Plant Tissue Culture

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Plant Tissue Culture
Written by Dennis Genovesi Class Coordinated by
Jim Heitholt
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Plant Culture
1. Pots 2. Greenhouse 3. Flower Beds 4. Field
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So, What Is Tissue Culture?
Or in vitro culture? Or micropropagation? Or
more recently Biotechnology?
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Plant Tissue Culture - Definition
Can be defined as the culture on nutrient media
under sterile conditions of plant seeds, organs,
explants, tissues, cells, or protoplasts.
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Characteristics of Plant Tissue Culture Techniques
1. Occur on a micro-scale. 2. Environmental
conditions optimized (nutrition, light,
temperature). 3. All microbes (fungi, bacteria,
viruses, and nematodes are excluded).
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Characteristics of Plant Tissue Culture
Techniques - 2
4. The normal pattern of plant development breaks
down giving rise to callus, adventitious roots,
shoots, and embryos. 5. Ability to grow as single
cells (protoplasts, microspores, suspension
cultures) 6. Plant cells are totipotent, able to
regenerate a whole plant.
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Glossary
1. Adventitious - development of organs or
embryos from unusually sites. 2. Agar - nutrient
medium component derived from algae for
solidification. 3. Androgenesis - haploid plant
production from pollen grains (microspores). 4.
Aseptic - free from microbes, i.e., sterile.
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Glossary
5. Autoclave - apparatus in which media,
glassware, etc. are sterilized by steam under
pressure. 6. Auxins - A class of plant hormones
which induces cell elongation often induces
adventitous roots, and inhibits adventitious
shoots. 7. Callus - actively dividing
undifferentiated cells often developing from
wounding or in tissue culture.
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Glossary
8. Clone - a group of cells, tissues, or plants
which are in principle genetically identical.
9. Contaminant - growth of microorganisms along
side of the plant tissue in vitro.
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Glossary
10. Culture Room - Room for incubating cutlures
with controlled light, temperature, and humidity.
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Glossary
11. Cytokinins - A class of plant hormone which
induce cell division and often adventitious buds
(shoots), while inhibiting rooting.
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Glossary
12. Differentiation - The development from callus
of readily recognized organs such as roots,
shoots, embryos.
high cytokinin, low auxin
equal cytokinin and auxin
low cytokinin, high auxin
cytokinin
auxin
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Glossary
13. Embryoid - Embryo-like structure that is
produced from a somatic cell in vitro. 14.
Explant - Plant part used to initiate a tissue
culture, e.g., embryo, shoot meristem,
microspores, leaf, flower petals, etc. 15.
Flaming - Sterilization of instruments (forceps,
scalpel) by a open flame after dipping in
alcohol. 16. Haploid induction - Stimulation of
plant formation from male or female gametes
without sexual fertilization.
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Glossary
17. Hardening off - Acclimation of in vitro
derived plants to ambient conditions. 18.
Laminar Flow Cabinet - A work space where
sterile air moves to allow the introduction of
explants aseptically. 19. Macronutrients -
Minerals needed in large amounts for normal plant
development (N, P, K, Ca, Mg). 20. Micronutrients
- Minerals needed in relatively small amounts for
normal plant development (Fe, B, Zn, Cu, Mo, Mn).
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Glossary
21. Mutation - Genetic change 22. Nutrient Medium
- Food for the explant which allows growth.
Contains macro- and micronutrients, vitamins,
sugar, and plant growth regulators. 23. Petri
dish - A flat dish made of glass or plastic with
sides turned up and an opposing cover plate. 24.
Solid media - Nutrient media solified with agar.
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Glossary
25. Somaclonal variation - Genetic changes which
occur during extended periods of tissue
culture. 26. Sterile - Medium or object with no
viable microorganism. 27. Subculture -
Transplanting in vitro grown tissues to
additional plates of fresh media. 29.
Totipotency - Each cell of a multicellular
organism contains all of the genes needed to form
the whole plant.
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Tissue Culture Uses
I. Embryo Culture A. Prevent (rescue) embryo
abortion B. Serves as a source of embryogenic
callus.
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Tissue Culture Uses - 2
II. Orchid Seed Culture A. Replace symbiosis
(mycorrhiza) B. Exclude microbes.
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Tissue Culture Uses - 3
III. Meristem Culture A. Elimination of
pathogens B. Vegetative propagation or cloning
(Stages I - IV)
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Tissue Culture Uses - 3
III. Meristem Culture, B. Stages
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Tissue Culture Uses - 4
III. Meristem Culture C. Meristem culture use by
micropropagation in the U.S for 1996.
Crop Type
Number
Percentage
Foliage Plants
64, 000,000
53
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15, 000,000
Trees and Shrubs
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13, 000,000
Vegetables
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Greenhouse Crops
9, 000,000
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Herbaceous Perennials
9, 000,000
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Fruits
4, 000,000
R. Zimmerman, USDA
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Tissue Culture Uses - 5
IV. Suspension Culture A. Cloning B. Creating
Genetic Variants
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Tissue Culture Uses - 6
V. Anther and Microspore Culture A. Doubled
haploid production
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Tissue Culture Uses - 6
V. Anther and Microspore Culture A. Doubled
haploid production
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Tissue Culture Uses - 6
V. Anther and Microspore Culture A. Doubled
haploid production
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Tissue Culture Uses - 6
V. Anther and Microspore Culture A. Doubled
haploid production
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Tissue Culture Uses - 6
VI. Protoplast Culture A. Somatic
Hybridization B. Transformation
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Tissue Culture Uses - 6
VII. Genetic Engineering A. Gene gun B.
Agrobacterium C. Protoplasts
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Tissue Culture Uses - 6
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What Kind of Working Space Is Needed for an
Efficient Tissue Culture Operation?
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Work Space
I. Greenhouse
A. Donor plant cultivation B. Grow out of
regenerates. C. Misting bench
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Work Space - 2
II. Laboratory for Media Preparation
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Work Space - 3
III. Storage Space for All Supplies, e.g.,
glassware and chemicals
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Work Space - 4
IV. Washing-Up Area for Dishwasher and
Autoclave
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Work Space - 5
V. Inoculation Room with Laminar Flow Hood
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Work Space - 6
VI. Incubation Room with Temperature Controls and
Clock for Adjusting Photoperiod
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Equipment Needs
I. Analytical balance for measuring in grams and
milligrams.
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Equipment Needs - 2
II. Glassware
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Equipment Needs - 3
III. Hot Plate with Magnetic Stirrer, pH meter
and Microwave
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Equipment Needs - 4
IV. Distilled or Deionized Water
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Equipment Needs - 5
V. Wide Range of Chemicals A. Mineral Salts B.
Vitamins C. Plant Growth Regulators D.
Sugars E. Agar
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Equipment Needs - 5
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Equipment Needs - 6
VI. Bleach and Alcohol for Surface Sterilization
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Equipment Needs - 7, 8
VII. Text References
VIII. Lab Notebooks
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Conclusions
Plant tissue culture is a useful experimental
tool in basic as well as applied research.
Basic 1. Plant Morphogenesis 2. Compounds with
hormonal activity 3. Mutagenesis 4. Genetic
Engineering
Applied 1. Micropropagation 2. Embryo rescue 3.
Producing virus-free plants 4. Doubled
haploids 5. Genetic Engineering