Doctoral Thesis
Application of biotechnology to improve frost tolerance of wheat
Doctor of Philosophy (PhD), Murdoch University
2023
Abstract
Cold or frost stress is a significant issue for wheat (Triticum aestivum) grown in Mediterranean environments. Antifreeze Proteins (AFPs) present in some frost-tolerant organisms can inhibit the growth of ice crystals, reducing frost damage. This project investigated whether two AFPs could protect tobacco and wheat plants from cold damage.
The two AFP genes were from wheat (5D-ScIRIP) and winter flounder (WfAFP) and were characterised in silico using bioinformatic tools. The physicochemical properties and protein structures indicated both AFPs possess properties to inhibit ice crystal growth.
In proof-of-concept experiments, the 5D-ScIRIP and WfAFP were each expressed in tobacco, and cold tolerance of transgenic plants was studied over two generations. Cold treatment (0℃, -1℃ or -2℃) of T2 plants enabled 47% and 28.6% of transgenic events carrying the 5D-ScIRIP and WfAFP, respectively, to survive and grow to maturity. Cold treatment of the T3 generation of cold-surviving plants recovered from treatments within one week and flowered earlier than wild-type plants. Much lower electrolyte leakage was detected in two of the best cold-tolerant tobacco events carrying the 5D-ScIRIP than the wild-type plants (<10% in 60–70% of the events studied), events 4-4T15 and 4-10T17, suggesting there was less cell damage after cold treatment. Gene expression analysis of the AFP transcripts correlated with reduced damage after cold treatment.
Mature and immature wheat embryos and embryo-derived calli were transformed with vectors containing the 5D-ScIRIP. Mature embryo treatment of wheat cultivar Scepter using particle bombardment plus Agrobacterium tumefaciens-mediated transformation gave 10.7% and 8.5% herbicide-tolerant T2 transgenic plants, respectively. After Agrobacterium treatment, 10.3% and 5.3% of herbicide-tolerant plants were obtained from immature embryos of cultivar Fielder and mature embryo-derived calli of Scepter. The transgenic plants had higher 5D-ScIRIP expression than the wild-type plants after cold treatment, and three transgenic lines, 3B-1, 25G-1 and 29A-1, had significantly low electrolyte leakage after cold treatment (-2℃).
This research indicates that increased expression of WfAFP and 5D-ScIRIP can improve cold tolerance in tobacco and wheat.
Details
- Title
- Application of biotechnology to improve frost tolerance of wheat
- Authors/Creators
- Keshala P Kuruwita Bandaralage
- Contributors
- Michael G. K. Jones (Supervisor) - Murdoch University, Centre for Crop and Food InnovationJohn Fosu-Nyarko (Supervisor) - Murdoch University, Centre for Crop and Food Innovation
- Awarding Institution
- Murdoch University; Doctor of Philosophy (PhD)
- Identifiers
- 991005626669507891
- Murdoch Affiliation
- Centre for Crop and Food Innovation; State Agricultural Biotechnology Centre
- Resource Type
- Doctoral Thesis
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