PLB 105 Developmental Plant Biology

CONTENTS:

• Study Objective List 5
• Laboratory 8 - Root Apical Organization and Primary Tissue Differentiation
• Laboratory 9 - Root Primary Growth, Lateral Root Initiation and Special Roots
• Laboratory 10 - Initiation of Secondary Growth in Roots and Anomalous Secondary Growth

Roots Read:

Fahn, Chapter 13

Secondary Growth Read:

Fahn, Chapters 14-16

Unusual Secondary Growth

Fahn, Chapter 17 (material on roots)

Periderm Read:

Fahn, Chapter 18

Student will be able to:

1. Describe several specific functions of roots. Be able to relate these functions to the structure, modifications, or cell types which make a particular function possible (e.g. storage functions in Daucus root facilitated by secondary growth with an abundance of storage parenchyma).

2. Recognize and describe the occurrence, structure, and function of the following:
   1. Tap root
   2. Fibrous root
   3. Adventitious roots
   4. Rhizome
   5. Lateral root
   6. Contractile roots

3. Diagram a generalized median longitudinal section (MLS) through a root tip. Draw the pattern of meristematic and derivative cell distribution for the following apical meristem types, and be able to discuss each in detail.
   1. Apical Cell Theory (Be able to discuss the concept of a merophyte as it applies to tissue development in Azolla roots.) (See figure 5-3)
   2. Histogen Theory (figures 5-1 and 5-2) Fully discuss the idea of a development spiral in roots with closed apical organization. Note the exercise in Laboratory 8.
   3. Discuss the idea of roots with closed and open apical organization. How are these organization patterns different?

4. Discuss the concept of a promeristem and quiescent center as proposed by Clowes.
   1. Describe the characteristics, structure and possible functions of the quiescent center.
   2. Describe the positions and functions of the proximal and distal meristems.

5. Diagram (MLS) a closed or open apical organization root. Show where the quiescent center and other components of the promeristem are located. Extend the diagram to show the positions of the primary meristems and mature tissues which form from them.

6. . Discuss the relationship between cell division, cell elongation and cell differentiation in a root tip (see the review paper by Rost [1994] in room 291 or on library reserve).
   1. Discuss the distribution of cell division in a root tip and differentiate between the terms "formative division" and "proliferative division".

7. Describe the root cap: include its structures, functions and role in perception of gravity.

8. Describe the structure, function and occurrence of the exodermis.

9. Describe and discuss the structure and function of the root cortex; include the innermost layer-the endodermis-and its special structure- the casparian strip.

10. Describe passage cells as they occur in both exodermis and endodermis.

11. Describe and recognize the pericycle in roots. Also, list the possible functions of the pericycle.

12. Discuss the direction of primary xylem and phloem initiation and maturation in roots. Define the term "stele" or "vascular cylinder."

13. Recognize, draw and describe the basic differences between roots of monocot and dicot plants.

14. Define:
    1. Diarch
    2. Triarch
    3. Tetrarch
    4. Polyarch

15. Describe the sequential formation of lateral roots---Initiation, Organization and Emergence. Also, discuss the factors which may influence the formation and distribution of lateral roots.

16. Describe and discuss the initiation of vascular cambium and secondary growth in roots.

17. Describe two examples of anomalous secondary growth in roots, indicating positions of vascular tissues and cambial layer(s).

18. Describe the formation of phellogen, phellem, and phelloderm in roots.

19. Discuss the differences between secondary growth in roots of dicots and monocots in terms of frequency of occurrence and types of meristems involved.

20. Diagram a root to show the precise path for the translocation of water through the cell layers of the root.

21. Discuss the anatomy and functions of the following roots or root structures:
    1. Endotrophic and ectotrophic mycorrhizae.
    2. Haustorial roots from parasitic plants.
    3. Root bacterial nodules.
    4. Actinorhizal roots.

22. In a series of transverse section diagrams, be able to show the sequential changes in a dicot root from the root cap through the initiation and growth of vascular cambium and phellogen.

23. Discuss the differentiation of adventitious roots.

1. Apical Organization.

   After reading the text material and reviewing your lecture notes on root apical meristem organization, it should be clear that no single concept of apical organization covers all the possibilities of cell organization found in roots. It should also be clear that some concepts (Apical Cell Theory and Histogen Theory) are not accepted as theories applied to all plant groups. Many plant anatomists, however, still analyze root apical meristems by the number of initial layers present and by the tissues which differentiate from them. It is useful, therefore, to look at several examples of root apical meristems and to attempt to analyze their organization critically using the theories available.

   For each root tip provided, determine the type of organization present by carefully examining a median longitudinal section (MLS) of a root tip. Some of the slides contain serial sections, so you will have to find the MLS by examining several sections. Note also the tissues which differentiate from the tiers or zones and make a diagrammatic drawing of each example. Determine in each instance if the root apex has open or closed organization.

   1. Allium cepa (onion-monocot)-type of open apical organization.

   2. Pisum sativum (pea-dicot)-type of open apical organization.

   3. Raphanus sativus (radish-dicot)-Closed apical organization- Most common histogen type in dicot plants-three initial tiers which give rise to 1) root cap and epidermis, 2) cortex, and 3) vascular cylinder. The idea of developmental spirals in roots with closed apical organization is completely new. Study figures 5-1 and 5-2. In figure 5-1 you will note that roots have the following levels of organization: cylinders, sectors, vertical cell files and cells. The accompanying diagram shows a developmental spiral made by connecting the T-junction cell divisions which occurred on the periphery of the histogen tier for the epidermis and root cap. The spiral diagram is for Arabidopsis roots, but it also explains the organization of many other plants with closed apical organization.

   4. Zea mays (corn-monocot)-Closed apical organization- Three tiers of initials giving rise to: 1) stele, 2) cortex and epidermis, and 3) root cap.

   5. Small seedlings of radish, pea, or a grass will be provided. Place the entire seedling on a microscope slide, crush it without smearing it, and stain with I2KI. Identify starch grains, especially in the root cap.

   6. "Typical Fern" - This prepared slide shows the apical cell organization. Identify the apical cell, first derivative cells and merophytes. (Another example of a type of closed apical organization)

      Working with your laboratory partner, make a drawing of a L.S. view of a fern root in median section. Label the apical cell and the different stages of merophytes. Compare what you see to the diagram in the laboratory manual (figure 5-3) and to the electron micrographs of Azolla fern roots. After you sort out the idea of merophytes, turn around and explain it to the person behind you.

   7. Pinus (pine) - Conifers generally have an open apical organization.

   8. Examine the demonstration slide (if it isn't clear on the microscope slide, look at slide #50 in the slide review module to see this) to show the position of the quiescent center in a root. Define these terms: quiescent center, distal meristem, proximal meristem, autoradiography.

2. Primary Growth Initiation and Maturation
   1. Pea seedling primary root - Make T.S. and stain with phloroglucinol or toluidine blue.

      1) Section as near the tip as possible. No differentiated cells should be apparent, although the outline of the stele may already be visible. Root cap may surround the root tissues.

      2) Section 1-2 cm basal to the apex. This region should contain mature protoxylem elements. Is the metaxylem visible? Identify the phloem, pericycle, and endodermis.

      3) Section 1-2 cm below the cotyledons. In what stage of development is the vascular tissue in this region? Do you see any cavities in the center of the root? Compare all three stages and see if you can mentally construct the entire root 3-dimensionally.

   2. Ranunculus sp. (buttercup-dicot)-Examine the slides of young and mature root T.S. The young root section has differenti-ated protoxylem. Note all cell types and tissues and compare with the same cells and tissues in the mature root. Make a drawing of each and label completely.

   3. Pyrus root - T.S. of developmental stages-root tip, protoxylem, maturing metaxylem, and mature root. Examine these slides comparatively and carefully follow the changes in each tissue.

   4. Smilax sp. - young and mature root T.S.-This monocot root has a multiseriate pericycle. Note the differentiation of cell types and tissues by comparing the young and mature sections. Make a drawing of each and label completely.

   5. Asparagus officinalis root T.S.-This is an example of a monocot root. Note the polyarch vascular cylinder, and be able to identify all cell types and tissues. The cortex in this root contains cells with bundles of raphide crystals. Can you identify the thick-walled cell layers on the periphery of the root? What is the cell layer just inside the thick-walled layers?

   6. Convolvulus sp. root (Morning glory)-Examine T.S. of young roots in the prepared slide provided. Note the excellent casparian strip in the endodermis. Be sure that you understand the function of this cell wall structure.

1. Mature Primary Roots

   The following series shows the major features of dicot and monocot roots. Pay particular attention to the organization of tissues within the stele (e.g. diarch...polyarch).

   1. Ranunculus (buttercup-dicot) mature root -- Note the conspicuous "passage cells" in the endodermis opposite the pro-toxylem poles. Protoxylem poles may vary from triarch to pentarch.

   2. Zea mays (corn-monocot) -- Protoxylem and phloem are on alternate radii, but metaxylem and phloem may be on the same one.

   3. Triticum (wheat-monocot) -- A monocot with no pith; make T.S. and stain. Also examine the prepared slides of mature and im-mature roots.

   4. Epidendrum (monocot-orchid) -- Cut a T.S. of this aerial root. Note green color and the velamen. Look carefully at the cell walls of the velamen; is there anything peculiar about them? The stele is typical of monocot roots. Look at prepared slides also, and identify the endodermis and exodermis. Notice that the secondary cell walls on these cell layers form only on three sides. Compare this to the demonstration slides of Vanilla orchid roots. Note the conspicuous exo- and endodermis in the older tissues. The exoder-mis sometimes has a casparian strip.

   5. Myriophyllum -- Examine prepared slides (T.S.) of rhizome for casparian strips.

   2. Lateral Root Formation
   1. Eichhornia (water hyacinth)-L.S. and T.S. prepared slides. Emerging lateral roots are seen. Follow the endodermis and pericycle and note what happens to them in the vicinity of the branch roots.
   2. Salix (willow) prepared slide-(Demonstration). Note the already emerged lateral roots. Consider this method of emergence. Do you see any flaw in this procedure with reference to the plant's ability to protect itself against potential pathogens?

   3. Vitis (grape) prepared slide-(Demonstration). Another example of lateral root formation.

   4. Pisum sativum (pea) prepared slide-Sectioned to show the origin of lateral roots.

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2. Experiments on root branching

   The TA will divide the laboratory into groups to conduct 3 different experiments. Each group will run their experiment for 1-3 weeks. During that time, you will collect data on the experiment and at the end, you will present your results to the class.

   1. Monstera group. You will be given a Monstera plant which has several aerial roots. Your objective will be to determine the effects of the contact medium on the branching behavior of these roots. (a) Figure out some way to have the aerial roots grow into soil, and some other liquid or solid medium. (b) After some period of time (you will design the experiment, but consult with your proctor), remove the root and determine if it has branched or not. Be sure to allow some aerial roots to remain as controls growing in air. (c) What would happen if you simply touched the aerial roots some other way? (d) When you complete the experiment you will have to think of how whatever happened was regulated.

   2. Pea group #1. You will be provided with sterile pea seedlings which are 3-5 days old. You will also be provided with a root growth apparatus that will allow you to grow seedlings in small chambers connected to an air line. Carefully excise the tips of the roots at different distances from the tip (1/4 mm to 2 mm). Then place the seedlings into the chambers and allow them to grow for about 1 week. At the end of that time you will collect the samples, section them and analyze their branching pattern. Do not forget to set up some un-treated controls. Be prepared to fully explain what you see in terms of the source of laterial roots, the angle of lateral root emergence, and how lateral root initiation and growth is regulated.

   3. Pea group #2. You will also be provided sterile pea seedlings plus some petri dishes which contain sterile agar, some of which contains the auxin transport inhibitor, triiodobenzoic acid (TIBA). Excise the root tips at different distances from the tip, and excise various other parts of the seedling. On some of them you should apply the TIBA agar blocks to various parts of the seedling. Be sure that one of your treatments involves excising the root tip and applying the TIBA at the base of the root, near the cotyledons. Allow these seedlings to grow for a week and then analyze the results. Your TA will explain how to keep these treatments sterile. Be prepared to fully explain your results.

1. Secondary Growth in Roots and Its Initiation
   1. Medicago (alfalfa)-Each slide contains four developmental stages, from just above the apical meristem to early secondary growth from vascular cambium. Trace the pattern of differentiation of proto- and metaxylem and phloem in the primary tissue. Note the direction of differentiation. Find the endodermis and pericycle. The pericycle is the outermost layer of the stele and can easily be located as the cell layer adjacent to the outermost protoxylem element. Carefully follow the initiation of vascular cambium in Medicago. What contribution is made by the pericycle? What is its source between the primary xylem and phloem?

   2. Helianthus (sunflower)-Each slide contains several developmental stages from early primary growth through the initiation and proliferation of secondary growth. Follow these stages from the youngest to the oldest section. Also follow the subsequent progression of the pericycle, endodermis, cortex and epidermis and the formation of the periderm.

   3. Pyrus (pear)-This set of 3 slides (mature root, early secondary growth, and secondary growth) is a third example showing the sequential development of primary to secondary tissues. By this time you should be adept at tracing this sequence and identifying the cell layers involved in the initiation and conduction of secondary growth. Follow the sequence of these slides, and be able to name all the cell types and tissues on each section.

   4. Glycine max (soybean)-Prepared slides (T.S.) with 3 stages of development - immature growth, early secondary growth, and secondary growth. Note the pattern of vascular tissues in each stage, including the status of the pericycle and the initiation of periderm.

   5. Quercus (oak) root secondary growth.

   6. Convolvolus (morning glory). Each slide contains several sections. Examine each section from starting at the top left of the slide with the youngest section and work along to the older sec-tions. Each section shows older tissues through the initiation and development of the secondary xylem and phloem.

2. Anomalous Secondary Growth in Roots
   1. Daucus (carrot)-Make T.S. and stain with toluidine blue. Find the position of vascular cambium, and name the cell types on both sides of it. Carrot is interesting because of the large amount of parenchyma in the secondary tissue.

   2. Beta (sugar beet)-Make T.S. and stain with toluidine blue. These roots have concentric rings of cambium, each containing discrete vascular bundles and parenchyma. Also examine the demon-stration slide of this material.

   3. Ipomoea (sweet potato)-Make T.S. Vascular cambium forms secondary vascular bundles and parenchyma. The secondary vascu-lar bundles in turn form a tertiary vascular cambium around indi-vidual xylem vessels or vessel groups. This cambium produces parenchyma-rich phloem away from the vessels and tracheary ele-ments toward them.

   4. Actaea root-This T.S. slide shows the primary root structure, the origin of vascular cambium, and secondary root structure. These roots are considered anomalous because of the very broad parenchyma rays and the presence of a discontinuous cambium which forms arcs and not a cylinder.

   5. Doxantha root-Secondary growth in this T.S. slide consists of alternating radii of mostly xylem and mostly phloem. The cambium forms at a different level in each arc.

   6. Dracaena draco root-Make T.S. of this monocot root if it's available. This is the only monocot plant with "true" secondary root growth, consisting of irregular arcs of multiseriate pericycle making parenchyma and amphivasal bundles to the inside and parenchyma to the outside. See if you can follow the pattern and make a drawing of it.

3. Special Roots.
   1. Pinus root T.S.-Demonstration slide showing an ectotrophic mycorrhiza. In this example, the fungus forms a weft of hyphae as a sheath around the entire root. Interestingly, roots with ectotro-phic mycorrhizae develop as shortened Y branched roots.

   2. Pisum root nodules-Many leguminous plants have a symbiotic relationship with the bacteria Rhizobium. The bacteria enters the root by penetrating a root hair with a tiny germination thread of Rhizobium. The bacteria then pass through into the cortex of the root and stimulate the proliferation of polyploid cortical cells. The bacteria are within the cells, but are surrounded by an inpocketing of the cell's plasmalemma, so the bacteria are actually outside the cells. Cut a T.S. through one of the nodules, stain with toluidine blue, and examine.

   3. Cuscuta (dodder)-Parasitic angiosperm. Dodder has haustorial roots which penetrate the host plant and tap the vascular tissue. Section and try to determine if the vascular connection is with the xylem, phloem or both. Also, look at the demonstration material on the anatomy of haustorial roots from Phoradendron (mistletoe).

   4. Iris rhizome-These are underground stems which perform the functions of roots. Cut a freehand section. Do you see any characteristics of roots? Stems? Rhizomes frequently have an intermediate organization of tissues.

   5. Zebrina (Wandering-Jew) stem. Cut T.S. of stem at node to include a median longitudinal section through one or more adventitious roots. Stain and determine the point of vascular connection (if possible) and the stelar type of the root. Is the root atypical anatomically? Other examples of adventitious roots, such as Ficus pumila, Monstera deliciosa, and Hedra helix, will be placed on demonstration.

   6. Taraxacum officinale (Dandelion)-Make L.S. of contractile (basal) portion of root; try to determine the mechanism of contraction. Look at "Big" Esau, pgs. 519-521.

   7. Examine the demonstration of the coralloid roots from the Cycad Encephalortus ferox. These roots occur near the soil surface. They are short and Y-branched. A thick layer of the nitrogen-fixing blue green alga Anabaena occurs in the outer cortex of the root.

   8. Examine fresh roots of Alnus (Alder) for actinorhizal nodules on young short roots. These are nitrogen fixing nodules, only the microorganism is the bacterium Frankia.