Plant Development Lab Schedule

Thursday 1-5 PM in Hulings 203

 

This is a tentative lab schedule for the term. While we will investigate the questions posed below, some of the approaches may change. At the end of last term the lower level of Hulings Hall flooded and our scanning electron microscope (SEM) drowned (literally). We are working on replacing it (the time frame is a major unknown right now). As an alternative I am working on purchasing a high resolution digital camera to use with a research grade stereomicroscope. We should be able to answer many of the same questions that we would with the SEM.

April 1

How is compound leaf morphology regulated at the genetic level?

  1. Using dissections and microscopy we will follow the initiation and development of wild-type leaves in N. tabacum (simple leaves) and Pisum sativum (compound leaves). This will also provide you with a review of basic plant structure.
  2. Gene interactions will be investigated by analyzing the F2 crosses leaf morphology mutants and wild type including the cross AF TL ST with af st tl. af (afila) mutants convert leaflets to tendrils, while tl (tendrilless) converts tendrils to leaflets. st (stipules reduced) results in very small stipules. We have five benches of segregating plants from five different crosses. Each group will collect information on the phenotypes of the plants in one group. We will share our data and try to determine the genotypes of the parental lines for all five crosses.

*At the start of lab on April 8, please turn in your answers to the five crosses and your reasoning (a brief paragraph for each cross is more than sufficient, 10 pts.).

April 8

Are there genes that affect stipules, but not the compound leaf or the compound leaf but not the stipules? [A written answer to this question with data is due April 15, 10 pts]

  1. Several mutant alleles of the gene COCH (COCHLEATA) that appear to control stipule development will be compared with wild type. We will determine whether or not it alters compound leaf development in any way. Cross sections of the base of stipules and leaflets will be compared using a phloroglucinol stain to focus on vascular patterns. Our leaf area meter will be used to compare area of stipules and leaflets. The affect of position on the complexity of the leaf will be investigated.
  2. The uni (unifoliata) muatant is reported to convert compound leaves to simple leaves. We will determine whether or not it alters the stipules in any way. Cross sections of the base of stipules and leaflets will be compared and our leaf area meter will be used to compare area of stipules and leaflets. The affect of position on the complexity of the leaf will be investigated.
  3. The mutation st (stipules reduced) that you saw last week, has its primary affect on the stipules. We will determine whether or not it alters the compound leaf in any way. Cross sections of the base of stipules and leaflets will be compared and our leaf area meter will be used to compare area of stipules and leaflets. The affect of position on the complexity of the leaf will be investigated.

April 15

Do regions of stipules and the compound leaf have specific homologies? [Turn in a written answer to this question with supporting data on April 22, 10 pts.]

  1. We will examine the interactions between the mutation sinuate (sil) which causes wavy leaf margins and the leaf morphology mutants you examined in week one. Look for specific effects of sil on the tips of the stipules.
  2. We will examine the interactions between sil and one or more of the coch mutants with a focus on both the tips of stipules and the overall stipule morphology. In both parts one and two you can utilize cross sections and phloroglucinol staining to see if the vascular patterns of the tips are altered.

April 22

Are there developmental, morphological, and/or evolutionary homologies between stipules and sepals? [Turn in a written answer to this question with supporting data on April 29, 10 pts.]

  1. Several coch mutants, as well as uni, affect both leaf and flower development. Use cross sections and glue imprints of abaxial and adaxial sides of the sepals and stipules of these plants to make comparisions between the two organs and also between the mutants and wild type plants. It will also be useful to compare the areas of stipules and sepals in different genetic backgrounds using the leaf area meter.
  2. Using the techniques described in part one, compare the sepals and stipules of wild-type plants, st plants, pim (proliferating inflorescence meristem), and pim st plants. The pim plants provide a useful contrast with the coch and uni plants because the mutation alters floral meristem development, but not leaf development.

April 29

How do I use the scanning electron microscope (SEM) to gain more detailed developmental information on the floral development in the mutants we have used this term?

  1. Today you will learn how to prepare specimens for observation with the SEM.
  2. Using specimens that are already prepared for you, we will go over the functioning of the SEM and how to grab digitized images that you may use in your final presentation.

May 6

What should I do for my independent research project?

 

For the rest of the term you will be working in small groups on a project using the mutants and methods that you have already been introduced to. During our last lab meeting, May 27, each group will use PowerPoint to present their findings. This is an opportunity to blend literature research and actual experiments. I would encourage you to look at your projects with some focus on how floral developmental pathways have evolved. We have plants ready for you to work with which places some limits on the types of experimenting you can do. Here are some sample questions. While this is an independent project, I expect you to arrive at the start of each lab meeting and confer with me about your project. I will also have lots of suggestions for you about readings for the projects you are working on that you can find on the library shelves or borrow copies from me.

  1. Not all coch mutants affect flower development. Can the effect of coch on floral development be influenced by gibberellins? We have set this experiment up for you by crossing coch into a line that produces more gibberellin. You may also want to try grafting these mutants onto wild-type plants to see if you can alter floral development through a graft transmissible signal. Do consider the appropriate controls in this case. Gibberellins do activate the homolog of the UNI promoter in Arabidopsis . You might want to read the article by Blazquez, M.A., R. Green, O. Nilsson, M.R. Sussman, D. Weigel (1998) Plant Cell 10: 791-800.
  2. We have characterized the pim mutant which has aberrant floral branching patterns, but do not know much about the pattern of floral development in uni flowers. This would make a great project as a comparison to pim . There is another allele of uni, uni-tac that alters leaf development but not flowers. You will want to consider this phenotype in developing a model for the role of uni in pea development. We do know that pim uni doubles have very leafy flowers and that pim coch doubles do not flower. This information might also be useful if you choose project three.
  3. The primary effect of coch and uni on leaf development is complementary, and both affect floral development. What would you predict to be the phenotype of the double mutant? We will have plants available for you to characterize. It will be important to interpret your results in terms of whether or not coch and uni are functioning in the same flowering pathway.

Your independent project is worth 60% of your grade for lab. This includes giving a group PowerPoint presentation and putting your PowerPoint folder into the class folder on Fabio. I will make arrangements with each group about how they want to approach the project and will recommend turning in different written and visual documentation over the course of the project for credit rather than just relying on your final presentation.

In preparing your talk, you may find it helpful to check out Sean Fox' "An Introduction to Using Power Point "

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