Publication Date

5-2015

Advisor(s) - Committee Chair

Shivendra V. Sahi (Director), Rob Wyatt, Chandrakanth Emani and Nilesh Sharma

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

AtPAP15 is one of the purple acid phosphatases expressed by Arabidopsis thaliana that has been extensively studied. Purified AtPAP15 has been shown to exhibit both phytase and phosphomonoesterase activities in acidic pH with maximal activity at pH 4.5. AtPAP15 is a phosphorus starvation inducible (PSI) gene that is expressed highly during phosphorus deficient conditions. In the current study, AtPAP15 was overexpressed in Nicotiana tabaccum under cauliflower mosaic virus (CaMV35S) constitutive promoter. After PCR confirmation of the gene, plants were transferred to the greenhouse and allowed to grow in pots. The pots contained Sta-Green potting mix (Lowe’s Inc., Mooresville, North Carolina,U.S.). Individual flowers were covered to ensure selfpollination. Seeds that were generated (T1 generation) were used for functional analyses. The T1 seeds were germinated in different phosphorus conditions- phosphorus deficient (0mM), optimum inorganic phosphorus (1.25mM), high inorganic phosphorus (20mM) and high organic phosphorus (20mM AMP and IHP)- and analyzed for total biomass, primary root length, soluble phosphorus (Pi) content and acid phosphomonoesterase (Apase) activity. Results demonstrated that under high organic phosphorus conditions, transgenic lines presented increased biomass, longer primary root length, high total soluble phosphorus and high Apase activity than the wild-type. These findings will have important implications for soils with high phosphorus content since most phosphorus in soil is available in organic form. As part of this thesis, Agrobacterium-mediated in planta transformation protocol for transformation of Medicago sativa (alfalfa) was standardized. The protocol would be used to develop transgenic alfalfa expressing AtPAP15. In this protocol, shoot apical position of 2d old seedling is excised followed by infection by Agrobacterium tumefaciens strain EHA 105 for TDNA transfer. The construct used was pCAMBIA1302 harboring mGFP reporter gene. The construct also contained kanamycin, rifampicin and chloramphenicol selection markers. Confirmation of mGFP expression in the transformed alfalfa seedling was performed using Zeiss microscope fitted with GFP filters. Results showed that this protocol is replicable and efficient. In planta transformation method, therefore, may be used for the development transgenic alfalfa.

Disciplines

Agronomy and Crop Sciences | Biology | Botany | Plant Biology

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