1. Lesson 6 Propagating Plants by Tissue Culture

2. Next Generation Science/Common Core Standards Addressed! zHS ‐ 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.] zHS ‐ 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. Bell Work! z1. Discuss the importance of tissue culture z2. Discuss a tissue culture method of propagation used in the greenhouse industry z3. Where can tissue culture be applied in the commercial field of agriculture? z4. Discuss what type of plants are best suited for tissue culture propagation

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

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

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

7. Advantages of Tissue Culture z1. Large numbers of plants can be produced from a single plant in relatively small space in a short period of time yThis reduces growing space, labor and plant maintenance requirements

8. Advantages Continued z2. Viruses and other systemic diseases are eliminated by propagating the quickly dividing cells of the shoot tip z3. The grower is able to produce plants with identical flowers z4. Horticultural cultivars can be improved by selecting plants, which vary slightly from the mother plant yExamples are leaf shape, disease resistance, growth habit and flower color z5. The growth of identically engineered plant cells

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

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

11. zB. Stage two - the cells of the explants multiply in one of two ways: y1. The cells may form a callus, which is a group of cells with no particular function xSupplied with the correct hormones in the medium, these callus cells can develop into a normal plant y2. The explant may produce many new explants if cytokinins, hormones responsible for cell division and differentiation, are placed in the medium

12. Stage Two: Explant Multiplication

13. yCytokinins encourage the increase in the number of buds on the explants to six or more per shoot xEach bud is capable of becoming a plant and producing more buds yBranching occurs as these buds develop into plant shoots, or plantlets xThese plantlets are divided and transferred to new containers yIn this way, a single explant can produce millions of plantlets in a year

14. zD. Stage four - the plantlets are removed form the glass container zC. Stage three - the plantlets have developed and are ready for root formation yShoots are transplanted to another medium containing auxins, a hormone that induces the growth of roots yThe plantlets are also given higher light intensity in preparation for stage four

15. yThey are divided, planted in a sterile medium, and placed in a greenhouse yCare must be taken during this transition to acclimatize the plant to their new environment

16. Tissue Culture Process Continued zOne of the most important aspects of tissue culture is sterile technique zSterile technique is the maintenance of an environment that is free of bacteria, fungi and viruses ySterilization of the agar media is essential yIn addition, the slightest air movement can stir spores of bacteria and fungi

17. zSpecial sterile work stations, called laminar hoods, are used when possible

18. zCleaning 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 zThe tissue culture agar medium and other materials used to prepare and place the explant must be sterilized yThis is usually done by an autoclave

19. zThe autoclave uses pressurized steam to sterilize medium, glassware, and instruments

20. zCultures are transferred from one container to another at various stages in their development yThis transfer must occur under sterile conditions to prevent contamination by microorganisms ySterilized equipment must be used for each transfer

21. Summary zWhy is tissue culture important in the horticulture industry? zWhat parts of a plant can be used in tissue culture? zDefine explant. zGive two advantages for using tissue culture. zWhat is a sterile agar medium? zWhat is the first stage in the tissue culture propagation method?

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

23. The End!