Optimization of oil palm protoplast isolation and transformation method for transient expression assays using DsRED reporter gene
Genetic engineering is highly regarded as a forefront technology in agriculture. Various crops have been improved via this technology to increase the overall yield or specific product of targeted crops. Genetic engineering has also been carried out to address multiple issues that cause production...
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| Format: | Thesis |
| Language: | English |
| Published: |
2021
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/98055/1/FBSB%202021%2024%20UPMIR.pdf |
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| Summary: | Genetic engineering is highly regarded as a forefront technology in agriculture.
Various crops have been improved via this technology to increase the overall
yield or specific product of targeted crops. Genetic engineering has also been
carried out to address multiple issues that cause production losses, including
drought conditions or susceptibility to pest and pathogen attacks. The steady
progress and maturation of genome editing technology have allowed scientists
to do this more precisely and efficiently. Nevertheless, multiple tools for the
target crop, such as an established transformation method for optimal DNA
delivery, a reliable transient expression system for preliminary evaluation of
the targeting efficiency, and a proven tissue culture routine for regenerating
genome-edited plantlets, are required. This current research was carried out
to develop a high-throughput transient expression system for oil palm by
utilizing protoplasts isolated from oil palm in vitro-derived leaves. First, seven
transformation vectors that carry DsRED protein-encoding genes, each
controlled by a different promoter, were constructed. Next, the isolation of
mesophyll protoplasts was optimized by identifying the best parameters
affecting protoplast yield and viability, such as enzyme combinations and
procedures to obtain clean and viable protoplasts. By doing this, an efficient
protocol for isolation of oil palm mesophyll protoplast that can produce up to
2.5 x 106 protoplasts/g FW with up to 94.78% viability was developed. Then,
optimization for isolation of protoplasts from the mesocarp of the age around
12 weeks after anthesis (WAA) was carried out with previously optimized
enzyme mixtures. After two hours of incubation time, 3.98 × 106 protoplasts/g
FW with 85% viability were recovered. Five parameters affecting the
polyethylene glycol (PEG)-mediated transformation efficiency were optimized,
including DNA and polyethylene glycol (PEG) incubation time, concentrations of DNA and PEG, and duration of heat-shock applied. This study has shown
an increment in transformation efficiency of almost 56%. The developed
transient expression system was tested with eight DNA constructs with DsRED
as a visual reporter gene. This experiment indicated that the CaMV35S
promoter drove significantly higher expression of DsRED in oil palm
protoplasts than other plants constitutive, oil palm constitutive and tissuespecific
promoter tested in this study. This advanced method will serve as a
high-throughput transient expression platform in the current pipeline for oil
palm genome editing. |
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