Theme: PHYTOHORMONES
Experiment 1: Phenotype effects of phytohormone application
Describe and characterize the effects of phytohormones application on germination and
development of Arabidopsis thaliana L.
Principle: Seeds of Arabidopsis thaliana L were
cultivated on agar media (0,2x MS medium) with
phytohormone enrichment. Seed germination and plant
growth is significantly affected by the presence of growth
regulators.
Plant material: Arabidopsis plants cultivated 2-3 weeks
on 0,2x MS medium in five treatments:
• control - no phytohormone addition
• auxin addition - medium enriched by indolyl-3-acetic acid (IAA)
• cytokinin addition - medium enriched by benzylaminopurine (BAP)
• gibberellin addition - medium enriched by gibberellic acid (GA3)
• abscisic acid addition - medium enriched by ABA
Observation:
1. Observe germination and plant growth
2. Compare plants affected by addition of phytohormones with plants cultivated on
control medium. Follow these criteria:
germination capacity
length of root system (measure the length of primary root), describe the
branching intensity
shoot growth
Experiment 2: Plant response to ethylene
Describe and characterize the effect of ACC application on growth and development of
Arabidopsis thaliana L. Compare wild type plants and ctr1 mutants with constitutive ethylene
response.
Principle: Ethylene is a stress hormone involved in regulation of various processes: fruit
ripening, leaf abscission, hypoxia response (aerenchyma formation) or rate of elongation.
Plants affected by ethylene enrichment show the triple response. The roots and shoots are
shorter; the formation of root hairs is stimulated. Ethylene is a gas hormone. Its enrichment
can be induced either by closing plant in a container with ripening fruits (apples, bananas) or
by an addition of ACC into media. ACC (1-aminocyclopropane-1-carboxylic acid) is the
immediate precursor of ethylene synthesis.
Ethylene action is mediated by ethylene receptors – proteinkinases ETR1 and ERS. The
response pathway involves also the negative regulator CTR1. Plants with the recessive ctr1
(constitutive triple response in absence of ethylene) mutation exhibit constitutively activated
ethylene responses – triple response, short roots, pronounced development of root hairs.
Plant material: Arabidopsis plants cultivated 2-3 weeks on control medium (0,2x MS
medium) and medium with ACC addition.
Observation:
1. Observe the growth of Col0 (wild type) plants and ctr1 mutants
2. Follow these criteria:
length of root system (measure the length of primary root), describe the
branching intensity
describe the development of root hairs (density, length of root hairs) –
use microscope
3. Compare phenotype of Col0 (wild type) plants and ctr1 mutants. Describe the effect of
ACC application.
Theme: ROOT GRAVITROPISM
Experiment: Role of auxin in gravitropic response of plant root
Visualise auxin distribution in root of Arabidopsis thaliana affected by change in spatial
orientation. Follow the effect of auxin transport inhibitor NPA on localisation of auxin
maxima in root and root gravitropic response.
Principle: Positive gravitropic response of root is coordinated by directional flow of
phytohormone auxin. Change in root orientation results in an accumulation of auxin on
bottom side of root apex, where it inhibits cell growth and causes asymmetry of root growth
and root curvature. Redistribution of auxin is prevented and gravitropic response is lost in
presence of auxin transport inhibitor NPA (1-N-naphtylphtalamic acid).
Transgenic plants with DR5:GFP and DR5:GUS reporter genes are useful tool for auxin
maxima visualisation. DR5 is auxin responsive promoter. GUS (beta-glucuronidase enzyme)
cleavages X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) into colourless glucuronic acid
and intense blue precipitate of chloro-bromoindigo.
Plant material: Arabidopsis plants cultivated in four treatments:
• control medium, no change in spatial orientation
• control medium, change in spatial orientation (135° rotation four hours before start of
GUS detection)
• medium with NPA, no change in spatial orientation
• medium with NPA, change in spatial orientation (135° rotation four hours before start
of GUS detection)
GUS activity detection:
1. Transfer several DR5:GUS plants from each treatment into separate hole in reaction
plate (marker each hole and pipette 1 ml of phosphate buffer before plant transfer)
2. Wash with 1 ml of fresh phosphate buffer (use 1ml pipette)
3. Add 0,5 ml of detection buffer into each hole.
The composition of detection buffer is following:
2ml of phosphate buffer
20µl of ferrocyanide
20µl of ferricyanide
20µl of X-gluc (substrate for glucuronidase activity)
4. Incubate 30 min at 37°C, check blue colour development and observe at microscope.
5. Compare blue colour distribution in root apex among four treatments.
6. Observe effect of NPA application on blue colour distribution
Observation of GFP localisation in plant in-vivo:
1. Transfer DR5:GFP plants from each treatment on microscopic slides into drop of
water.
2. Observe localisation of green fluorescent protein in root apex under blue light
excitation.
3. Compare among treatments and compare with GUS localisation.