Colorado Agriscience Curriculum Section: Plant Reproduction Unit 4: Plant Reproduction & Genetics Lesson Number 6: Asexual Propagation III Colorado Agricultural Education Standards: AS 11/12.4 The student will demonstrate an understanding of physiological processes in agriculturally important plants HRT 11/12.2 The learner will understand and describe the anatomy of horticulture plants. Colorado Science Standards: SCI 1.1 Asking questions and stating hypotheses, using prior scientific knowledge to help guide their development SCI 3.1 Students know and understand the characteristics of living things, the diversity of life, and how living things interact with each other and with the environment. SCI 3.3.5 Using examples to explain the relationship of structure and function in organisms. SCI 3.3.6 Describing the pattern and process of reproduction and development in several organisms. Student Learning Objectives (Enablers) As a result of this lesson, the student will … Objective 1: Understand the types and application of grafting. Objective 2: Understand tissue cultures and their application. Time: Instruction time for this lesson: 55 minutes. Tools, Equipment, and Supplies Power Point or Colored Overheads Agriscience: Fundamentals and Application Text Key Terms Grafting Stock Root Stock Graft Union Scion Bud Grating T-Budding Budding Rubber Tissue Culture Agar Interest Approach Much of the seed stock that is used to produce potatoes is replaced with better cultivars over a period of a few years. Farmers who produce seed potatoes are constantly seeking plant that are resistant to diseases and pests. Individual potato plants that are identified in the field as being superior to other potato plants are often selected as parent stock. From the plant materials obtained from these plants, many new potato plants are cloned. These valuable young plants are initially raised in a greenhouse environment, and the supply of seed potatoes is expanded in the field until an adequate supply is available for commercial planting. The seed potatoes are then cut into pieces for planting. A field of potatoes is a perfect example of massive cloning efforts that have been advanced to a commercial scale of operation. How many of you know what cloning is? What is the result of cloning? Do you think we can clone in this agriscience class? Today, we are going to explore grafting and tissue cultures. This information will build the ground work we need for tomorrow. Because, tomorrow we are going to complete a lab on cloning African violets or potatoes. So let’s begin exploring the fascinating world of grafting, tissue cultures, and plant cloning. Summary of Content and Teaching Strategies Objective 1. Understand the types and application of grafting. Slides 1 – 7 Slide 2: Grafting is a procedure for joining two pant parts together so they grow as one. This method of asexual propagation is used when plants do not root well as cuttings, or when the root system is inadequate to support the plant for good growth. Grafting will allow the production of some unusual combinations of plants. For instance, several varieties of apples can be grown on one tree. Some nut trees can be made to grow varieties other than their own. Some unusual foliage plants can also be made by grafting. Finally, dwarf fruit trees are created by grafting regular varieties on dwarfing root stock obtained from related trees with similar, yet different genetics. Slide 3: The top part of the plant that is to be propagated is called the scion. The rootstock, or stock, will be the new plant’s root system and will supply the nutrients and water. The graft union is where the two parts meet. Slide 4: To ensure successful grafting, the following conditions are necessary. One, the scion and the rootstock must be compatible. Two, each must be at the right stage of growth. Three, the cambium layer of each section must meet. And fourth, the graft union must be protected from drying out until the wound has healed. There are many types of grafts. Some common grafts are the whip, or tongue graft, bark graft, cleft graft, bridge graft, and bud graft. Each type is used for a special purpose. Lets take a look at two types, the T-graft and the whip graft, so that we may have a better understanding of the process of grafting. Go through slides 5,6,&7 with the students. Explain each step and have them duplicate the diagrams in their notebooks. Objective 2 Understand tissue cultures and their application. Slides 8 – 13 Slide 8: A relatively new method of plant propagation is micro-propagation, or tissue culture. Instead of using a large part of the plant as in other types of vegetative or asexual propagation, a small and actively growing part of the plant is used. The result is that many new plantlets ma be obtained from a section of a leaf. The process must be done in a very clean atmosphere, and it is not successful in the greenhouse or other traditional propagation areas. Tissue culture requires the use of sanitary conditions. There are many commercial tissue-culture laboratories currently producing a large variety of plants. The greatest advantage of tissue culture propagation is that numerous plants can be propagated from a single disease-free plant. Plants can be propagated more efficiently and economically than with traditional methods of asexual reproduction. The main disadvantage is that the work area must be very clean. All of the equipment must be sterile. In commercial production by tissue culture, there is more expense in equipment and facilities than there is with traditional methods of propagation. Slide 9: The materials necessary for tissue culture are; a clean, sterile area in which to work; clean plant tissue; a multiplication medium; a transplanting medium; sterile glassware and tools; a scalpel, razor, or an X-acto knife; and tweezers. Slide 10: The first step in preparing for tissue culture is to prepare the medium in which the tissue will grow. The medium is called agar, which is available commercially from many of the scientific supply houses. The Virginia Cooperative Extension Service offers the following formula and procedure for preparing media for experimentation in tissue culture on a small basis. Use a quart jar to mix the following materials: 1/8 cup of sugar; 1 teaspoon of soluble, all- purpose fertilizer, The label will indicate that all of the major and minor elements are present, it is especially important that the soluble fertilizer contain ammonium nitrate, If it is lacking, add 1/3 teaspoon of 35-0-0 soluble fertilizer; 1 tablet (100mg) of inositol (myo-inositol), this can be obtained from most health food stores; ¼ of a pulverized tablet containing 1 to 2 mg of thiamine; 4 tablespoons of coconut milk, the source of cytokinin, this is obtained from a fresh coconut and freeze the remainder for later use; 3 to 4 grains of rooting hormone containing 0.1 active ingredient Indole-3-butyric acid (IBA0; fill the jar with purified, distilled, or deionized water; finally shake the jar to dissolve all ingredients. Slide 11: After the medium is dissolved, prepare the culture tubes using either test tubes with lids or other suitable glass containers. Fill the culture tubes one quarter of the way with sterile cotton balls. Use one or two per tube. They do not need to be packed tightly. Pour the prepared medium into the culture tubes to just below the top levels of the cotton. Place the lids on loosely. After the medium is placed in culture tubes, it is ready to be sterilized. Sterilization may be done in two ways; one, heat in a pressure cooker for 30 minutes; or two, heat in an oven for four and half hours at 320 degrees. After they are removed, place the culture tubes in a clean area and allow them to cool. If several days will go by before using all of the tubes, wrap them in small groups in plastic wrap or fail before sterilizing. The tools and equipment used for tissue culture must also be sterilized. This can be done as the medium is sterilized by placing the tweezers, razor blade scalpel, or knife in the pressure cooker or oven. After the initial sterilization, they may be cleaned by dipping them in alcohol before and after each use. The work area must be thoroughly cleaned and sterilized. Wash the area with a disinfectant. Keep a mist bottle filled with a mixture of 50 percent alcohol and sterilized water to spray work areas and tools, as well as the hands and arms of the propagator. Slide 12: After the growing medium is properly prepared and cooled and the work area properly cleaned, the next step is to prepare the plant tissue. Various parts of the growing plant may be tissue cultured. For the production of vigorous plantlets, use only actively growing portions. With some species of plants, only a small, quarter inch square section of the leaf is used, where as for others, a half-inch of the shoot tip is used. With ferns, a quarter inch of the tip of the rhizome is used. Remove the parts of the plant to be used and discard the excess plant material. Submerge the plant part in a solution of one part commercial bleach and nine parts water for ten minutes. This will disinfect the pant tissue. Remove the tissue with sterile tweezers and rinse the material in sterile water. When the plant part has been disinfected in the bleach solution, it can be handled only with sterile tweezers and must not touch any non-sterile surface. When the plant material has been disinfected and rinsed, remove any damaged tissue with a sterilized scalpel or razor blade. Remove the lid from a properly prepared culture tube or jar and place the plant material on the agar. Take care that the plant material is not completely submerged. Recap quickly to avoid contamination from the air. It is best if the material is placed in front of the propagator so that work is not done over the uncapped culture tube. It is important that all work and transferring of materials be done quickly and in a clean environment. Scrub all areas with disinfectant and clean all tools with a disinfectant solution. Any contamination may lead to unsuccessful work. Bacteria and fungus will grow in unclean culture tubes and will overtake the new plant growth. Slide 13: After all plant material has been cultured, put the cultures in an evenly warmed (70-75 degrees F) and well-lighted area. The plant tissue will NOT do well in direct sunlight. If any contamination has occurred, it will be evident in 48 to 96 hours as mold or rotting on the medium. If contamination occurs, remove the contaminated tubes and wash them for reuse. When the plantlets have grown to a satisfactory size, take them out of the culture tubes and transplant them into a good growing medium. Remember, the plantlets are fragile, so handle them carefully. As each plant is removed from its culture tube wash the plant thoroughly and transplant it into its own culture container. When the plant is well established in it own culture container with viable roots and top growth, transplant the plant again into a suitable potting mix. Place the plant inside a protected area with high humidity. The plants are coming out of a well- protected environment with plenty of humidity and light. After they have adapted to the pot and are growing well, they may be treated like any other growing plant. This process will take about three to six weeks from the beginning to a successfully growing plant. Review/Summary Grafting is simply joining of two plant parts together so that they can grow as one. This process is used when plants do not root well as cuttings and it allows for some unusual combinations such as several colors of roses on one plant. The scion is the top part of the plant that is to be propagated while the rootstock is the bottom of the plant that is to be propagated and the graft union is the portion where the two meet. Some conditions that must be present for grafting to be successful are; scion and rootstock must be compatible; each must be at the right stage of growth; the cambium layer of each must meet; and the grafting union must be protected to prevent drying out. Tissue cultures are another way of producing plants asexually. In a tissue culture a small and actively growing portion of a plant is used. Many plants can be produced very quickly but extremely sterile conditions are required for it to be successful. Tissue culture utilizes a growing medium called agar and is started in test tubes. Application Extended classroom activity: Conduct an experiment in tissue culturing utilizing the information of this lesson. If it is spring, softwood cuttings and grafting could also be done. FFA activity: Use LifeKnowledge AHS 12 to illustrate the following. Although tissue culture is now a common form of plant reproduction, at one time it took some very forward thinking scientists to conceptualize the usefulness of this technology. By using the LifeKnowledge lesson, help students realize that their concepts and ideas may someday become standard practices. SAE activity: Research careers in the field of horticulture and floral culture. See what careers are available in the areas of tissue cultures and grafting. What are the educational requirements, what is the starting pay, what is the top end pay?
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