Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Phytochrome and Light Sensitive Seed Germination

Objectives:  Upon completion of this lab you should be able to:

1. describe the characteristics of a phytochrome-mediated response

2. calculate seed germination percentages

3. describe the effect of red and far-red light on light sensitive seed germination

4. describe the effect of light intensity, quality and duration on lettuce seed germination

5. perform a chi square 2 x 2 contingency analysis

Introduction: 

     Phytochrome is a water soluble, blue-green pigment that serves as the receptor for a wide variety of plant responses to light.  Some phytochrome-mediated responses in plants include the flowering of photoperiod sensitive species, de-etioloation responses, nyctinastic leaf movements, stimulation of enzyme synthesis and synthesis of anthocyanins.

     Phytochrome exists in two forms that are interconvertible, Pr and Pfr.  Red light from 600-700 nm will convert phytochrome in the red form (Pr) to the far-red form(Pfr).  Far-red light, 700-800 nm, will convert phytochrome in the far-red form to the red form.  Pfr is somewhat unstable and slowly reverts back to Pr or is destroyed.  Pfr is generally considered to be the active form of phytochrome.

     A physiological responses is considered to be mediated by phytochrome if:  (1) actions caused by red light can be reversed by exposure to far-red light; and (2) low irradiances of red or far-red light cause the response.

Exercise 1:  Light Sensitivity in Lettuce Seeds

                Light sensitivity was first observed in the Grand Rapids variety of lettuce.  It has since been observed  in other varieties including Salad Bowl and Black-seeded Simpson.  However, not all varieties of lettuce have light sensitive seeds.  In fact, the light sensitivity may even vary within a cultivar.  Not surprisingly, plant breeders have tried to eliminate this trait since it is undesirable from a grower's standpoint.  Light also stimulates the germination of seeds of other plants including dandelion (Oxlade and Robinson, 1986).  Temperature plays a role in light sensitivity, but will not be considered further here.  In this exercise we will examine varieties of lettuce to determine if they exhibit light sensitivity.

Protocol:  Place about 50 lettuce (Lactuca sativa) seeds of each available cultivar in each of two petri dishes (60 x 15 mm) lined with filter paper.  Label the dishes with the variety, lot number, seed age, date, your name and treatment (light vs. dark).  Add enough water to the dishes until the seeds just fail to float.  Seal with a strip of parafilm.  Wrap the dark dish with aluminum foil.  Place both dishes in a lighted area.  Score for germination after 48-72 hours.  Use a 2 x 2 contingency table to determine if any of these seeds are light-sensitive.

Data: 

Variety: __________________  Start date: ______________  Start time: ________________

Analysis date: ________ Analysis time:  _________  Time since beginning of experiment (h) _____

Questions:

1.  What is the null hypothesis for the chi square test?

2.  Calculate chi square and p for each variety.

3.  Is the germination of any of the lettuce cultivars that you tested affected by light?  Explain.  Cite your data to support your answer.

Exercise 2:  Effect of Light Quality on Germination

Protocol: Obtain 9 petri dishes (60 x 15 mm) lined with filter paper and label them "dark control, non-imbibed red, red, fr, r-fr, r-fr-r, white control, green, blue".  Working in the dark under a green safe light, place about 50 hydrated lettuce seeds (Lactuca sativa cv Grand Rapids) and then add just enough water that the seeds fail to float.   Using the light apparatus provided, expose the seeds to the following treatments:  red light (5 min) using imbibed or non-imbibed seeds; far-red (10 min); red (5 min) then far-red (10 min); red (5 min) then far-red (10 min) then red (5 min); white light (5 min); green (5 min) and blue (5 min).  Add water to the seeds until they just fail to float.  Seal the dishes with parafilm, put the dishes in a dark location and score for germination 48-72 hours later.

Data:

Variety: __________________  Start date: ______________  Start time: ________________

Analysis date: ________ Analysis time:  _________  Time since beginning of experiment (h) _____

1. Plot a histogram of your results.

2.  For each of the following pairs, determine if there is a statistically significant difference in results using a chi square contingency analysis.  Give the null hypothesis, chi square value, p value and your conclusion.

                a.  imbibed vs. un-imbibed

                b.  dark vs. white light

                c.  red vs. far red

                d.  blue vs. dark

                e.  green vs. dark

                f.  red vs. dark

3.  Is the seed germination response in Grand Rapids lettuce phytochrome-mediated?  Explain citing your data. 

4.  What was the effect of imbibition on light sensitivity? 

5.  In general, were the results what you expected?  Explain. 

6.  Why is the safe light green? 

7.  Why were red-treated seeds exposed for 5 minutes but the far-red seeds treated for 10 minutes?

Exercise 3:  Sensitivity of Lettuce Seeds to Light

     In this exercise we will observe the response of light-sensitive lettuce seeds to varying amounts of light.  We will try to discern the minimal light requirement for a response. Kodak Wratten neutral density filters will be used to reduce the light.  These filters are made of colloidal carbon and dyes dispersed in gelatin.  They allow uniform passage of light in the 400-700 nm range.  These filters should be protected from overheating.  The spectral qualities are provided below.

                A LiCor quantum radiometer will be used to measure the photon flux density received by the seeds.  This is expressed as the number of photons in the 400-700 ranges incident per unit time on a unit surface.  The units are usually expressed in mEinstein's sec ^-1m^-2 where mE = 1mmol photons which equals 6.02 x 10^-17 photons sec^-1  m^-2.

Protocol:  Working under the safe light, place about 50 hydrated lettuce seeds into each of 8 pre-labeled (dark, light, 0.1, 0.4, 1.0, 2.0, 3.0, 4.0) petri dishes (60 x 15 mm) lined with filter paper.  Transfer the dishes to the appropriately labeled box and expose the seeds to fluorescent light for 5 minutes.  Seal the dishes with parafilm.  Determine the photon flux density of light in the "light" control box with the LiCor Quantum radiometer.  Using the physical characteristics reported for each filter (see table), calculate the flux density for each filter.  Measure the photon flux density of room light and outdoors (sunlight).  Plot % germination vs. photon flux density.  Plot % germination vs. log photon flux density.  Calculate total exposure (mE m^-2) for each treatment.  Plot % germination vs. log exposure (mE m^-2).

Data:

Variety: __________________  Start date: ______________  Start time: ________________

Analysis date: ________ Analysis time:  _________  Time since beginning of experiment (h) _____

(measured for control, calculate for others)

1.  Did light intensity influence the germination of the lettuce seeds?  Explain.  Cite your evidence?

2.  What units is photon flux density expressed in?  What does this unit represent?

3.  How did the minimal amount of light required for the response  compare to full sunlight? room light?

4.  Do the neutral density filters transmit far-red light?  ultra-violet? green?

Exercise 4:  Exposure Time and Lettuce Seed Germination

     In this experiment we will determine the duration of light exposure required to initiate light-sensitive seed germination.

Protocol:  Working under a safe light, place about 50 hydrated seeds (L. sativa cv. Grand Rapids) in each of 5 petri dishes lined with filter paper.  Label the dishes 0, 1, 10, 100, 300 seconds.  Expose the seeds to the appropriate duration of white light.  Add enough water until the seeds just fail to float.  Measure the irradiance of the light source with LiCor Quantum Radiometer.  Place seeds in the dark and score for % germination 48 hours later.  Plot % germination vs. exposure time.  Plot % germination vs. exposure (mE m^-2)

Variety: __________________  Start date: ______________  Start time: ________________

Analysis date: ________ Analysis time:  _________  Time since beginning of experiment (h) _____

1.  Does duration of the light exposure have an effect on light sensitive lettuce seed germination?  Explain. Cite your evidence.

Last updated:  01/07/2009     � Copyright  by SG Saupe