NIEjr01: Who is faster, 2n or 4n in Growth and Cell Fate - - PowerPoint PPT Presentation

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

Background Information

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Background Information

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Diploid 2n

Tetraploid 4n

• 2 pairs of

homologous chromosomes

• 48 chromosomes
• 4 pairs of

homologous chromosomes

• 92 chromosomes

Same genotypes may exhibit different phenotypes

Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Background Information

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Key terms to be defined CIM Shetty K., Asano Y., Oosawa K. (1992)

• Callus inducing medium

SIM Schaller G.E., Bishopp

• A. et al. (2015)
• Shoot inducing medium

Callus Morel G., Wetmore R.H. (1951)

• Formation
• f

unorganized cell mass

What is Cell Fate Reprogramming?

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Background Information

Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Background information

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Cell fate: The ultimate differentiated state to which a cell has become committed.

undifferentiated pluripotent stem cell Target genes

regulates regulates

Epigenetic mechanisms

• eg. DNA methylation,

histone modification

Cell fate reprogramming

Gene transcription

Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Background information

What does it mean to reprogram cell fate?

When cell fate is reprogrammed, the developmental processes that define cell fate are reverted. Cells dedifferentiate from totipotency into pluripotency.

How?

→ Epigenetic memory is erased. The epigenetic marks on each allele from both chromosomes are reset.

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Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Background information

Cell fate can be reprogrammed by (1) Artificially Induced pluripotent stem cells (IPS)

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Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Background information

Cell fate can be reprogrammed by (2) Production of germ cells (eg. egg/sperm cell) (3) Regeneration

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(2)

Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

The ability to reprogram cell fate is especially important for wound induced regeneration in plants, or De novo organogenesis

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Background Information

De novo organogenesis: The ability of plants to regenerate lost body parts when wounded.

Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Mechanism of De novo organogenesis

Wound response

WIND1 WIND1 WIND1

A

Cellular dedifferentiation

B

Callus formation

C

What has been researched on ▫ Developmental and experimental factors that impact plant regeneration

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Background information: Previous research

Less is known about genetic factors such as variation in ploidy number within the same species can cause significant differences in the regenerative response.

Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Aim

Find the ploidy that regenerates the fastest and is most suitable and receptive to cell fate reprogramming

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Wound Induced Callus Formation

Background Information

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

General Methodology

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Model plant: Arabidopsis Thaliana

Universally recognised model plant in experiments Response and development is similar to most crops Smaller genome

Wound Induced Callus Formation

General Methodology

Background Information Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Varying ratios of auxin and cytokinin in medium to trigger In vitro De novo organogenesis

Shoot regeneration

Callus formation at the tip of the shoot

Cytokinin Auxin

Cytokinin

Auxin Cytokinin

Auxin

Root regeneration Leaf regeneration

Balanced ratio of C and A Rich in Cytokinin Rich in Auxin

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Types of experiments carried out

• 1. Lateral root

formation experiment

• 1. Hormone

induced shoot regeneration protocol

• 1. Wound

induced callus formation experiment

• 1. CIM-induced callus

formation for leaf explant experiment

• 2. CIM-induced callus

formation of root explants experiment

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Statistical tools used for analysis

Real time polymerase chain reaction (qpcr). Mean and Standard Deviation One way analysis of variance hypothesis testing tool (ANOVA)

Individual experiments and Results

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We excised the tip of a hypocotyl. Observed the callus formation of the wounded explant 2 days,

3 days and 4 days after wounding by using a

microscope

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Methodology - Wound induced callus formation

Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

2n callus observed after 4 days

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4n callus observed after 4 days It was observed that the callus formed on 4n plants are larger than the callus formed on the 2n plants

Results - Wound induced callus formation

Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Results - Wound induced callus formation

Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Results - Wound induced callus formation

Statistical test: One-way Analysis of Variance (ANOVA) test → Test whether there are statistically significant differences between 2n and 4n results

Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Results - Wound induced callus formation

If P-value < α = 0.05, reject null hypothesis Null Hypothesis

Alternative Hypothesis

• No statistically

significant difference

• Any observed

differences may be due to chance and sampling error.

• There is a

statistically significant difference

• The observed

differences are not due to chance or sampling error.

Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Results - Wound induced callus formation

ANOVA

Source of Variation SS df MS F P-value F crit Between Groups 1893.779 1 1893.78 139.17

0.000000000184 4.35124

Within Groups 272.159 20 13.608 Total 2165.937 21

Since our P-value < α = 0.05, we reject the null hypothesis. Thus, we confirm that our results are statistically significant.

Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Results - Wound induced callus formation

CONCLUSION 4n has higher regenerative capacity in callus formation than 2n

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Background Information

Wound Induced Callus Formation

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Methodology - Hormone induced shoot regeneration

Background Information

Hormone Induced Shoot Regeneration

General Methodology Wound induced callus formation Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

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Background Information

Hormone Induced Shoot Regeneration

General Methodology Wound induced callus formation Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Results - Hormone induced shoot regeneration

51.3 4.36 10 20 30 40 50 60

2n 4n

Rate of regenerated shoot explants (3sf)

Rate of regenerated shoot explant for 2n and 4n explants

1.55 0.557 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

2n 4n

Number of regenerated shoots per explant (3sf)

Shoots per regenerated explants for 2n and 4n explants

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2n shoot explants regenerated many more meristems, than 4n shoot explants

Background Information

Hormone Induced Shoot Regeneration

General Methodology Wound induced callus formation Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Results - Hormone induced shoot regeneration

CONCLUSION 2n has higher regenerative capacity in hormone induced shoot regeneration than 4n

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Results - Hormone induced shoot regeneration

Background Information

Hormone Induced Shoot Regeneration

General Methodology Wound induced callus formation Lateral root formation CIM induced leaf and root regeneration Discussion and Conclusion

Recorded the number of lateral roots formed by 2n and 4n plants after 6 days, 8 days and 10 days

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Methodology - Lateral root formation

Background Information

Lateral Root Formation

General Methodology Wound induced callus formation Hormone induced shoot regeneration CIM induced leaf and root regeneration Discussion and Conclusion

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Results: Lateral root formation

Background Information

Lateral Root Formation

General Methodology Wound induced callus formation Hormone induced shoot regeneration CIM induced leaf and root regeneration Discussion and Conclusion

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Background Information

Lateral Root Formation

General Methodology Wound induced callus formation Hormone induced shoot regeneration CIM induced leaf and root regeneration Discussion and Conclusion

2n plant had a had a more complex root system compared to the 4n explant.

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Results - Lateral root formation

Background Information

Lateral Root Formation

General Methodology Wound induced callus formation Hormone induced shoot regeneration CIM induced leaf and root regeneration Discussion and Conclusion

CONCLUSION 2n has faster lateral root growth rate than 4n

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Results - Lateral root formation

Background Information

Lateral Root Formation

General Methodology Wound induced callus formation Hormone induced shoot regeneration CIM induced leaf and root regeneration Discussion and Conclusion

Methodology - CIM-induced callus formation of leaf explant

The blade parts of 2 weeks old Arabidopsis Thaliana plants were cultured on CIM induced mediums

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Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

4n explants had a higher regenerative capability compared to 2n explants

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Methodology - CIM-induced callus formation of leaf explant

2n 4n

Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

LBD16: LATERAL ORGAN BOUNDARIES DOMAIN16

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Results - CIM-induced callus formation of leaf explant

WOX11: WUSCHEL RELATED HOMEOBOX11

4n 2n

Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

CONCLUSION

4n has higher regenerative capacity in CIM induced leaf regeneration than 2n

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Results - CIM-induced callus formation of leaf explant

4n’s higher rate of regeneration is due to upregulation of gene transcription factors WOX11 and LBD16

Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

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Root explants of 7 day old Arabidopsis thaliana plants are incubated to CIM induced mediums and cultured under day long condition. Callus formation is recorded on the 7, 14, 21, 28 days.

Results - CIM induced callus formation of root explant

Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

Results - CIM-induced callus formation of root explant

2n Arabidopsis root explants regenerated a lot more significantly larger callluses than 4n Arabidopsis shoot explants.

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Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

CONCLUSION

4n has greater root regeneration capacity compared to 2n

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Results - CIM-induced callus formation of leaf explant

Background Information

CIM induced Leaf and Root regeneration

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation Discussion and Conclusion

Discussion and Conclusion

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Discussion of results

▫ Lateral root experiment ▫ Shoot regeneration experiment 2n explants had a faster growth rate compared to 4n explants ▫ Wound induced callus formation experiment ▫ CIM-induced leaf regeneration experiment ▫ CIM-induced root regeneration experiment 4n explants had a faster regenerative capacity compared to 2n explants

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Background Information

Discussion and Conclusion

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation CIM Induced leaf and root regeneration

Implications of our study

Our study adds to the scientific pool of knowledge of plant regeneration to advance the field of tissue culture Improving Agriculture and Biotechnology ▫ Improving crop yield by cultivating 4n crop ploidies that can regenerate organs faster ▫ Using 4n in genetically modified food

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Wound Induced Callus Formation

Discussion and Conclusion

General Methodology Hormone induced shoot regeneration Lateral root formation CIM induced leaf and root regeneration Background Information

Conclusion

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Conclusion

(4n) Tetraploid explants exhibit greater: Cell regenerative capacity in ▫ Wound induced callus formation ▫ CIM-induced leaf regeneration experiment ▫ CIM-induced root regeneration experiment. (2n) Diploid explants exhibit greater: ▫ Root cell proliferation capabilities ▫ Cell regenerative capacity in the hormone induced shoot regeneration

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Background Information

Discussion and Conclusion

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation CIM Induced leaf and root regeneration

Limitations

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Limitations

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Limitations Elaboration

Insufficient amount of data to carry

• ut qPCR
• The results obtained may be

inaccurate Only experimented on 1 plant species

• Effects of different ploidy

numbers on different plants may differ

• Future work: experiment with
• ther species of plant (such as

maize)

Contradicting definitions of a callus

• Recent studies: tissue is not

dedifferentiated

• Previous study: tissue is

dedifferentiated

Background Information

Discussion and Conclusion

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation CIM Induced leaf and root regeneration

Future work

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Future research and Extensions Future areas of research: ▫ Gene signalling pathways in 4n versus 2n that cause 4n to regenerate faster than 2n for the wound induced callus experiment and CIM induced leaf and root explant regeneration, but exhibit slower regeneration rate for shoot regeneration and ▫ Experiment on 4n and 2n ploidies of other plant species like maize and rice

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Background Information

Discussion and Conclusion

General Methodology Wound induced callus formation Hormone induced shoot regeneration Lateral root formation CIM Induced leaf and root regeneration

Acknowledgements

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Acknowledgements Special thanks to our mentor Associate Professor Chen Zhong and Dr. Yan An, and our school based mentors Mrs May Liow and Mr Lim Lee

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Thank you!

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Q&A