Radish genetics I: plant parentals

Overview

In this exercise, we will work as a class to investigate whether stem coloration in radish hypocotyls (stems of recently germinated seeds that are between the embryonic leaves and roots) follow rules of Mendelian inheritance. To investigate this question, we are starting with true-breeding families of seeds (called lineages or lines). In a true breeding line, all of the seeds are genetically identical for the trait of interest. One line is true breeding for purple hypocotyls. The purple color is caused by the presence of a reddish-purple pigment called anthocyanin. The other line is true breeding for the absence of anthocyanin in the hypocotyl. As a result, stems in this line appear green or yellow instead. Our goal is to determine whether production of anthocyanins in hypocotyls is a dominant or recessive trait. We will investigate this by looking at traits of 3 generations of radish seeds.

In the paper "Genetics" bag in your lab kit, you have 4 different types of seeds on paper discs. Two of the discs contain seeds labeled P1 and P2 and you will be starting only these two today. These are the parental generation, and each is a different true breeding line. Before the semester began, plants with both of these traits were already grown to flowering, and crosses were made between the P1 and P2 lines by transferring pollen from flowers of one plant to stigmas of the other type. The seeds from these crosses were collected and represent the offpsring, called the first filial generation (F1). All seeds in this generation have one gene copy from one parent that had a purple stem and one gene copy from the other parent that had a green or yellow stem. By definition, a dominant trait is one that appears in individual that has two different forms of the gene. In contrast, a recessive trait is one that can be present in a true breeding line, but that is not expressed when an individual has one copy of that form of the gene and one copy of a different form of the gene. The F1 plants have two different forms of the gene, one form from each parent. Thus, depending on the stem colors of the F1 plants, you should be able to figure out which form of the gene is dominant and which form is recessive.

Before the semester began, F1 seeds were planted and grown to flowering. Then pollen from F1 plants was used to pollinate flowers on other F1 plants. Seeds were collected from these crosses and represent the second filial (F2) generation, or offspring of the F1 plants. You have F2 seeds in your kits as well. As we will study later in the semester, we expect a particular ratio of F2 plants with each of the two parental traits, depending on which trait is dominant and which is recessive. Later in the semester, we will determine the ratio of purple stems and green/yellow stems in the F2 generation in order to further test which trait is dominant and which is recessive.

Even though presence of anthocyanin is partly under genetic control, it is also influenced by the environment. Anthocyanin is made to help the plant deal with bright light, much in the same way that we put on clothes or sunscreen to protect oursevles against the light. In dark areas, plants will not make anthocyanin whether they have the gene copy to make it or not. Thus it is important in our experiment that the petri dishes you use are placed in a very bright location.

A diagram of the crossing scheme is shown here:

P1 X P2

no anthocyanin with anthocyanin

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F1

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F2

Raddish genetics quiz

Students next complete a Blackboad quiz on the background material to make sure they've read and processed it. Once completed, they are allowed to continue to next part of assignment.

Growing radish seedlings

Locate the "Genetics" bag in your supplies. You should have 4 petri dishes with labeled sheets of paper. There are seeds embedded in the paper. Each sheet should specify P1, P2,, F1 or F2. Today, we will only grow the two dishes with parental seeds. As explained in the overview, these are the dishes labeled P1 and P2.

1. Use your permanent marker to label the outside of each petri dish with the same label as on the paper (in case the paper becomes illegible).

2. Hold each petri dish on it’s side, and use your ruler to measure 1 cm up from the bottom. Draw a horizontal line with your permanent parker. You will stand your dishes up in a bowl or saucer, and this line will be the depth to which you should fill your dish. If you have half-sheets of paper in your dishes rather than full sheets, then arrange the paper inside the dish so that part of the paper is below the line and most of it is above the line. This arrangement is necessary because the underwater seeds will probably not survive, but part of the paper needs to be immersed in water in order to pull water upward to the seeds above.

3. Open each dish (only the P1 and P2 dishes today!) and drip water onto the paper until it is soaked.

4. Close the dishes and stand them up in the provided bowl with part of the paper below the water line. Fill the dish with water only to the fill line you just drew.

5. Place the dish under bright direct light, . Anthocyanin is produced as a response to high light levels, so if light levels are too low, all stems will look yellowish or greenish, despite their genotype. We want to avoid that problem. You can put them under a desk lamp with a compact fluorescent or LED bulb.

6. Photograph the two petri dishes P1 and P2 standing in a bowl with water in a window (best), outside or under a lamp. In your photo, we should be able to see the window or other light source AND that part of the paper is below the water line and most of it above. You will turn this in to the genetics Padlet linked here.

7. Keep the bowl filled to the appropriate depth. Seeds should germinate and grow to a size where stem color can be determined in about 3-4 days. You will post photographs once they are ready to do so.

[Note to instructors: materials may be purchased as kit from Carolina Biological]

See also follow-up activities over next 4 weeks.