Research Ideas, for students working with professor Mione

Link to Jaltomata home page
professor Thomas Mione, Central Connecticut State University
Biology Department, Copernicus Hall, 1615 Stanley Street, New Britain, CT 06050-4010

Bio 327                                    Questions We Are Asking

 

1a.  Self-compatible versus self-incompatible. 

Tag and hand-pollinate 20 flowers with pollen from same plant.
Tag and hand-cross-pollinate 20 flowers.

Use day 1 flowers as pollen recipients, flowers that, if you looked at them the previous day, had never been open.  For pollen donors, use any flower having dehisced anthers.

Cross-pollination involves the pollen from one plant being applied to the stigma of a flower of a different plant of the same species.

1 b. How many days from pollination to ripe fruit?
You will be able to use the same flowers/fruits you are using for 1a.

1 c. Are fruits produced without pollination, or is pollination required for fruit production?

Tag 10 more flowers and don’t do anything to them.  Don’t let anyone physically bump into them after they are tagged because just bumping into flowers could self-pollinate them. We had better not see pollinators in our greenhouses (if we do, the results are void).

1 d. If the species is self-compatible, is the number of seeds per fruit and the fruit size significantly different for self-pollinated versus cross-pollinated plants?

Note that if the species is self-compatible, you will be able to answer this questions using the exact same flowers you used for question 1a.

Pot size may affect fruit size, so the pots must all be the same size.

This requires you to be sure, when you pollinate, to completely cover a stigma with pollen.  You must look at a stigma with a hand lens after you pollinate, to make sure the stigma is covered with pollen.

The results of 1d may not be available until after the semester ends.  If you choose to address this questions your grade will not be affected if you don’t have data (at the end of classes).  I will ask you to meet with me to show me exactly which tags go with what (if 1d is one of your experiments, don’t disappear during finals week, without showing me which tags are for what!).

1 e. Agamospermy is the production of fruit without fertilization, and is unlikely but we must test for it.  Before anthers open (dehisce), emasculate flowers (remove anthers) with forceps, being very careful to not kink or damage the style.  Do not pollinate.  Tag the flowers.  Do not kink or otherwise damage the peduncle or pedicel while you are emasculating.

 

2. What part of the flower makes nectar? 


Are you ready for surgery on a living flower?  Remove all petals and stamens (with a sharp razor blade and a steady hand) while the flower remains attached to the plant (do not kink the flower’s peduncle or pedicel while you do surgery).  

Come back in a few hours and twice the next day.  If you see nectar, we know that it is at least made by something other than petals and stamens (but may also be made by petals and stamens).

Try removing half of the corolla/androecium on the same side but not the other.

Do this to multiple flowers, or figure out another way to determine exactly which part of the flower is making the nectar.

You will need my magnification visor. Both species,  Jaltomata calliantha and J. sinuosa, may be used, but the nectar of J. sinuosa is nearly clear so it may be more difficult to see the nectar.

 

3. Nectar volume.                                   J. calliantha

I will lend you very fine glass tubes, micropipettes, that each hold 20 microliters.  To determine nectar volume, you hold these tubes, one at a time, to the nectar, and count how many you fill to the red line.  Empty the flower as much as possible without damaging it.  One has to learn to pull these away from the flower the second the nectar fills to the red line.

Flowers have to be tagged before they open. The first day a flower is open it is called a day 1 flower, and the 2nd day it is called a day 2 flower. 

You have to know the age of the flower (a day 1 flower, a day 2 flower, a day 3 flower) and the only way to know this is to tag flowers before they open. If you tag a flower (that is not open yet) on a Friday and come back on Monday and it is open you can’t assume it is a day 1 flower, because Saturday or Sunday may have been its day 1.  In other words, once you tag a flower you must make observations every day.


3a. One question that comes to mind: how many days in a row will nectar refill after daily removal, and will the flower’s life exceed the number of days, such that the flower has no nectar during one or more of its last days?  In Peru, hummingbirds are after this nectar like it is candy.  If a plant can save energy by not producing any floral nectar during a flower’s last days, the plant is duping the pollinator into visiting (being a courier) when there is no reward, a very interesting situation.

3b. Does pollinating the flower result in a change in nectar production?

Use pairs of observations.

One flower of each pair will not be pollinated; measure the volume of the nectar every day.

The other flower of the pair will be pollinated the day it opens, with pollen from a different plant consistently, and nectar is removed and the volume measured every day.  

Adding all nectar produced in the flower’s lifetime, does the one that is pollinated produce less nectar?

Consider adding taking down floral longevity data while doing this!

If you want to color code tags, one color for not pollinated, the other color for pollinated, you are welcome to color half your tags red.

 

4. Nectar color.                                       Jaltomata calliantha

Nectar starts out clear or amber in color, and changes to orange or red in a few days.

a) If you take nectar from day 1 flowers and put the nectar in a very small tube, does it turn orange or red if the tube is open?  If the tube is closed?  If the tube is in the refrigerator and open?  If the tube is in the refrigerator and closed?  Nectar turning color suggests oxidation.

b) Is refill nectar orange or red when it comes in? 
In a day 2 or a day 3 flower or a day 4 flower, remove all nectar in the morning: after removing nectar with micropipettes (as described in 3 above).  Gently stick a twisted corner of a kimwipe in the flower, into all five nectar troughs, removing all the nectar.  Come back in one hour, in two hours, in three hours and four hours and report the color of the refill nectar. Keep track of whether each flower was a day 2, a day 3 or a day 4 flower.

 

5. Nectar’s sugar concentration.  This is estimated with a refractometer.

Your challenge is to read nectar literature and then figure out which question(s) you want to ask.  Here is one question:

How does the sugar concentration change if nectar is:

a) not removed (record whether or not the nectar comes from a day one flower, a day two flower, a day three flower, a day four flower), versus

b) removed and refills (be consistent: take readings 22 to 25 hours after nectar is completely removed). 

I will show you a certain kind of very small plastic disposable pipette you can use to remove the nectar from the flower.

Record the temperature at each reading because my refractometer does not have built-in temperature compensation.  The temperature in a greenhouse varies with elevation above the floor, and a thermometer that is a meter higher than your flower will give you an incorrect reading.

 

6. Floral longevity. How many days do flowers last?

Tag flowers before they open.  Visit the flower every day.  Score a flower as zero before it opens.  The first day it is open, 1.  The second day it is open, 2.  When the corolla withers (senesces), the flower’s life is over. 


Score a flower as P (pistillate) when it is open but all anthers are closed (have not yet dehisced). 
Score a flower as H (hermaphroditic) when one or more of the anthers have dehisced.

Compare unmanipulated (control) flowers with one or more of the following:
a) flowers pollinated on day one (but not emasculated),
b) flowers from which you remove nectar every day,
c) flowers that are pollinated on day 1, and nectar is removed every day.

Your observations must be paired, on the same plant.  In other words, every experimental (a or b or c) gets a control on the same plant at the same time.  Paired observations allow a paired T-test.

When it is warmer, flowers don’t last as long, so we need to record the temperature.  Report the temperature range in your materials and methods.

If you miss an observation on a given day, say Sunday, and then see when you return (say Monday) that the flower’s life is over, you can’t use that flower because you don’t know when it senesced. For floral longevity flowers need to be observed every day.

Does a plant have a built-in physiological mechanism to drop a corolla-androecium that it no longer needs?  If flowers that are pollinated the first day they are open don’t last as long as unpollinated flowers, the answer will be yes.

 

7. Flower color change. 
Notice that the corolla changes color.  Look at the buds before the petals open and record the petal color.  Look at the petals the next day, the first day they are open, and record your observations about color.  Look at flowers every day.  Record color changes.  Color charts are used by artists and botanists, and if you can get your hands on one (on a smart phone while you are working?), all the better.

 

8. Calyx size change.                              

Measure the length of sepals every 4 or 5 days, starting when the flowers are not yet open. Pollinate the flowers with which you are working, so that you are sure to get fruits.  Gently flatten a sepal to a metric ruler.  Measure from peduncle to lobe-tip.  Graph your results after the fruit is no longer growing.  Let your X axis be days, starting from 1.  Each data point could be the average length of the sepals of 10 flowers. 

There is no hypothesis testing with this one, it is merely descriptive.  It will give your group practice managing data and graphing, and you will be the first to see the unique curve of the growth of this floral appendage on this species.  This is interesting because sepals first surround and presumably protect a flower bud.  Later they are like a hat over the fruit possibly (in J. calliantha) helping hide the fruit so that birds don’t take it away? 

In J. sinuosa the “hat” over the fruit (the calyx) probably advertises the fruit to dispersers.

 

9a. Changes in the stamen length and angle of stamens, documented with photography and measurements.  When stamens angle out, positioning the anthers away from the stigma, outcrossing is promoted. 


We need to measure the stamens, or just the filament, every half hour starting very early in the morning, on a flower’s day 2.  Simply hold a metric ruler up to the stamens and record the length.  Pool data of several different flowers of the same species, and graph your data.  Ask me for help with graphing if needed.  You may take a J. sinuosa plant home, so that you can set a timer for yourself and start early in the morning, and record the length every half hour.

It is really difficult to get good (in focus) photos of small objects.  Work with me using a research camera and / or the microscope in my office if needed. You may use your own equipment. 

The smaller the camera’s aperture opening, the greater the depth of field.  Greater depth of field means that more of your subject will be in focus.  A higher F number means a smaller aperture opening.  In other words, F16 gives greater depth of field than F8.  The smaller you make the aperture opening, the slower the shutter needs to be under the available light. Slow shutter speed leads to blurry images, but if we use a tripod we can use a slow shutter speed. 

 

10. Do ripe fruits drop off and land on the ground? Or, do the ripe fruits remain attached to the parent plant?

If ripe fruits drop as soon as they become ripe, do they drop with the calyx attached to the fruit or do they drop without the calyx?

If ripe fruits remain attached to the plant, for how many days do they remain attached before they finally drop? 

Do Jaltomata flowers close for the night, and if they do, what is the environmental variable that triggers closure? We have two different races of Jaltomata procumbens flowering, and one closes its corolla in the afternoon, and the other half-closes its corolla much later (but I don’t know when). Also study J. sinuosa, flowering now.

You could put a plant in a dark box at noon, and come back and check the flower in a half hour and an hour to see if the corolla closed. Starting at the same time of day, repeat with a different race. If the flower closes in the darkness of a box, and it is still bright outside, will the same flower re-open? If you put bright lights on a plant that has open corollas, will they stay open past when they would have closed without the lights?

You would make regular observations to see if the corolla of one race closes with cloud cover and the other does not.

You would need to tag each flower you work with, because a flower closing at the end of its life is different from temporary closure for the night and then opening the next day. For example, if a flower does not open on Tuesday we know that it is done, and the fact that it closed on Monday becomes irrelevant

You would have to visit the greenhouse at various times of the 24 hour day, including some observations at 8 or 9 or 10 pm. We might be able to set you up with a plant to take home to study, but growing these indoors in an apartment or house is not easy (J. sinuosa grows really well in a very sunny window in our winter and spring).

You would need to do a literature search to see the published reasons why natural selection may have favored temporary closure of the corolla for the night. Does humidity (dew) go up at night and would high humidity damage pollen or dilute the nectar? Would nocturnal pollinators (moths) removing nectar be less effective for accomplishing cross-pollination than a diurnal animal such as bees?

 

12. Time lapse photography.  One species we are growing angles its stamens out (an attempt at outcrossing) before angling its stamens in for self-pollination. 

Can we capture this floral behavior in time lapse?  Can we make two to three days last only two minutes in a video? 

If one of you has a GoPro you can try.   The following is from Gopro support: “Once recording is stopped the camera will automatically compile the frames into a cohesive video clip that you can playback … on your … computer.”


Also, at iTunes, there is a program called “Lapse It.” 

 

13. If you remove flowers and put them in a vial, and you have an identical vial that is empty, and you blind test someone, can they tell which jar or vial contains the flowers?  

Both vials have to be cleaned at the same time with the same soap and then very thoroughly rinsed, and have the caps removed the same number of seconds before the blindfolded person takes a whiff.

Flip a coin to decide if you will first give the person a smell of the empty vial or the vial containing flowers.  

If yes, and if you use at least five people (more if you get inconsistent results) you can report that the flowers have a scent that is detectable by humans.

 

14. If we have access to Photoshop, there is an amazing technique that I hope someone will try. You take several pictures of the exact same thing, adjusting the focus knob slightly between photos.  Now you have one photo that is in perfect focus up at the front of the object, another photo of the same thing that is in focus in the middle of the object, and a third that is in focus near the back of the object.  Give all the photos to Photoshop and it amalgamates them into one photo, taking only the parts of each that were in focus.   I can’t teach you because I have never done it, but it is explained in a YouTube video. I have the cameras; your challenge will be to follow the instructions in the video.  Search the internet with “focus stacking” and/or “focus stacking in Photoshop.”
Project 14 can’t be your only project this semester, because it is not botany.

 

Keep in mind

We are sharing plants.  A flower is yours if you tag it first. 

Water plants as needed, when you get to the greenhouse. We will have plants that become dry.  We must notice this, look for this, every time you work!  A dry plant will not make much nectar, potentially throwing off someone’s results!

Wet floor, easy to slip and fall!

Remove tags of flowers that are no longer providing data.

You should temporarily place a plant on top of an upside-down pot to raise your plant higher while you work on it, and then when you are done remove the pot.  This makes it easier to see into flowers or pollinate or photograph.

We have to be so very careful about accidentally breaking a branch.  If someone else’s tags are on a branch, and I accidentally break the branch, that person’s experiment is ruined.

We need to know, for any statement you make, your sample size.  If you say “The pistillate phase lasts seven days” you would need to report after your statement, in parenthesis or in your Materials and Methods, the number of flowers and the number of plants.    Keep in mind that cuttings having the same number are not to be considered different plants!   One must say something like, “7 plants, 3 of which were cuttings of the same plant.”

If a plant needs support, please ask me or Dr. Don Blume for help.  A baggy tie (thin flexible metal) is tightened first to the stake then loosely wrapped around a plant, and then the two ends of the baggy tie are twisted to each other. 

On J. sinuosa, you may choose to remove all of the flowers but one from each inflorescence.  If you decide to do this, simply carefully cut (with scissors) the pedicels of the flowers you want to remove.  If you decide to do this, be consistent and do it for all of your observations or experiments.  This makes your life easier for some experiments, allowing you to tag a peduncle, instead of tagging a pedicel.  Watch out: very small buds you did not remove become flowers, and you don’t want to confuse these with the one you are actually considering.

Your lab report, about your research, must be carefully crafted (spelling, organization, proofreading). 

J. calliantha was discovered only recently, by my colleague, and named for the first time in 2010. 

 

 

Statistics

1) State the null hypothesis (H0) and the alternative hypothesis (HA).
Example:

H0: The number of seeds per fruit is about the same in fruits that result from self-pollination and fruits that result from cross-pollination.

HA:: The number of seeds per fruit is different in fruits that result from self-pollination and fruits that result from cross-pollination

Our null hypothesis is that the means of the two groups are equal. Explain this in your own words.

You should report the means in your results.  Also mention the P-value and whether or not you rejected the null hypothesis.

Use the following web site or any statistics software such as Minitab or Sas: http://www.graphpad.com/quickcalcs/ttest1.cfm

2) The software uses our sample data to test whether the null hypothesis is unlikely to be true.

3) If P < 0.01 reject H0
If P > 0.1 do not reject H0
If  0.01 < P < 0.1 the significance is borderline.

The P-value quantifies exactly how unusual the observed results would be if the H0 were true.

The smaller the P value the more unusual the result if, indeed, the H0 is true.

 

 

How to make a cross:

Bag a branch having one or more flowers that will open the next day. The bag prevents pollinators from pollinating the flowers. Skip this step if plants are in a pollinator-free greenhouse.

Use a hand lens, preferably 14X, to view stigma making sure there is no pollen on it (even in a "pollinator-free" greenhouse pollinators occasionally get in). Skip this step if you tagged a flower while closed, covered it with a pollinator exclusion bag the day before, and now it is open.

Remove extra flowers from the inforescence you are working with. Usually this involves leaving one.

Remove anthers (undehisced) from the flower that will receive pollen, making sure you don't break the style (this is called emasculating the flower).

Number a tag (use pencil) and attach it around the peduncle of the flower to receive pollen.

Record the cross, including the date. Number crosses consecutively, starting the numbering over again each year, and recording all crosses in your notebook. I record the state of the flower receiving the pollen (for example, filaments short & anthers undehisced) with a symbol, and the state of the flower providing the pollen. The accession number of the flower receiving the pollen is written first, followed by X, followed by the accession number of the plant providing the pollen.

Apply pollen to stigma, and then view stigma with a hand lens to make sure you have covered the stigma with pollen.