NIEjr01: Who is faster, 2n or 4n in Growth and Cell Fate Reprogramming? NTU x MGS Loh Kay-En Elizabeth, Faith Sze Xin Rei, Associate Professor Chen Zhong, Dr Yan An, Mr Lim Lee, Mrs May Liow
2 Background Information
Background Background Information Information General Methodology Diploid 2n Tetraploid 4n Wound Induced Callus Formation 4 pairs of 2 pairs of ● ● homologous Hormone homologous induced shoot chromosomes chromosomes regeneration 92 chromosomes 48 chromosomes ● ● Lateral root formation CIM induced leaf and root regeneration Discussion Same genotypes may exhibit different phenotypes and 3 Conclusion
Background Information Key terms to be defined CIM Shetty K., Asano Y., ● Callus inducing medium Oosawa K. (1992) SIM Schaller G.E., Bishopp ● Shoot inducing medium A. et al. (2015) Callus Morel G., Wetmore ● Formation of unorganized R.H. (1951) cell mass 4
Background Background Information Information General Methodology What is Wound Induced Callus Formation Cell Fate Reprogramming? Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and 5 Conclusion
Background Background information Information General Cell fate: The ultimate differentiated state to which a cell has Methodology become committed. undifferentiated Wound pluripotent stem cell Induced Cell fate reprogramming Callus Formation Hormone regulates induced shoot regeneration Epigenetic mechanisms regulates Gene eg. DNA methylation, transcription Lateral root histone modification formation Target genes CIM induced leaf and root regeneration Discussion and 6 Conclusion
Background Background information Information General Methodology What does it mean to reprogram cell fate ? Wound Induced Callus When cell fate is reprogrammed, the developmental Formation processes that define cell fate are reverted. Hormone induced shoot Cells dedifferentiate from totipotency into pluripotency. regeneration Lateral root formation How? → Epigenetic memory is erased. CIM induced leaf and root The epigenetic marks on each allele from both regeneration chromosomes are reset. Discussion and 7 Conclusion
Background Background information Information General Methodology Cell fate can be Wound Induced reprogrammed by Callus Formation Hormone induced shoot (1) Artificially regeneration Induced Lateral root pluripotent formation stem cells (IPS) CIM induced leaf and root regeneration Discussion and 8 Conclusion
Background Background information Information General Methodology Cell fate can be Wound (2) Induced reprogrammed by Callus Formation Hormone induced shoot (2) Production of regeneration germ cells (eg. Lateral root egg/sperm cell) formation CIM induced leaf and root (3) Regeneration regeneration Discussion and 9 Conclusion
Background Background Information Information General Methodology The ability to reprogram cell fate is especially Wound Induced Callus important for wound induced regeneration in Formation plants, or De novo organogenesis Hormone induced shoot regeneration De novo organogenesis: The ability of Lateral root formation plants to regenerate lost body parts when CIM induced wounded. leaf and root regeneration Discussion and 10 Conclusion
Mechanism of De novo organogenesis A Wound response B Cellular dedifferentiation C Callus formation WIND1 WIND1 WIND1 11
Background Information Background information: Previous research General Methodology What has been researched on Wound Induced Callus Developmental and experimental factors that ▫ Formation impact plant regeneration Hormone induced shoot regeneration Less is known about genetic factors such as variation in ploidy Lateral root number within the same species can cause significant formation differences in the regenerative response. CIM induced leaf and root regeneration Discussion and 12 Conclusion
Background Aim Information General Methodology Find the ploidy that regenerates the fastest Wound Induced and is most suitable and receptive to cell fate Callus Formation reprogramming Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and 13 Conclusion
14 General Methodology
Background Information Model plant: Arabidopsis Thaliana General Methodology Universally Wound recognised model Induced Callus plant in experiments Formation Hormone induced shoot Response and regeneration development is Lateral root similar to most crops formation CIM induced Smaller genome leaf and root regeneration Discussion and 15 Conclusion
Varying ratios of auxin and cytokinin in medium to trigger In vitro De novo organogenesis Auxin Cytokinin Cytokinin Auxin Auxin Cytokinin Balanced ratio of Rich in Cytokinin Rich in Auxin C and A Root Callus Shoot regeneration formation regeneration at the tip of Leaf the shoot regeneration 16
Types of experiments carried out 1. CIM-induced callus 1. Wound 1. Hormone 1. Lateral root formation for leaf explant induced callus induced shoot formation experiment formation regeneration experiment 2. CIM-induced callus experiment protocol formation of root explants experiment 17
Statistical tools used for analysis One way analysis of Real time polymerase Mean and Standard variance hypothesis chain reaction (qpcr). Deviation testing tool (ANOVA) 18
19 Individual experiments and Results
Methodology - Background Information Wound induced callus formation General Methodology Wound We excised the tip of a hypocotyl. Observed the Induced Callus Formation callus formation of the wounded explant 2 days, 3 days and 4 days after wounding by using a Hormone induced shoot regeneration microscope Lateral root formation CIM induced leaf and root regeneration Discussion and 20 Conclusion
Results - Background Information Wound induced callus formation General Methodology It was observed that the callus formed on 4n plants are larger than the Wound callus formed on the 2n plants Induced Callus Formation Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and 21 2n callus observed after 4 days 4n callus observed after 4 days Conclusion
Results - Background Information Wound induced callus formation General Methodology Wound Induced Callus Formation Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and 22 Conclusion
Results - Background Information Wound induced callus formation General Methodology Wound Induced Callus Statistical test: Formation One-way Analysis of Variance (ANOVA) test Hormone induced shoot regeneration → Test whether there are statistically significant Lateral root formation differences between 2n and 4n results CIM induced leaf and root regeneration Discussion and 23 Conclusion
Results - Background Information Wound induced callus formation General Methodology Null Hypothesis Alternative Hypothesis Wound Induced Callus Formation ● ● No statistically There is a significant statistically Hormone induced shoot difference significant regeneration ● Any observed difference Lateral root ● differences may The observed formation be due to chance differences are not and sampling due to chance or CIM induced leaf and root error. sampling error. regeneration Discussion If P-value < α = 0.05, reject null hypothesis and 24 Conclusion
Results - Background Information Wound induced callus formation General Methodology ANOVA Wound Induced Callus Source of Formation Variation SS df MS F P-value F crit Hormone Between induced shoot regeneration Groups 1893.779 1 1893.78 139.17 0.000000000184 4.35124 Lateral root Within formation Groups 272.159 20 13.608 CIM induced Total 2165.937 21 leaf and root regeneration Since our P-value < α = 0.05, we reject the null hypothesis. Discussion and 25 Thus, we confirm that our results are statistically significant. Conclusion
Results - Background Information Wound induced callus formation General Methodology CONCLUSION Wound Induced Callus Formation Hormone induced shoot regeneration 4n has higher regenerative capacity in Lateral root callus formation than 2n formation CIM induced leaf and root regeneration Discussion and 26 Conclusion
Methodology - Hormone induced Background Information shoot regeneration General Methodology Wound induced callus formation Hormone Induced Shoot Regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and 27 Conclusion
Results - Hormone induced shoot Background Information regeneration General Methodology Rate of regenerated shoot Shoots per regenerated Wound explant for 2n and 4n induced explants for 2n and 4n callus explants explants formation 60 1.8 Rate of regenerated shoot explants Number of regenerated shoots per explant Hormone 51.3 1.55 1.6 50 Induced Shoot 1.4 Regeneration 1.2 40 1 30 Lateral root 0.8 (3sf) (3sf) formation 0.557 0.6 20 0.4 CIM induced 10 4.36 0.2 leaf and root regeneration 0 0 2n 4n 2n 4n Discussion and 28 Conclusion
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