RESUMEN
Premature leaf fall of apple caused by Marssonina coronaria is economically very important apple disease and all the commercially available apple cultivars are susceptible to this disease. The non-availability of an efficient transformation system for this fungus hinders the functional genomics research. Herein, we report for the first time, the successful Agrobacterium-mediated transformation in apple leaf blotch fungus M. coronaria by transferring T-DNA harbouring the genes for hygromycin phosphotransferase (hpt), ß-glucuronidase (uidA) and green fluorescent protein (gfp) under the control of CaMV 35S promoter. The key factors that affect the transformation efficiency including type of recipient fungal material, acetosyringone concentration, the conditions for co-cultivation, Agrobacterium concentration, Agrobacterium strains and membrane types as support were investigated. The present results have recommended that 250 µM concentration of acetosyringone, 24 °C temperature and 48 h time, 0.5 OD600 of A. tumefaciens, EHA105 Agrobacterium strain and Whatman filter paper were the optimal co-cultivation conditions for the transformation of M. coronaria by using fragmented mycelia suspension and mycelial plugs. We observed that conidia were tedious to transform as compared to the fragmented mycelia and mycelial plugs of this slow growing fungus. These optimized parameters yielded 54 and 70 average transformants per 60 mycelial plugs and 104 fragmented mycelia, respectively. Fungal transformants were analysed for T-DNA integration, gus gene expression and gfp gene expression. Strong gus histochemical staining and green fluorescence expression indicated that the CaMV 35S promoter can drive gene expression in M. croronaria. Some mutants showed difference in the morphology of the colony as compared to the wild type control. This report will be very useful to inspect molecular basis of apple-M. coronaria interactions by deciphering the functional roles of various genes in this pathogenic fungus.
Asunto(s)
Agrobacterium tumefaciens , Ascomicetos , Marcadores Genéticos , Transformación Genética , Agrobacterium tumefaciens/genética , Ascomicetos/genética , Marcadores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Malus/microbiología , Regiones Promotoras Genéticas/genética , Esporas FúngicasRESUMEN
Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagentâ¯+â¯RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15â¯day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus.
Asunto(s)
Ascomicetos/genética , Calmodulina/genética , ADN Complementario , Malus/microbiología , Biología Molecular/métodos , ARN de Hongos/aislamiento & purificación , Ascomicetos/crecimiento & desarrollo , Ascomicetos/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Pruebas Diagnósticas de Rutina/métodos , Regulación Fúngica de la Expresión Génica , India , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 28S/genéticaRESUMEN
In the present study, an attempt was made to eliminate apple chlorotic leaf spot virus, apple mosaic virus, apple stem grooving virus and apple stem pitting virus from apple cultivar 'Oregon Spur-II'. Thermotherapy was carried out at 37-40 °C for 4 weeks followed by culturing of meristems of different sizes. During establishment of explants, highest survival percentage (62.35%) and proliferation (30.68%) was recorded during summer season. However, size of meristems and position of buds from where meristems were excised also influenced their survival. The meristems of size 0.6-0.7 mm were found to be the most appropriate for maximum establishment. Meristems excised from buds positioned on distil portions of actively growing shoots showed better results. MS medium supplemented with BA (1.0 mg/l), IBA (0.05 mg/l) and GA3 (0.1 mg/l) resulted in 56.62% establishment of explants, while maximum number of meristems proliferated with low BA (0.5 mg/l), IBA (0.08 mg/l) and same GA3 concentration. Two to fourfold multiplication was observed. Virus indexing of shoots raised from different sizes of meristems was carried out and found that 0.3-0.6 mm size was able to eliminate ACLSV, ApMV, ASGV and ASPV. However, some of 0.5-0.6 mm sized shoots were found infected with ACLSV. Larger meristems could not completely eliminate the viruses under study.