Molecular and physiological responses of recalcitrant Indica rice to pluronic F-68 during callus regeneration
Pluronic F-68 (PF-68) is a non-ionic surfactant commonly used as a growth additive in plant tissue culture. However, there are limited studies on the effects of PF-68 in rice. Therefore, this study was undertaken to evaluate the growth promoting effects of PF-68 on callus proliferation, shoot gro...
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| Format: | Thesis |
| Language: | English |
| Published: |
2021
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/104594/1/FBSB%202021%2034%20IR.pdf |
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| Summary: | Pluronic F-68 (PF-68) is a non-ionic surfactant commonly used as a growth additive in
plant tissue culture. However, there are limited studies on the effects of PF-68 in rice.
Therefore, this study was undertaken to evaluate the growth promoting effects of PF-68
on callus proliferation, shoot growth and root growth of recalcitrant MR219 rice. MR219
calli and shoot apices were cultured on Murashige and Skoog (MS) medium
supplemented with different concentrations [0 %, 0.02 %, 0.04 %, 0.06 %, 0.08 %, 0.10 %
(v/v)] of PF-68. Calli grown on MS medium supplemented with 0.04 % PF-68 improves
callus proliferation rate by 1.59-fold (fresh weight), 1.24-fold (dry weight) and enhanced
root induction from the calli by 1.29-fold. However, increasing frequency of brown and
black calli was observed when 0.10 % PF-68 was used. In shoot growth study, PF-68 did
not exhibit any growth promoting effects on MR219. On the other hand, optimum root
growth was observed in shoot apices treated with 0.04 % PF-68. Growth of the roots was
increased significantly by 1.43-fold and root length by 1.19-fold compared to the control.
In order to evaluate the underlying mechanism of growth promoting effects of PF-68,
callus was used as a study model and three different concentrations were selected for
further analysis; namely control, optimum (0.04 % PF-68) and high concentration (0.10 %
PF-68). Biochemical analyses revealed high accumulation of sugar (1.77 mg/mL) and
protein (0.17 mg/mL) contents in 0.04 % PF-68-treated calli. Similarly, quantitative realtime
reverse transcription polymerase chain reaction (RT-qPCR) also revealed that high
expressions of sucrose synthase (2.65-fold) and NADH-dependent glutamate synthase
(1.86-fold) transcripts, which correlated with the high sugar and protein contents
detected in 0.04 % PF-68-treated calli. Besides, calli treated with high concentration of
PF-68 (0.10 %) recorded increased accumulation of phenolic (0.74 mg/mL), flavonoid
(0.08 mg/mL), and phenylalanine ammonia lyase (PAL) activity (0.28 U/μg protein),
which implied enhanced secondary metabolites biosynthesis in 0.10 % PF-68-treated
calli. Further gene expression quantification also recorded an increased in 4-
coumarate:CoA ligase 3 (1.28-fold) and chalcone-flavonone isomerase (1.65-fold)
transcripts in 0.10 % PF-68-treated calli. Subsequent biochemical analyses revealed high
H2O2 activity (0.10 mg/mL), malondialdehyde content (0.024 U/μg protein) and
peroxidase activity (0.15 U/μg protein) in 0.10 % PF-68-treated calli. Consistently, high
expression level of ascorbate peroxidase (1.61-fold) was observed in 0.10 % PF-68-treated calli, suggesting activation of the plant defense mechanism against increasing
stress induced from high concentration of PF-68. However, a decrease in esterase activity
(34,204.50 nmol/ng protein) was recorded at 0.10 % PF-68, which implied increasing
stress induced by PF-68 to trigger programmed cell death. Further comparative
proteomic analysis revealed an upregulation of alpha-amylase and NADH-dependent
glutamate synthase proteins detected in 0.04 % PF-68-treated calli. This indicates PF-68
enhances callus proliferation via enhanced carbon and nitrogen metabolism in 0.04 %
PF-68-treated calli. In contrast, upregulation of PAL protein was detected in 0.10 % PF-
68-treated calli. These results suggest that secondary metabolite biosynthesis was
enhanced in 0.10 % PF-68-treated calli. In addition, nutrient ion analysis revealed an
increased uptake of K, Mg, Ca, Fe, Zn, Cu and Mn ions were also observed in 0.04 %
PF-68-treated calli. Among these nutrient ions, K had the highest increment of nutrient
content detected in 0.04 % PF-68-treated calli. The increased K uptake plays an
important role in plant growth and development such as protein synthesis and
carbohydrate metabolism. Overall, the results from this study showed that the growth
promoting effects of PF-68 on in vitro MR219 rice cultures were concentration
dependent. Taken together, at optimum concentration, PF-68 improves recalcitrant rice
callus proliferation via enhanced sugar metabolism, amino acid biosynthesis and nutrient
uptake which are crucial towards plant growth and development. However, at high
concentration, PF-68 induces stress response in plant as evidenced by the increased
secondary metabolites content, H2O2 activity, malondialdehyde content and peroxidase
activity. Hence, optimum concentration of PF-68 has potential to be utilized as an
additive for plant growth and development in tissue culture of rice cultivars. |
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