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Chikmagalur District, India

Divakara S.T.,University of Mysore | Santosh P.,Central Coffee Research Institute | Aiyaz M.,University of Mysore | Venkata Ramana M.,Defence Food Research Laboratory | And 3 more authors.
Journal of the Science of Food and Agriculture | Year: 2014

BACKGROUND: Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp. RESULTS: All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters. CONCLUSION: The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities. © 2013 Society of Chemical Industry.

Mishra M.K.,De Montfort University | Mishra M.K.,Central Coffee Research Institute | Devi S.,De Montfort University | McCormac A.,De Montfort University | And 4 more authors.
Biologia | Year: 2010

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants. © 2010 Versita Warsaw and Springer-Verlag Wien.

Devi S.,De Montfort University | Mishra M.K.,Central Coffee Research Institute | Elliott M.,De Montfort University
Tropical Life Sciences Research | Year: 2012

Transgenic rice plants were generated using particle bombardment to introduce the Agrobacterium cytokinin biosynthesis gene driven by Arabidopsis (Arabidopsis thaliana) senescence specific promoter (SAG12) for delaying leaf senescence. Using two plasmids we co-transformed one week old embryogenic calli derived from mature Japonica rice (Oryza sativa) variety Chin Guang. The selectable marker hygromycin phosphotransferase (hph) gene and the reporter gene B-ß-glucuronidase (uidA), both under the control of cauliflower mosaic virus (CaMV) 35S promoter were placed on the same co-integrate vector whereas the cytokinin biosynthesis gene, isopentenyl transferase (ipt) driven by the SAG12 promoter is supplied in another plasmid. A total of 32 transgenic rice plants were regenerated of which 27 plants were randomly selected for histochemical ß-glucuronidase (GUS) assay, PCR and Southern blot analysis. Co-integration frequencies of 88% and 69% were obtained for two linked genes (uidA and hph) and two unlinked genes (hph and ipt gene) respectively in R0 plants. Southern blot analysis confirmed the results of histochemical GUS assay and PCR amplifications. A complex integration pattern for all the transgenes including the multiple copies integration was predominantly observed. © Penerbit Universiti Sains Malaysia, 2012.

Bharatha Nandhini R.M.,Madurai Kamaraj University | Rahul R.N.,Andhra University | Thilaga S.,Manonmaniam Sundaranar University | Rao N.S.P.,Central Coffee Research Institute | Ganesh D.,Madurai Kamaraj University
South African Journal of Botany | Year: 2013

Interspecific C. ×. R hybrid (Coffea congensis×. Coffea canephora) in India is cultivated as mixed population with male parent C. canephora as this species is an efficient pollen donor for enhanced yield. But distinction of C. ×. R hybrid from C. canephora in old plantation is difficult due to varying plant sizes of C. ×. R hybrid and often resembles with C. canephora. C. ×. R hybrid cultivated under different agroclimatic conditions show distinct vegetative growth pattern with varying yields. Thus development of DNA marker for identification of C. ×. R hybrid is important for clonal propagation and seed preparation from selective individuals. In this study, two DNA bar coding loci of chloroplast genome (rbcL and matK) of parents, F1 hybrid and its back cross progeny were partially sequenced to identify SNPs as DNA marker for distinction of C. ×. R hybrid from C. canephora. Seven SNPs in the matK gene sequence and three nucleotides in the rbcL gene sequence were identified as DNA markers for the genetic identity of C. congensis. These SNPs were found in F1 and advanced progenies of C. ×. R hybrid due to maternal inheritance. Large number of samples of C. ×. R hybrids with varying morphological features revealed no polymorphism among C. ×. R hybrid and C. congensis. Thus, the SNPs in C. congensis can be used as DNA markers for precise identification of C. ×. R hybrid for production of clones besides tagging the chloroplast inheritance in advanced progenies. © 2013 South African Association of Botanists.

Garcia C.A.,CIRAD - Agricultural Research for Development | Garcia C.A.,French Institute of Pondicherry | Bhagwat S.A.,University of Oxford | Ghazoul J.,ETH Zurich | And 6 more authors.
Conservation Biology | Year: 2010

The new approaches advocated by the conservation community to integrate conservation and livelihood development now explicitly address landscape mosaics composed of agricultural and forested land rather than only protected areas and largely intact forests. We refer specifically to a call by Harvey et al. (2008) to develop a new approach based on six strategies to integrate biodiversity conservation with sustainable livelihoods in Mesoamerican landscape mosaics. We examined the applicability of this proposal to the coffee agroforests of the Western Ghats, India. Of the six strategies, only one directly addresses livelihood conditions. Their approach has a clear emphasis on conservation and, as currently formulated risks repeating the failures of past integrated conservation and development projects. It fails to place the aspirations of farmers at the core of the agenda. Thus, although we acknowledge and share the broad vision and many of the ideas proposed by this approach, we urge more balanced priority setting by emphasizing people as much as biodiversity through a careful consideration of local livelihood needs and aspirations. ©2009 Society for Conservation Biology.

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