Lesson 6

1. Lesson 6 Propagating Plants by Tissue Culture

2. Next Generation Science/Common Core Standards Addressed! • HS‐LS1‐1. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells. [Assessment Boundary: Assessment does not include identification of specific cell or tissue types, whole body systems, specific protein structures and functions, or the biochemistry of protein synthesis.] • HS‐LS2‐5. Develop a model to illustrate the role of photosynthesis and cellular respiration in the cycling of carbon among the biosphere, atmosphere, hydrosphere, and geosphere. [Clarification Statement: Examples of models could include simulations and mathematical models.] [Assessment Boundary: Assessment does not include the specific chemical steps of photosynthesis and respiration.] WHST.9‐12.7 Conduct short as well as more sustained research projects to answer a question (including a self generated question) or solve a problem; narrow or broaden the inquiry when appropriate; synthesize multiple sources on the subject, demonstrating understanding of the subject under investigation. (HS‐LS1‐3)

3. Agriculture, Food, and Natural Resource Standards Addressed • PS.03.01.03.a. Summarize optimal conditions for asexual propagation and demonstrate techniques used to propagate plants by cuttings, division, separation, layering, budding and grafting.

4. Bell Work! • 1. Discuss the importance of tissue culture • 2. Discuss a tissue culture method of propagation used in the greenhouse industry • 3. Where can tissue culture be applied in the commercial field of agriculture? • 4. Discuss what type of plants are best suited for tissue culture propagation

5. Terms Cultivar Resistance Explants Auxins Callus Cytokinins Explants Plantlet Sterile agar medium Sterile technique Tissue culture

6. Interest Approach • We’ve talked about how you can produce African violets through leaf cuttings. • But, what if you had to produce 1,000 of them that are identical to each other in a short period of time? • What problems/challenges would this produce for the grower? • How can tissue culture help solve this problem?

7. What Is Tissue Culture and Why Is It Important? • Tissue culture is the practice of growing plant cells on artificial media • It involves the culture or growing of small pieces of plant tissue • It is performed on artificial medium under sterile conditions • Foliage plants, pot plants and cut flowers are propagated by this method

8. Advantages of Tissue Culture • 1. Large numbers of plants can be produced from a single plant in relatively small space in a short period of time • This reduces growing space, labor and plant maintenance requirements

9. Advantages Continued • 2. Viruses and other systemic diseases are eliminated by propagating the quickly dividing cells of the shoot tip • 3. The grower is able to produce plants with identical flowers • 4. Horticultural cultivars can be improved by selecting plants, which vary slightly from the mother plant • Examples are leaf shape, disease resistance, growth habit and flower color • 5. The growth of identically engineered plant cells

10. What Process Is Used For Tissue Culture Propagation? • The tissue culture propagation process can be defined in four main stages: • A. First stage - small pieces of plant material, called explants, are carefully removed form the parent plant • Explants are obtained from the actively growing part (shoot tips, sections of leaves, stems and roots, embryos, etc) of a desired plant

11. The explants are cleaned and placed on sterile agar medium in glass bottles or test tubes • The sterile agar medium is a gel that contains water, sugars, nutrients, and plant hormones to support and promote plant growth • Tiny leaves, stems and roots make tissue culture possible Removing explants

12. B. Stage two - the cells of the explants multiply in one of two ways: • 1. The cells may form a callus, which is a group of cells with no particular function • Supplied with the correct hormones in the medium, these callus cells can develop into a normal plant • 2. The explant may produce many new explants if cytokinins, hormones responsible for cell division and differentiation, are placed in the medium

13. Stage Two: Explant Multiplication

14. Cytokinins encourage the increase in the number of buds on the explants to six or more per shoot • Each bud is capable of becoming a plant and producing more buds • Branching occurs as these buds develop into plant shoots, or plantlets • These plantlets are divided and transferred to new containers • In this way, a single explant can produce millions of plantlets in a year

15. D. Stage four - the plantlets are removed form the glass container • C. Stage three - the plantlets have developed and are ready for root formation • Shoots are transplanted to another medium containing auxins, a hormone that induces the growth of roots • The plantlets are also given higher light intensity in preparation for stage four

16. They are divided, planted in a sterile medium, and placed in a greenhouse • Care must be taken during this transition to acclimatize the plant to their new environment

17. Tissue Culture Process Continued • One of the most important aspects of tissue culture is sterile technique • Sterile technique is the maintenance of an environment that is free of bacteria, fungi and viruses • Sterilization of the agar media is essential • In addition, the slightest air movement can stir spores of bacteria and fungi

18. Special sterile work stations, called laminar hoods, are used when possible

19. Cleaning of the plant before removal of the explant is usually accomplished by a brief soaking in a bleach solution, followed by a rinse in sterile water • The tissue culture agar medium and other materials used to prepare and place the explant must be sterilized • This is usually done by an autoclave

20. The autoclave uses pressurized steam to sterilize medium, glassware, and instruments

21. Cultures are transferred from one container to another at various stages in their development • This transfer must occur under sterile conditions to prevent contamination by microorganisms • Sterilized equipment must be used for each transfer

22. Summary • Why is tissue culture important in the horticulture industry? • What parts of a plant can be used in tissue culture? • Define explant. • Give two advantages for using tissue culture. • What is a sterile agar medium? • What is the first stage in the tissue culture propagation method?

23. Summary Continued • What is a callus? • What must be added to a callus in order for it to continue to develop? • What is a plantlet? • How do auxins help an explant? • What are some practices of sterile technique?

24. The End!