Title: “Tater Tots or Not” (Will potato cells turn into tiny potatoes or some other plant part?)
How do potato cells show traits of living things?
Introduction: Teachers! This lab is a great way to introduce t he “ WOW” factor into you classroom early in the year. By requiring a relatively sophisticated lab procedure, essential team collaboration, inquiry and critical thinking, this lab sets the stage for an engaging and rigorous year of study in the biological sciences.
If the advanced preparation seems a bit daunting at first, DO
NOT BE INTIMIDATED!
The actual preparation of the medium is as easy as preparing hot chocolate in the microwave, the ingredients are easily ordered and the student benefits are well worth the effort.
This is definitely not a “fluff” lab. It covers several important key concepts that are typically covered in the beginning of the year
(See objectives) and several NYS Science & Technology Standards (See below).
Overview: In this activity students cut sections of potato plants, place them on two slightly different mediums and observe them growing in two different conditions. Grown in the dark with an anti gibberellin, the plant tissue should actually produce small tubers (potatoes!); grown in the light they will grow leaves, roots, and stems. The activity as presented here is best done early in the year when Traits of Living Things is taught. This activity allows students to

practice tissue culture lab techniques

examine how plant cells develop and differentiate into specialized plant tissues

to observe life functions such as o Growth & Development, o Response to the Environment, o Cell Reproduction, and o Obtaining & Using Energy
Extensions of this lab can also be used to illustrate cloning, vegetative propagation, plant cell response to hormones and external stimuli, and scientific method.
NYS Standards:
MST Standard 1: Students will use mathematical analysis, scientific inquiry, and engineering design as appropriate to pose questions, seek answers and develop solutions
MST Standard 4.2 Organisms inherit genetic material in a variety of ways

MST Standard 4.4 Continuity of life is sustained through reproduction and development
MST Standard 4.5 Living organisms perform basic biochemical processes that are essential to maintaining a dynamic equilibrium and sustaining life
MST Standard 4.7 Human decisions and activities have an impact on the physical and living environment.
Estimated time:

Teacher Prep : 1 to 2 hours before lab to make medium, pour plates, set up lab materials for students. Directions appear under procedure

Class Time : o Depending on source of plants (See Background ) o 45 minute class period to for students to perform procedure o One 10 minute observation session every three days. This can be
Objectives:
WOW factor done during bell work or while other independent work is going on in the class room. This will continue approximately 2-3 weeks o 45 minute class period for teams to compare results, reflect and formulate conclusions. Followed by class discussion.
Grabs the students’ attention early on in the year
 by doing “real” lab work and work that includes lot’s of hands-on
Sets a tone of academic rigor by
 challenging the students with work that is different and more complex than middle school.
 requiring collaboration, critical thinking, and scientific inquiry

LAB SKILLS
CURRICULUM
CONTENT

Practice with aseptic technique

Practice with dissection tools

Practice with making detailed scientific observation (make at least 3 observations each time)

Practice with scientific drawing

Develop team cooperation and collaboration skills

Practice with dissecting microscope
Traits of Living things
First hand observation of how living cells exhibit traits of living things such as
Obtaining & Using Energy for

Growth

Development

Cell reproduction

Response to the environment
Tools of a Biologist
Students get practical experience with tools of a biologist

Why is a dissecting scope more useful than a cmpd. light microscope in this case?
Scientific Method

This experiment contains several variables. Which is really responsible for the formation of tubers? How can you design an extension that will help answer that question

Quantitative vs. qualitative data
Asexual Reproduction

How is asexual reproduction advantageous to plants? To humans?
Background:
Tissue culture involves growing new plants from small pieces of plant tissue in a sterile nutrient medium. By slightly altering the medium, the plant pieces can produce more undifferentiated cells or roots and leaves that can eventually grow into mature plants. Several resources are listed at the end of this lab for more background information.
The largest limiting factor with this lab is a source of plant material. These can be supplied by a local university or biological supply. They can also be grown by the teacher if started about six weeks prior to when they are to be used in the classroom. ( See Step # 1 of the procedure Plant Potatoes)
This lab would be nicely preceded by a classroom visit from a research scientists who deals with tissue culture, plant propagation, cloning or other related topics since one of the main objectives is to capture student attention and give them an opportunity to envision themselves doing the job of a scientist.
If there is a lot of air flow in your classroom, you may consider constructing a few sterile “hoods” in which students can do plant transfers. See Appendix D
Materials:
Teacher Materials: potatoes
100 ml of 10% bleach solution with one drop of dish detergent
75 mL of 70% ethanol
70% ethanol in spray bottle paper towels sterile scalpel
60 mL Petri plates (1 per team of four)
Student Materials: Per team of four
4 pairs of goggles smooth cutting surface (cutting board, plate, aluminum foil, etc.) paper towels
2 sterile scalpels
2 sterile forceps waterproof label pens
2 sterile potato plants

Vocabulary:
Tissue culture
Agar differentiation
Extension vocab: cloning, stem cells, vegetative propagation, totipotent
Procedure:
Six weeks prior to lab
Plant Potatoes
1. Several weeks (6) before you plan to do the lab with the class, you must obtain actual potato plants. a. First, o btain potatoes that have some “eyes” starting to bud on them. You may get potatoes from a garden or whole foods store. If you get them from a grocery store, it’s a good idea to wash them
(just like the dishes) in soapy water to remove any chemicals that inhibit budding that may have been sprayed on the potatoes.
Remember to rinse them well after washing. b. The potatoes can be used in two ways to get plants. One way is to stick the potato half submerged in water in a dark place until roots have started to develop. Then put them in the light so the shoot part will grow. c. The other method is to cut potatoes in large pieces so that each chunk has at least 2-3 eye buds and plant them in sterile potting soil in a few plastic containers or plant boxes. (I used plastic bins that were 30x20 cm and about 12-15 cm deep.) d. Grow plants in warm, well lighted environment until they are 3-4 inches tall.

Two weeks prior to lab
Make Rooting Medium
2. When potato plants are about 3 inches tall it is time to make rooting medium. Follow the directions in Appendix A. This is the same rooting medium that the students will use.
Sterilize the Cuttings
3. When potato plants are about 3 inches tall it is time to sterilize cuttings and transfer them to a sterile rooting environment. a. Use a sharp knife or scalpel to remove a 2 – inch portion of growth stem from the plant b. Rinse the cutting in soapy water and then in clear water. c. Place the cutting in 70% ethanol for 15-20 seconds d. Prepare 100 mL of 15% bleach solution that has one drop of liquid soap added. Then remove the cutting from alcohol and place cutting in bleach solution for 7-10 minutes e. Place cutting in clean beaker and cover with about 50-75mL of sterile deionized water. Swish beaker then drain. Repeat two more times. (Don’t skimp on the rinsing. It is important) f. Once cuttings are sterile, they can be cut into pieces and transferred to culture tubes containing rooting medium. The procedure is the same as the student lab but is re-printed here for your convenience
Transfer Nodes to Rooting Medium
4. Spray cutting surface with 70% ethanol solution and wipe with a sterile
(clean) paper towel. a. Sterilize forceps and scalpel by soaking them in 70% ethanol and either allowing them to air dry or tap them on a clean paper towel.
Use forceps to place sterilized cutting onto cutting surface. b. Cut node sections from plant using sterile scalpel and place one each on the medium in the culture tubes. c. Cover tubes with loose fitting plastic caps or aluminum foil. Seal w/ micropore tape and label. Allow to grow in bright sunny place.
They should be ready for the students to use in about two weeks.
Two days prior to lab
5. Make rooting and tuber medium by following directions in Appendix A and B. a. 10 mL of both rooting and tuber medium per team of four b. Pour apx. 10 mL of rooting medium into each Petri plate and cover. c. Pour apx. 10 mL of tuber medium into each culture tube and cover w/ plastic lid. d. Let cool until gel sets

Student Activity: See attached student version
Assessment: Written assessment through follow up questions and summary.
Oral assessment through classroom analysis and discussion of data.

PHYTOTECH LAB - Technical Literature (Easy articles on plant propagation media prep, etc) http://www.phytotechlab.com/TABSTYLE/Technical.asp?CartId={36C4162A-9CC9-
4066-BDAA-9B114FEVERESTCEFC06}
PHYTOTECH LAB - Tissue Culture Techniques http://www.phytotechlab.com/TABSTYLE/Tissue.asp?CartId={B9FEFA59-A276-44C0-
A238-7AF0F071EVEREST0B41}
BIOTECHNOLOGY ONLINE - Many pdf Worksheets that are labs or activities that can be used for Environment, Biotechnology, Human Uses,and Food &
Agriculture http://www.biotechnologyonline.gov.au/resources/res_worksheet.cfm
BIOTECHNOLOGY ONLINE
–What is Biotechnology? Gives many web sites that have interactive or animated clips for making DNA, Proteins, splicing and inserting genes, etc http://www.biotechnologyonline.gov.au/teaching_support/tsp_s4_whatis_biotechnology.c
fm
BIOTECHNOLOGY ONLINE
– Plant Tissue Culture in the Classroom http://www.biotechnologyonline.gov.au/pdf/biotech/plant_tissue_culture_in_class.pdf
NEW YORK STATE STANDARDS
– www.NYSed.gov
HORTICULTURE SCIENCE PAGE –Plant Tissue Culture for Home Gardeners http://www.une.edu.au/~agronomy/AgSSrHortTCinfo.html

Rooting medium
Component
MS Salts*
Sucrose
Quantity for 1 L Quant. Req.
4.43 g
20 g
Added pH
1. Put beaker w apx 400 ml of de-ionized water on stir plate w/ stir bar
2. Weigh out and add MS Salts and Sucrose to beaker
3. Add D.I. water bring volume up to 1 liter
4. Adjust pH using meter or pH paper
5. Use KOH to bring up to 5.7 + .
5.7 + .03
6. Next measure out Agar
Component
AGAR-Agar**
(Plant cell culture tested)
Quantity for 1 L
8g/L
7. Pour directly into beaker of medium solution and stir. Cover with saran wrap and place in microwave for 15 minutes. (10 minutes for 500mL)
8. Add 2.5 mL of PPM biocide
Component
PPM***
Quantity for 1 L
2.5mL/L
9. Carefully dispense hot medium to culture tubes (Apx. 10 mL each) and cover immediately with plastic caps
10. Allow to cool until gel sets
* Product # M519-10L MURASHIGE & SKOOG BASAL MEDIUM w/ VITAMINS; CONTAINS
THE MACRO- AND MICRONUTRIENTS, AND VITAMINS AS DESCRIBED BY MURASHIGE &
SKOOG (1962). PLANT TISSUE CULTURE TESTED. www.phytotech.com for order information
**Product # A111-100G AGAR, PLANT TC/ MICROPROPAGATION GRADE www.phytotech.com for order information
***PPM Order information http://www.ppm4plant-tc.com/references.htm

Tuberization medium
Component
MS Salts*
Quantity for 1 L
4.43 g
Quant. Req. Added
Sucrose 80 g
1. Put beaker w/ apx 400 ml of de-ionized water on stir plate w/ stir bar
2. Weigh out and add MS Salts and Sucrose to beaker
3. Add De-ionized water to bring volume up to 1 liter
4. Adjust pH using meter or pH paper
5. Use KOH to bring up to 5.7 + .
5.7 + .03 pH
6. Next measure out Agar
Component Quantity for 1 L
AGAR-Agar**
(Plant cell culture tested) 9g/L
7. Pour directly into beaker of medium solution and stir. Cover with saran wrap and place in microwave for 15 minutes. (10 minutes for 500 mL)
8. Add 2.5 mL of PPM biocide
Component Quantity for 1 L
PPM*** 2.5 mL/L
9. Allow medium to cool to apx. 68 0 C
10. Add A-Rest ( Ancymididol)
Component
A-Rest****
Quantity for 1 L
9mL/L
2 week old tubers
11. Carefully dispense hot medium to sterile petri plates (Apx. 10 mL each) holding lid at 45 0 angle then covering immediately.
12. Allow to cool until gel sets
* Product # M519-10L MURASHIGE & SKOOG BASAL MEDIUM w/ VITAMINS; CONTAINS
THE MACRO- AND MICRONUTRIENTS, AND VITAMINS AS DESCRIBED BY MURASHIGE &
SKOOG (1962). PLANT TISSUE CULTURE TESTED. www.phytotech.com for order information
**Product # A111-100G AGAR, PLANT TC/ MICROPROPAGATION GRADE www.phytotech.com for order information
***PPM Order information http://www.ppm4plant-tc.com/references.htm
**** Product # 03-0100-1
A-Rest permits producing plants to any desired height. This linear response is related to dosage so that heavier applications produce more compact plants, while lighter applications produce plants nearer to normal growth heights. Order Info http://www.hummert.com/catalog.asp

July 17, 2006
Joyce VanEck
1. Place sample in 70% ethanol for 30 seconds
2. Place sample in Clorox/soap solution for 10 minutes a. 10% - 15% Clorox b. 1 drop (.1%) dish detergent (Use fume hood if you use Joy brand detergent)
3. Rinse three times with sterile* water before placing on medium a. To rinse place sample in beaker. Completely cover with sterile water b. Swish, then cover beaker with cheese clothe and completely drain c. Repeat this process two more times with clean sterile water each time. d. Don’t skimp on the rinsing. It’s important
* To make sterile water, I boiled the water in a glass jar for 10 minutes in the microwave. After it had cooled a bit, I covered it with a tight fitting screw cap. I let it cool completely (24 Hrs) before using.

1. Purchase clear plastic containers that are meant for file folders.($1.29 each) Dimensions 41x28x25cm. These are usually deep enough to be effective.
2. Purchase clear vinyl sheeting from a fabric store ($3.00/yd). Cut flap large enough to cover front opening.
3. Secure top of flap w/duct tape.
4. Sterilize by spraying dowm with 70% alcohol before using.