Showing papers by "Andy Ganapathi published in 2016"

Involvement of exogenous polyamines enhances regeneration and Agrobacterium-mediated genetic transformation in half-seeds of soybean.

Abstract: The present work demonstrates the participation of polyamines (PAs) to improve direct regeneration and Agrobacterium-mediated transformation in soybean half-seeds. The inclusion of PAs to culture medium along with optimal plant growth regulators (PGRs) enhanced shoot induction [98.3 %; 4.44 µM N6-benzyladenine (BA) and 103.27 µM spermidine] and elongation [90.0 %; 1.45 µM gibberellic acid (GA3) and 49.42 µM spermine]. The polyamine putrescine (62.08 µM) alone greatly enriched root induction (96.3 %). The influence of PAs on transformed plant production was assessed by comparing optimized protocol (comprising PAs and PGRs) with a regeneration system involving only PGRs. Plant transformation was performed in half-seeds of cultivar DS 97–12 using strain EHA105 harboring pCAMBIA1301. Transgene expression and integration was confirmed by GUS staining, PCR, and Southern hybridization. The transformed explants/materials successively cultured on co-cultivation (BA and spermidine), shoot induction (BA and spermidine), shoot elongation (GA3 and spermine), and rooting medium (putrescine) showed enhanced transformation efficiency (29.3 %) compared with its counterparts (14.6 %) with respective PGR alone [BA, GA3, or indole-3-butyric acid (IBA)]. Overall findings of the study suggest that involvement of PAs improved T-DNA transfer during co-cultivation, and delivered most suitable condition for efficient regeneration/survival, which led to enhanced transformation efficiency in soybean.

High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper)

Abstract: In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA® (4 mg l−1) containing MS medium followed by BASTA® foliar spray on 15-day-old black gram plants (35 mg l−1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.

Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell.

Abstract: Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD600, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.

Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335.

Abstract: Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0-6 mg l-1) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l-1 ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T1 plants. Southern analysis of T1 plants confirmed the integration of bar gene into the genomic DNA and the bar positive T1 plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot node-glufosinate system in an Indian soybean genotype.

Elicitation Approaches for Withanolide Production in Hairy Root Culture of Withania somnifera (L.) Dunal.

Abstract: Withania somnifera (L.) Dunal is a versatile medicinal plant extensively utilized for production of phytochemical drug preparations. The roots and whole plants are traditionally used in Ayurveda, Unani, and Siddha medicines, as well as in homeopathy. Several studies provide evidence for an array of pharmaceutical properties due to the presence of steroidal lactones named "withanolides." A number of research groups have focused their attention on the effects of biotic and abiotic elicitors on withanolide production using cultures of adventitious roots, cell suspensions, shoot suspensions, and hairy roots in large-scale bioreactor for producing withanolides. This chapter explains the detailed procedures for induction and establishment of hairy roots from leaf explants of W. somnifera, proliferation and multiplication of hairy root cultures, estimation of withanolide productivity upon elicitation with salicylic acid and methyl jasmonate, and quantification of major withanolides by HPLC. The protocol herein described could be implemented for large-scale cultivation of hairy root biomass to improve withanolide production.

RETRACTED ARTICLE: Overexpression of thaumatin gene confers enhanced resistance to Alternaria brassicae and tolerance to salinity and drought in transgenic Brassica juncea (L.) Czern

Abstract: RETRACTED ARTICLE: Overexpression of thaumatin gene confers enhanced resistance to Alternaria brassicae and tolerance to salinity and drought in transgenic Brassica juncea (L.) Czern