CRISPR/Cas-9 mediated genome editing in polyploidy potatoes
PLSC 460 Final Presentation
1. Growth Regulating
Hormones in Maize
Dr. Burkhart Schulz
Peter English
Catherine Hoover
Jennifer Johnson
Jake Lefler
Yingyu (Winnie) Liu
Mike Shen
Mei Zhao
2. NAM
● Nested Association Mapping
● Genomes searchable
● Basis for experiment
● Testing genetic differences among seeds
3. Seed Selection
● 27 lines of seed selected
● Different geographical areas of the world
● Sweet corn, “pop” corn, feed corn, bush
corn, tropical corn, etc.
4. Seed Selection cont.
● Different genetic variability within different
seeds
● Geologically different to test different
environmental conditions and factors
5.
6. Two Experimental Treatments
● Three groups of seeds
o Water-control (H2O)
o Gibberellic Acid (GA)
o Propiconazole (PCZ)
● Genetic susceptibility and resistance to
different chemical treatments
8. Hypothesis
Our purpose in this experiment is to measure
how much each line responds to GA and PCZ
treatments.
9. Measurements (in mm)
● Primary Root
Length
● Number of
secondary roots
● Mesocotyl length
● Length of true leaf
● Calculate
percentage
compare to the
water treatment
10. Materials and Methods
Materials:
● 27 lines: maize seeds from USDA; genome
sequenced
● Three treatments:
o Water (Control)
o Gibberellic Acid (GA)
o Propiconazole (PCZ)
● Vermiculite
11. Materials and Methods
Methods:
● Pre-germinate seeds
● Plant seedlings with similar growth
● Grow seedlings under a dark cloth
● Harvest and photograph
● Generate data using Image J
12. Methods
● Pre-germinate 35 seeds
for each line
● Plant evenly germinated
seedlings one inch deep
(root downwards)
● Treatment
● Incubate in dark for 3-4
days
13. How we did the treatments
● Stock solutions for PCZ and GA
● Pour 2.5mL of the stock
solution into 1 liter of buffered
water
● 1.5 L of solution per flat
27. Experimental Design
Problems
● Small sample size
● Uneven germination:
○ The different species have various gene on germination.
○ the different day length for the seeds germination.
● Seed contaminations:
○ Presence of fungus on water and GA groups.
● User input error using ImageJ
28. Experimental Design
Improvements
● Triple sample size
o Increased sample size to decrease standard deviation
● Fungal infection:
o 70% ethanol solution on water and GA groups.
● Growth Chamber:
o to keep the moisture and the temperature constant for the pre
germination.
o to keep environment void of light during growth
29. Experimental Design
Improvements
● Input error
o Using droppers to accurately add hormones to water solution
o Use of droppers to apply specific water amounts per cell
o Have multiple people measure same data in ImageJ
● Repeat trials on Tzi 8:
o PCZ hypersensitive
30. Data Application
● Plant Breeding
o selecting for plant height
o Increased growth for silage production
o Reduced growth for weather resistance
● Genetic isolation related to plant
growth
o Nested Association Mapping
o GA and/or Propiconazole effectiveness
31. References
● Hartwig, T., Chuck, G., Fujioka, S., Klempien, A., Weizbauer, R., Potluri,
D., . . . Schulz, B. (2011). Brassinosteroid control of sex determination in
maize. Proceedings of the National Academy of Sciences, 19814-19819.
● Hartwig, T., Corvalan, C., Best, N., Budka, J., Zhu, J., Choe, S., . . .
Grebe, M. (2012). Propiconazole Is a Specific and Accessible
Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize.
PLoS ONE, E36625-E36625.
● Poland, J., Bradbury, P., Buckler, E., & Nelson, R. (2011). Genome-wide
nested association mapping of quantitative resistance to northern leaf
blight in maize. Proceedings of the National Academy of Sciences, 6893-
6898.
33. Presentation Structure (20 min)
● Introduction, general (why, what)
● Important background literature (NAM), two sets
● Material and Methods (what and why the lines, why
use vermiculite, hormones, pre-germinate, different
background), two sets
● Results (what has been measured, why, visual overkill,
corn of different background), two sets
● Discussion (Interpretation, tissue specificity,
correlation, striking lines, usability for breeding), two
sets
34. Introduction
Growth regulators (phytohormones)
Simplified version of signal transduction
The figure of corn seedling
NAM population
Mutant phenotype
Excessive Deficient
GA tall, pistil in
tassel (tassel
seed)
dwarf, anthers in
ear
BR dwarf,
feminization of
male parts
35. Rationales
● GA: growth, sex determination
● GA-deficient: dwarf, anthers in
ear
● GA-excessive: tall, pistil in
tassel (tassel seed)
● BR:growth, sex determination
● BR-deficient: dwarf,
feminization of male parts
36. Overarching Goal
● Use NAM to identify gene(s)
regulating BR and GA
● Sex determination
● Hybrid seed production and
yield
Nelson, 2011
37. Data
● Image J software
● Generate data of
o Primary root length
o # of secondary roots
o Mesocotyl length
o True leaf length
38. Results
Primary Root Length
- B97 – Very high response to GA, PCZ responded just as much as the control group did
- MS71 – GA responded higher than control group (expected), but seedlings in PCZ group were hypersensitive (not expected)
- Tzi8 – GA seedlings hyposensitive, but seedlings in PCZ group even more hyposensitive – don’t even show on graph!
Number of Secondary Roots
- B97 – Hypersensitive in both GA and PCZ groups!
- MS71 – Both very hyposensitive – surprising considering it did very well in primary root length
- Il14H – Hyposensitive to GA, hypersensitive to PCZ
Mesocotyl Length
- M16W – Exactly how we anticipated the growth hormones/inhibitors to work
- CML103 – GA group hyposensitive, PCZ group hypersensitive
- Tzi8 – Consistently hyposensitive to PCZ, no matter the tissue
True Leaf Length
- CML52 – Responded strongly to PCZ, but also GA
- Tzi8 – Consistently hyposensitive to PCZ, no matter the tissue
- Il14H – Hypersensitive to PCZ, hyposensitive to GA - unexpected
If the PCZ group is tall, the seedling was
hyposensitive to it
If the PCZ group is short, the seedling was
hypersensitive
39. Result
Primary root length: H2O group has the
longest roots, M37W most confirmed our
hypothesis, B97 most sensitive to GA, Tzi8
most sensitive to PCZ, CML228 least sensitive.
40. #of Secondary Roots: Tzi8 most confirmed our
hypothesis, Tzi8 most sensitive to GA, Tzi8
most sensitive to PCZ, a couple lines are
insensitive.
41. Mesocotyl: GA group has the longest
Mesocotyl, CML228 most confirmed our
hypothesis, M162W most sensitive to GA, Tzi8
most sensitive to PCZ, HP301 least sensitive.
42. True leaf: NC350 most confirmed our
hypothesis, Ki11 most sensitive to GA, Tzi8
most sensitive to PCZ, a couple lines are
insensitive.
Editor's Notes
Purpose of NAM: making it easier to track beneficial genes in a species population.
These are two different corn lines under the same treatments.
