al. 2017

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“…Leaf explants of one month old culture were excised and placed at the middle of Petri plate containing regeneration medium. Leaf tissue was bombarded at standardized biolistic parameters i.e 6 cm flight distance and 650 psi pressure were employed for the transformation (Srinivas et al 2016). Each plate containing 5 explants, totally twenty-five explants were bombarded and cultured for two days on the same medium at 25 ± 2°C, photoperiod is 16/8 hrs.…”

Section: Biolistic Transformation Of Chloroplast Vectormentioning

confidence: 99%

“…In our lab, a reliable regeneration, Agrobacterium and biolistic mediated genetic transformation using uidA, hpt and gus and nptII genes respectively in S. dulcis (Aileni et al 2011a;Srinivas et al 2016). In Scoparia dulcis, efficient plant regeneration system comparable to model plant system like tobacco using leaf explant made more convenient to achieve reliable chloroplast transformation by targeting rpl32/trnL and trnR/trnN insertional sites of SSC and IR regions of plastid genome using pFaadAII and KNTc heterologous vectors respectively (Muralikrishna et al 2016;Narra et al 2018a).…”

Section: Introductionmentioning

confidence: 99%

Improved plastid transformation efficiency inScoparia dulcisL.

et al. 2019

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The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicumspecific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.

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“…Plasmid coating on to gold microcarriers was carried out according to prescribed protocol and 7 ll of this suspension was used for bombardment (Dheeraj et al 2008). The optimized bombardment parameters for stable nuclear transformation with 650 psi pressure and 6 cm of distance from stopping plate to target tissue with fixed vacuum pressure of 25 mm of Hg (Srinivas et al 2016), was employed for plastid transformation.…”

Section: Plastid Transformation Proceduresmentioning

confidence: 99%

“…Stable genetic transformation in S. dulcis was achevied through Agrobacterium using uidA and hpt genes (Aileni et al 2011) and efficient biolistic transformation using uidA and npt II genes (Srinivas et al 2016). Development of chloroplast transformation in this plant system will provide new opportunities towards plastid based metabolic engineering of certain valuable natural product entities and to delve this technology towards enhanced metabolite production and extension in engineering of biosynthetic pathways by plastid transformation.…”

Section: Introductionmentioning

confidence: 99%

Stable plastid transformation in Scoparia dulcis L.

Narra

Kota

Kumar

et al. 2016

Physiol Mol Biol Plants

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In the present investigation we report stable plastid transformation in L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring as a selectable marker and as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats,/ of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

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Biolistic transformation of Scoparia dulcis L.

Cited by 13 publications

References 28 publications

Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment

Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment

Improved plastid transformation efficiency inScoparia dulcisL.

Stable plastid transformation in Scoparia dulcis L.

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