Publication Date

7-1-2003

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

This study describes rapid propagation of Sesbania drummondii using nodal explants isolated from seedlings and young plants. The nodal segments proliferated into multiple shoots on Murashige and Skoog (MS) medium supplemented with 22.2 /M benzyladenine. Murashige and Skoog medium containing 2.2 and 4.5 //M thidiazuron induced 5 - 6 healthier shoots per axillary node from 3-month-old plants. Nodal segments cultured on MS medium containing combinations of benzyladenine (8.8 and 11.1 /JM) and either indole-3-butyric acid (0.24 - 2.46 //M) or indole-3-acetic acid (0.28 - 2.85 //M) produced fewer shoots. Callus when subcultured on 2.2 /LihA thidiazuron containing medium resulted in its mass proliferation of calli having numerous embryoid-like structures. Indole-3-butyric acid (0.24 - 2.46 |aM) was found suitable for root induction. In vitro regenerated plants were acclimatized in greenhouse conditions. This report is a first report on studies of in vitro regeneration of S. drummondii. grobacterium tumefaciens, bearing the plasmid pCAMBIA 1305.1, was used to develop a genetic transformation system for S. drummondii. This plasmid contains a GUS (P-glucuronidase) reporter gene and a gene conferring resistance to the antibiotic hygromycin. The genes are placed under the control of the 35S CaMV (cauliflower mosaic virus) promoter and a NOS terminator. A plant catalase intron is inserted into the GUS reporter gene. The presence of GUS enzymatic activity, as a result of Agrobacterium-mediated transformation, was indicated by a GUS histochemical assay: the appearance of blue color in transformed tissue in the presence of substrate X-Gluc (5-bromo-4-chloro-3-indoly glucuronide). Polymerase Chain Reaction (PCR) was used to demonstrate the presence of the GUS reporter gene in the plant genome. Genomic DNA extracted from the transformed tissue was used to amplify the GUS gene by gene- specific primers. The PCR products were visualized by agarose gel-electrophoresis.

Disciplines

Medical Sciences | Plant Sciences