Regulation of Rice Germin-like Protein 2 Gene Promoter in Response to Some Signaling Molecules and Fungal Infection in Potato

Germins and germin-like proteins (GLPs) characterize a major plant proteins family that participate in several stress-linked and developmental activities. Molecular cloning and genetic evaluation of germins and GLPs have assisted well assessment of their regulation and elevated proficiency in the field of biotechnology. Moreover, the plant signaling molecules have fundamental role in the regulation of growth-linked activities and signaling systems in plants under adverse environmental conditions. The ~1100-bp Oryza sativa (rice) root germin-like protein 2 (OsRGLP2) gene promoter fused to a ?- glucuronidase (GUS) reporter gene was used to generate transgenic plants. Solanum tuberosum var. desiree was selected as an experimental plant for Agrobacterium tumefaciens mediated transformation. The present study was focused to analyse the regulation of OsRGLP2 promoter in response to some plant signaling molecules and fungal infection in transgenic potato. Further, an optimized nodal based regeneration protocol was established for potato. Potato micropropagation is valuable in resolving various difficulties associated with cultivation and productivity and it is a useful approach to generate multiple copies of the genetically modified plants. Callus culturing was optimized at different hormonal concentrations and combinations. It was observed that best (90 %) callus growth was found at 2.5 mg/L 2,4dichlorophenoxyacetic acid (2,4-D) while minimum growth (35 %) was observed at 1.5 mg/L zeatin and 1.0 mg/L 6-benzylaminopurine (BAP) combination. Moreover, Murashige and Skoog basal media supplemented with 1.0 mg/L BAP and 1.5 mg/L indole acetic acid (IAA) exhibited best regeneration potential from callus tissue used as explants. Microtuberization in potato plants is a valuable technique for obtaining germfree seed tubers, healthy plantlets, for germplasm exchange and understanding the physiological and morphological biology of tubers. Furthermore, research was conducted to analyse the effect of variable concentrations of carbon (C) source, nitrogen-based (N) supplementary nutrients, plant growth regulators (PGRs), hydrogen peroxide (H2O2) pre-treatment and different in vitro temperatures to reveal the best microtuberization conditions. Microtuberization productivity (growth percent (%) and fresh weight (mg) data records were statistically significant. After the initial optimization of tissue culture conditions, the Agrobacterium tumefaciens mediated transformation protocol was optimized for potato with the OsRGLP2 gene promoter construct. Transgenic potato plants produced were analysed in response to different plant defense signaling molecules, the relative quantification data records revealed that the OsRGLP2 promoter activity was enhanced by jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), hydrogen peroxide (H2O2) and under salt stress but up to a certain limit of salt concentration. It was observed that the OsRGLP2 promoter regulated differently towards various plant signaling molecules. Moreover, the tuberous samples from transgenic potato plants were analysed for the OsRGLP2 promoter functioning as a result of abiotic stress signals. The quantitative real time PCR (qRTPCR) analysis indicated that the GUS gene transcript accumulation detected in tubers in response to JA, SA, ABA, and H2O2 but the OsRGLP2 promoter activity in tubers was fairly low in comparison to non-tuberous samples. On the basis of quantitative results it was revealed that the OsRGLP2 promoter is linked with plant growth and developmental processes, cell signaling, seed germination, seedling development, regulation of plant water balance and osmotic stress as associated with different plant signaling molecules applied. In addition the OsRGLP2 promoter activity was also evaluated in response to potato specific fungal pathogens that were Fusarium solani (Mart.) Sacc. and Alternaria solani Sorauer. The qRT-PCR results revealed 4-5 fold increase in promoter activity after every 24 hrs following infection. There was 15 fold increase in OsRGLP2 promoter activity after 72 hrs of Fusarium solani (Mart.) Sacc. treatment and a 12 fold increase was observed with Alternaria solani Sorauer. The data confirmed that the OsRGLP2 promoter activity was enhanced under fungal stress. Moreover, there was a hyper accumulation of H2O2 in transgenic plants which is a clear signal for the presence of H2O2 responsive regulatory elements in OsRGLP2 promoter responsible for triggering particular defense related genes in the potato genome. The present analysis is valuable for the characterization of a monocot promoter activated under fungal stress in a dicot plant. The OsRGLP2 promoter evidently harbours certain biotic stress responsive cis-acting regulatory factors that function under fungal infection. On the basis of the present study, the data signifies the regulation of OsRGLP2 promoter in response to plant signaling molecules and biotic stress conditions. Therefore, it can be stated that the OsRGLP2 promoter is highly significant for future research studies in valuable crops by cloning several genes linked with abiotic and biotic stress resistance under its control