Spring.wmf (18300 bytes) 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

Hormones and Fruit Development in Rapid-Cycling Brassica rapa (RCBr)

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

  1. grow an RCBr ("Fast Plant") from seed to seed
  2. describe the life history of Brassica rapa using terms such as radicle, hypocotyl, cotyledon, epicotyl, node, internode, pedicel, receptacle, sepals, petal, androecium (or stamen); filament, anther, gynoecium (or pistil), stigma, style, ovary, nectary, pollination, silique (pod), raceme, inflorescence, fertilization
  3. cross pollinate a flower
  4. describe the effect of plant hormones on RCBr fruit expansion
  5. prepare computerized bar graphs and scatter plots
  6. perform simple statistical tests (mean, standard deviation, correlation, t-test)

Introduction:
    The purpose of this lab is two-fold.  First, this lab will provide the opportunity for you to study the growth of a plant throughout its entire life cycle.  To learn more about the history and biology of RCBr visit the visit the Wisconsin Fast Plants� web site.  This site provides a wealth of information, including the on-line version of Dr. Williams article and slides/images of the plants.  The second major purpose of this lab is to use Fast Plants� as a model experimental organism to study the physiological mechanisms involved in fruit growth.  

Pre-Lab Assignment:
    Before coming to lab, print a copies of this exercise, RCBr Growth Techniques, and the Pre-lab assignment.  Read the article by Nitsch (1950).  If daily measurements (MOD's) are going to be collected, also print a copy of the data sheet. 

EXERCISE 1:   Rapid-Cycling Brassica rapa Pod Structure

  1. Sketch (in the box below) the external features of the pod (label stigma, style, ovary, receptacle) in Fig. 1.
  2. Measure the length of the pod (mm) ________ (Note: what will you measure?) and record your data in Table 1.

    3.  
     

     

     

     

     

     

     

     

    		  
    Fig 1. External Features of the Pod Fig 2. Internal Features of the Pod
  3. Dissect the pod carefully, pry off one of the halves (carpels). Sketch the arrangement of seeds in the pod in Fig 2. Locate and label the funiculus (stalk that attaches seed to pod), septum (partition between fruit halves), placenta (region where the funiculus attaches the seed to the fruit wall).
  4. Record the number of seeds in the pod in Table 1. Collect the seeds on the sticky tape provided.
  5. Record the class data in Table 1.

Questions:

  1. Who is responsible for the color of the seed? Mom, dad or junior (seed)?
  2. Do the seeds in a single pod have the same mother? the same father? same grandmother?
  3. What is the relationship of the seeds in a single pod to one another? siblings, cousins, etc.?
  4. Can the mother of the seeds in a pod also be the father?
  5. Will the seedlings that develop from these seeds all look alike? Explain.
  6. The production of this pod including seeds required (insert appropriate number):

    7. _____ flower(s) ______ ovary(s) ______ ovule(s)

    _____ egg(s) ______ sperm(s)   ______ pollen grain(s).

  7. Calculate the mean (� standard deviation) for pod length and seed number.
  8. Plot a frequency distribution of # observations vs. pod length. What do you conclude from this graph? Is our population "normal"? Should it be? What factors could influence the size of the pod?
Table 1: Size and seed number of wild type RCBr pods
  Pod Length (mm) Seed Number   Pod Length (mm) Seed Number   Pod Length (mm) Seed Number
1     11     21    
2     12     22    
3     13     23    
4     14     24    
5     15     25    
6     16     26    
7     17     27    
8     18     28    
9     19     29    
10     20     30    

EXERCISE 2:   Factors Influencing Pod Size in Rapid-Cycling Brassica rapa

  1. What relationship do you predict to exist between seed number and pod length?  Explain.
  2. Test your hypothesis by preparing a scatter graph (plot pod length vs. seed number).  Do your data support your prediction?

Questions To Consider:
     What is the maximum number of seeds that could be produced per pod? per plant? What factors (variables) effect the number of seeds that are produced by a pod/plant? Describe an experiment to determine these values. Which will have the most seeds, the uppermost or lowermost pods?

EXERCISE 3:  Experiment to Test Hypotheses Concerning Fruit Development in Rapid-Cycling Brassica rapa

Background Information:
    In most plants, fruits develop only after pollination and fertilization. These processes, in conjunction with signals from the developing seed, provide the trigger for fruit development. Thus, fruits contain seeds and these are required for normal fruit development. However, many plants can produce fruits without seeds. Such fruits, which are termed parthenocarpic, may develop: (1) without pollination (as in banana and pineapple); (2) after pollination but without fertilization (as in some orchids); or (3) after fertilization but following embryo abortion (as in grape) (Salisbury and Ross, 1992).

    Parthenocarpic fruits can sometimes be induced to develop by treating the carpel (pistil) with a hormone such as auxin (indole-3-acetic acid, IAA) or gibberellic acid (GA). For example, auxin released by the achenes stimulates receptacle (fruit) formation in strawberry (see Nitsch, 1950) and GA content is associated with seed size in bean fruit (see Skene and Carr, 1961). Plants are often more responsive to exogenously applied GA than auxin. It has also been shown that applying an extract of pollen to some plants can induce parthenocarpic fruit development (Salisbury & Ross, 1992).

Questions:  Do plant hormones stimulate fruit expansion in B. rapa? Which, if any, hormones induce parthenocarpy in RCBr? Would a combination of hormones and pollination have a similar effect? Will the application of hormone antagonists prevent parthenocarpy? Does pollen from other species have any effect on parthenocarpy in RCBr? Will an extract of pollen or immature ovules induce parthenocarpy?  

Hypothesis: e.g.:  GA stimulates fruit expansion in B. rapa.

Predictions: e.g.:  If GA treatment stimulates fruit expansion, then the carpels of an unpollinated flower treated with lanolin paste containing GA should elongate and expand as much as a cross-pollinated control. Unpollinated carpels treated with lanolin paste alone should exhibit little expansion.

Protocol:

  1. Design your experiment. With your research team brainstorm possible ideas to test with your group.  We will share these with the class and then select one to study in more detail.
  2. Working individually, prepare 4 quads (or film cans) for planting, according to information provided in the Directions for Growing Rapid-Cycling Brassica rapa.   In addition, there are a variety of references in the file cabinet in the botany lab (PENGL 345) including a copy of the " Fast Plant Manual".
  3. Label the quads - include your name, date and seed strain (wild type seeds are designated +/+). Use label stakes or flags made from a toothpick and piece of tape - don't write on the quads.
  4. Plant two wild type seeds (+/+) in each cell of the quad.
  5. Grow your plants according to the directions provided.  It is your responsibility to keep the plants alive! Check them daily for the first few days. When the seedlings are about five-days old, thin to one plant per quad (which ones will you remove?). The remaining plants should be of uniform size and appearance.
  6. After 12-15 days, flowers will appear. This is the time to begin your treatments.
  7. In a typical experiment you will have four groups (quads) of plants to treat. You will treat six flowers on each of the four plants in each quad. Thus, you will treat a total of 96 flowers (6 flowers/plant x 4 plants/treatment x 4 treatments) during the experiment. In most cases your four treatments will include a pollinated control, unpollinated control, hormone treatment, and hormone control (see below for details).
    1. Pollinated Control Quad - Transfer pollen from the stamens of a flower on one of the plants to the carpels of the flowers on each of the other plants using a bee stick. For more details, check out the directions provided or in the Fast Plants Manual. Keep these plants separate from the others to avoid accidentally cross pollinating any of the other treatments. After cross-pollinating the sixth flower on each plant, cut off the growing tip to remove any unopened flower buds.
    2. Unpollinated control Quad - when a flower buds open, remove the stamens with a pair of fine-tipped forceps. After removing the stamens from the 6th flower on each of the four plants in the quad, cut off the growing tip to remove any unopened flower buds. For this treatment and all of the others except the pollinated control, be careful to avoid accidentally pollinating the flowers.
    3. Control (Lanolin or aqueous extract) Treatment Quad - remove the stamens as described above. Then, either apply a thin coating of lanolin paste to the sides of each carpel with a toothpick or place a drop of water on the carpel. Treat six flowers and then cut off the tip as above. Avoid cross-pollinating these.
    4. Hormone (or pollen) Treatment - remove the stamens. Then, either apply a thin coating of lanolin paste containing hormone (GA, IAA, NAA, or IBA) to each carpel or apply a drop of pollen extract to the carpel. You will need to prepare the extract so save all the stamens that you remove from your plants. After the sixth flower has been treated on each plant cut off the growing tip. Again, avoid cross-polllinating these. When the siliques (pods) reach maturity, measure the length and width of each pod and record your data (Table 1).

    8.  Count and collect the expanded seeds in each ovary (fruit). Please return the seeds to me. Record these data in a facsimile of Table 2.

Data:

Table 2: Length, width and seed number for RCBr pods after treatment with _______ (GA, IAA or other).
Rep Unpollinated Control Pollinated Control Control Treatment Hormone Treatment
Pod Length (mm) Pod Length (mm) # Seeds Pod Length (mm) Pod Length (mm) # Seeds Pod Length (mm) Pod Length (mm) # Seeds Pod Length (mm) Pod Length (mm) # Seeds
                         
                         
                         
                         
                         
                         
                         
                         
                         
                         
                         
Mean +/- SD                        

Post-Lab Results:  At the conclusion of the experiment, write an abstract summarizing your results.  Among the questions to consider in your abstract: Did the treatments influence pod elongation?  Do your results suggest which hormone is involved in fruit elongation and where it is made in cross-pollinated carpels, or how it is transported, or what tissues have target cells?  Finally, append to your abstract: 

  1. A completed version of Table 2, with mean and standard deviation calculated for each treatment.
  2. A histogram (bar graph) summarizing the results (pod length, pod width & seed number) for your experimental data (Table 2).
  3. A scatter graph (y vs. x; don’t connect individual data points) of pod length (mm) vs. seed number for the cross-pollinated plants.  Determine the correlation coefficient for these data.
  4. A histogram showing the frequency distribution of pods of varying lengths for the cross-pollinated treatment.
  5. Suitable statistical test(s) to determine if any of the treatments had a significant effect on pod elongation. Show your data/work.

References:

| Top | SGS Home | CSB/SJU Home | Biology Dept | Biol 327 Home | Disclaimer |

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