Exploiting induced genetic diversity through using mutagenesis is particularly important in giant miscanthus (Miscanthus x giganteus; Mxg) due to its restricted genetic variability. Experiments were conducted to develop an efficient in vitro propagation protocol for Mxg, induce mutations in Mxg using a chemical mutagen, and select Mxg in vitro for heat tolerance. To optimize in vitro propagation of Mxg, five explant types [i.e. immature inflorescences, shoot apex (in vitro), shoot apex (greenhouse), leaf explants (in vitro), and leaf explants (greenhouse)] were tested on five media. Shoot forming calli from immature inflorescences, an excellent source of explant in Mxg, grown in media with 13.6 microM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.44 microM 6-benzylaminopurine (BA) resulted in greatest shoot regeneration rate. Optimization of explant and callus type and media resulted in efficient in vitro proliferation of Mxg and the developed protocol was utilized in consecutive experiments of mutation induction and in vitro selection of Mxg for heat tolerance. Immature inflorescence explants (1-2 mm) were treated with 0.6%, 1.2%, and 1.8% of ethyl methanesulfonate (EMS) whereas the calli (1-2 mm3) were treated with 1.2%, 2.4%, and 3.6% of EMS for 90 min. Results of inter simple sequence repeat PCR analysis revealed polymorphisms indicating presence of genetic differences in Mxg putative mutants. In vitro callus cultures (mutagen treated and non-treated) of Mxg subjected to temperature treatments of 45±2°C for 12 hrs or 40±2°C for 7 days were selected for heat tolerance. Assessment results of electrolyte leakage and photosystem II (PS II) efficiency tests indicated a significant difference in percent membrane damage among Mxg clonal lines whereas PSII was weakly affected by the heat stress. The results suggest that in vitro derived Mxg clonal lines may be utilized for further studies of Mxg heat tolerance in developing potential Mxg ecotypes to adapt to different thermal environments. These studies provided the first investigation of in vitro induced mutagenesis in Mxg using a chemical mutagen. Genetic analysis results presented in this study indicates the potential use of developed Mxg putative mutants in future research programs, although significant morphological alterations were not observed during preliminary screening in the greenhouse.
| Identifer | oai:union.ndltd.org:MSSTATE/oai:scholarsjunction.msstate.edu:td-4993 |
| Date | 17 August 2013 |
| Creators | Perera, Dinum |
| Publisher | Scholars Junction |
| Source Sets | Mississippi State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
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