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Care of Arabidopsis thaliana at the
Center for Plant Lipid Research
Care of Arabidopsis thaliana at the
Center for Plant Lipid Research
By Matthew Cotter
University of North Texas
April 2005
Abstract
Care of Arabidopsis thaliana at the Center for Plant Lipid Research
By Matthew Cotter
The purpose of this manual is to introduce new lab members to the care of the model
research plant Arabidopsis thaliana at the Center for Plant Lipid Research at the
University of North Texas. This manual is intended for undergraduate lab assistants
working in the Center’s research labs. It is assumed that these students have little
experience in plant research. These students are assumed to have had basic high school
biology and chemistry. Although the manual is intended for beginners, some advanced
users may find the information helpful.
This manual covers the basic care of Arabidopsis. It includes steps for planting, growing
and harvesting seeds. In addition, it includes a brief introduction to Arabidopsis and its
use in the research lab.
iii
Table of Contents
Table of Contents
Abstract......................................................................................................................................... iii
List of Illustrations...................................................................................................................... vii
Chapter 1: Introduction to Arabidopsis ..................................................................................... 3
Arabidopsis in Research ........................................................................................................... 3
Structure of Arabidopsis ........................................................................................................... 4
General growth of Arabidopsis................................................................................................. 5
Growing Arabidopsis for Research Uses.................................................................................. 7
Chapter 2: Planting Arabidopsis............................................................................................... 11
Sterilizing Soil ........................................................................................................................ 11
Sterilizing seeds ...................................................................................................................... 13
Preparing flats ......................................................................................................................... 14
Planting Seeds......................................................................................................................... 15
Chapter 3: Growing Arabidopsis .............................................................................................. 19
Watering.................................................................................................................................. 19
Growth Support....................................................................................................................... 20
Pest Control............................................................................................................................. 21
Chapter 4: Harvesting Arabidopsis........................................................................................... 25
Collecting Plants ..................................................................................................................... 25
Harvesting Seeds..................................................................................................................... 26
Storing Seeds .......................................................................................................................... 28
Chapter 5: Cleaning Arabidopsis Materials............................................................................. 31
Disposing of Plant Tissue ....................................................................................................... 31
Cleaning Planting Equipment ................................................................................................. 31
Works cited.................................................................................................................................. 35
Photo Credits............................................................................................................................... 39
Glossary ....................................................................................................................................... 43
Appendix A: Using Micropipettes ............................................................................................. 47
Appendix B: Sterilization Solutions .......................................................................................... 51
v
List of Illustrations
List of Illustrations
Figure 1: Scanning electron micrograph of Arabidopsis seeds......................................................4
Figure 2: Structure of Arabidopsis.................................................................................................5
Figure 3: Arabidopsis Growth Chamber.........................................................................................7
Figure 4: Biology growth room ......................................................................................................7
Figure 5: ARASYSTEM components...........................................................................................11
Figure 6: Autoclave.......................................................................................................................11
Figure 7: Autoclave settings .........................................................................................................12
Figure 8: Seeds for sterilization ...................................................................................................13
Figure 9: Sterilizing seeds............................................................................................................13
Figure 10: Prepared flat................................................................................................................14
Figure 11: Seeds ready to be planted ...........................................................................................15
Figure 12: Planting seeds.............................................................................................................15
Figure 13: Dried Plants ................................................................................................................19
Figure 14: 6 week old plants........................................................................................................20
Figure 15: Thrips larvae................................................................................................................21
Figure 16: Plant material in envelope ..........................................................................................25
Figure 17: Finished envelope.......................................................................................................25
Figure 18: Arabidopsis freezer ....................................................................................................26
Figure 19: Crushed plant stems ...................................................................................................26
Figure 20: Seed harvesting setup .................................................................................................27
Figure 21: Straining seeds............................................................................................................27
Figure 22: Cleaned seeds .............................................................................................................27
Figure 23: Finished seeds ............................................................................................................27
Figure 24: Seed desiccator...........................................................................................................28
Figure 25: Disposal of soil...........................................................................................................31
Figure 26: Cleaned tubes .............................................................................................................32
Figure 27: 1000 µL settings .........................................................................................................47
Figure 28: Micropipette stop positions ........................................................................................47
vii
Chapter 1: Introduction to Arabidopsis
Chapter 1: Introduction
3
Chapter 1: Introduction to Arabidopsis
Arabidopsis thaliana is one of the most important research models in plant biology. At
the Center for Plant Lipid Research, Arabidopsis is used to explore basic plant
biochemistry principles. This information can then be applied to other, more useful
plants, such as cotton and tobacco.
To understand how to care for Arabidopsis, you need to know why it is important. Once
you know how it is used, certain techniques used make more sense. In addition, you need
some general information on the plant itself.
There are many excellent sources of information on Arabidopsis.
The most
comprehensive source of information is The Arabidopsis Information Resource (TAIR)
website. This website is administered by the Carnegie Institute for Plant Biology and the
National Center for Genome Resources. The website offers detailed information on
almost all aspects of Arabidopsis. Several books are also available for further
information. The Center for Plant Lipid Research has two books on hand, Arabidopsis
Protocols and Methods in Arabidopsis Research, which both provide advanced
information on Arabidopsis research protocols.
Arabidopsis in Research
Arabidopsis thaliana was first used for plant research in the mid 1940’s. Initially, the
plant was used for genetics experiments because mutants could be created easily
(Martinez-Zapater, v). By the mid 1980’s, the plant was being used for more in-depth
studies of plant physiology and biochemistry. This switch in uses was because the small
genome allowed relatively simple studies of the plant’s processes (Martinez-Zapater, v).
Today, Arabidopsis is the model plant research system. There Tip: Words in red
text are defined in
are six main reasons why this simple plant is used (TAIR).
the glossary
1.
Rapid life cycle which produces seeds in about 8 weeks.
2.
Each plant produces many seeds.
3.
Arabidopsis has a small genome that has been completely sequenced. This
completed genome allows researchers to search for genetic sequences of interest.
4
Care of Arabidopsis
4.
There are extensive genetic maps of all five Arabidopsis chromosomes. These
maps show were genes are located in the chromosomes and allow researchers to
find gene groups to study.
5.
There are many existing mutant seed lines for study. The many mutant lines
allow researchers to study the effects of missing or extra genes.
6.
Arabidopsis can be easily transformed. The plants are transformed by the bacteria
Agrobacterium tumefaciens, a common bacterium used to insert new genes into
the plant cells.
All of these properties are exploited in the Center’s lab.
Structure of Arabidopsis
Structurally, Arabidopsis thaliana is a
relatively simple plant.
Arabidopsis
seeds are about 1 mm in diameter. There
are about 1250 seeds in 25 mg. These
small seeds range in color from light to
dark brown, as shown in Figure 1.
The grown plant is shown in Figure 2.
Each seed produces a single stem. The
stem rises up from the leafy base, called
the rosette. The rosette consists of about Figure 1: Scanning electron micrograph
20 large leaves. On the stem are the
of Arabidopsis seeds
siliques (seed pods) and the flowers.
There are also a few smaller leaves, usually found on the lower portion of the stem.
The flowers are small and white. They are found on the stem, typically clustered around
the tip. There are several other flowers spaced about 20-25 mm apart along the stem. A
typical plant will have 10-12 flowers.
The siliques are small, green pods that hold the maturing seeds. Each pod holds about 20
seeds. Depending on the plant ecotype, a single Arabidopsis plant can have over 30
siliques on a single stem, producing about 600 seeds.
Chapter 1: Introduction
5
Flowers
Siliques
Stems
Leaves
Figure 2: Structure of Arabidopsis
General growth of Arabidopsis
Arabidopsis growth has four major stages: germination, rosette production, bolting, and
senescence (TAIR).
Stage 1:
Germination. In this stage, the radicle emerges from seed coat and begins to
grow. The radicle is the root tip and grows down into the soil to bring
nutrients up to the growing plant. The radicle emerges about 4 to 5 days after
planting.
Stage 2:
Rosette production. In this step, the plant begins to produce large leaves just
above the surface of the soil. The plant start putting out these leaves after 7 or
8 days and rosette growth is complete by day 21.
6
Care of Arabidopsis
Stage 3:
Bolting. In this stage, the plant produces a large stem. On this stem, the plant
produces flowers and the siliques (seed pods). This step is called bolting
because the stem grows very rapidly, up to 5 mm per day. The stage lasts
from about day 21 to about day 50. While the stem is growing, the plant is
producing small white flowers and green siliques.
Stage 4:
Senescence. In this stage, the plant begins to senesce. Senescence is
programmed cell death, in which the plant cells stop carrying the basic
functions of life. This step is necessary to open the seed pods and allow the
seeds to be dispersed. This step begins around day 45 to 50. Once senescence
begins, the plants are allowed to dry out.
Stage 1: Germination
Stage 2: Rosette Production
Stage 3: Bolting
Stage 4: Start of senescence
There are many different varieties, or ecotypes, of Arabidopsis thaliana. Each type has
specific properties, such as leaf size, maximum growth height, or number of seeds
produced. The varieties are usually named after the location they were first found in. In
this lab, we mainly use the Columbia ecotype. This type has very robust growth and
average growth characteristics. These plants are usually considered to be the wild-type,
or the plant type found in nature.
Chapter 1: Introduction
7
The Center has recently started researching plants from other ecotypes. The Landsberg
erecta (Ler) ecotype has shorter stems and smaller siliques than Columbia. The
Wassilewskija (WS) ecotype has very tall stems and flowers earlier than Columbia. The
Cape Verdi Islands (Cvi) ecotype has much smaller leaves and grows much slower than
Columbia. Each of these ecotypes has a specific use in the lab.
Growing Arabidopsis for Research Uses
Because this is a research lab, the plants must be grown under precise conditions. This
helps ensure that experimental results can be repeated by others. To ensure the
conditions are the same each time, protocols have been developed for the care of the
plants. These protocols must be followed every time, with no deviation.
The majority of the plants grown in the growth room are for seed production. Generally,
an entire flat of a single seed line is grown so plenty of seed is available for experiments.
You must be extremely careful to avoid getting seeds from one seed line mixed up with
seeds from another. A few wrong seeds in a full can give imprecise results in
experiments, or they could be grown for seed production and contaminate an entire flat of
seeds.
The plants are grown in either specially designed growth chambers (Figure 3) or in
growth rooms (Figure 4). The growth chambers can be set to different temperatures and
lighting conditions, depending on the experiment. These chambers can only hold a
maximum of four flats of plants, so they are generally not used for growing Arabidopsis
for seed production. Currently, there is only one growth chamber set up for the Center’s
use.
Figure 3: Arabidopsis Growth Chamber
Figure 4: Biology growth room
8
Care of Arabidopsis
The growth rooms are located on the third floor of the Biology building. These rooms
are set up for growing plants specifically for seed production. There are two Arabidopsis
rooms, each capable of holding around 40 flats. These rooms are kept at a constant 69°F
and 70% humidity, the optimal growing conditions for most of the Arabidopsis lines the
lab uses. The plants are grown using a fourteen hour daylight cycle.
Tip: When grown for seed production, Arabidopsis needs 200 µmol/m2/s of
light. This amount is provided by six fluorescent lights at a distance of 50 cm.
Even though the care of Arabidopsis is precisely determined by existing protocols, there
may be times where something goes wrong at some point in the growth. This could be
due to a power outage in the growth room, improper watering, or an insect infestation. If
anything unusual happens to the plants, even if you think it is insignificant, you MUST
make a note of the changes. These seemingly minor details could help explain why an
experiment did not work as expected or gave conflicting results later on.
Many of the techniques used in growing Arabidopsis may seem difficult at first glance.
Don’t panic - after working on several flats, they will become easier. Relax and enjoy
taking care of some simple plants and learn some excellent fundamental techniques for
your future research activities.
Chapter 2: Planting Arabidopsis
Chapter 2: Planting
11
Chapter 2: Planting Arabidopsis
The first step in growing Arabidopsis is to plant the seeds. This is the most time
consuming task. In this step, the soil is autoclaved, the seeds are sterilized, the
Arabidopsis flats are prepared, and the seeds are added to the soil.
At the Center for Plant Lipid Research, we use
the ARASYSTEM from Lehle Seeds to grow
Arabidopsis. These materials, shown in Figure
5, provide a simple method for growing the
plants. We also use potting media from Lehle
seeds. This soil is specially formulated for
Arabidopsis. Arabidopsis controlled release
fertilizer also helps ensure optimal growth.
Sterilizing Soil
The soil must be sterilized by autoclaving before
preparing the flats. The autoclave (Figure 6)
uses high pressure and steam to kill any living
cells in the soil. This destroys any bacteria or
fungus living in the soil. It also serves to kill
any seeds that may be present. The autoclave
also dries material using a slight vacuum. This
ensures consistent moisture content, allowing
greater control of the water in the soil.
1.
2.
3.
Add soil to a clean foil roasting pan. The
pan should be full level to the top.
Cover the pan with aluminum foil. It
usually takes two sheets to fully cover
the soil.
Place a small piece of autoclave tape on
the top of the foil. This tape will show
“Autoclaved” in dark brown when it
comes in contact with hot steam. This
allows a visual check if the autoclave
worked properly.
TUBE
CAP
ARABASKET
ARAFLAT
ARATRAY
Figure 5: ARASYSTEM components
Figure 6: Autoclave
12
Care of Arabidopsis
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Place the pan of soil in the autoclave. To autoclave more than one pan, use the
large metal tray in the autoclave room to hold all pans.
Close the door to the autoclave. To lock the door, turn the wheel clockwise until
the door is securely closed.
Set the autoclave to the correct settings (Figure 7).
The timer should be set for 20 minutes. If it is not, turn the silver dial until it
is set properly.
The mode selector dial should be set to <Fast Exhaust and Dry>. If it is not,
turn the dial until it is set properly.
All other settings should be left alone.
Start the autoclave by turning the start switch to “Ster.” The entire autoclave
procedure takes 45 minutes. The autoclave is finished when the orange timer
moves past 0 to 15.
Turn the start switch to “Off.” Wait a few minutes for the pressure to reach 0.
Put on the orange autoclave gloves.
Open the autoclave door by turning the wheel
Caution: Steam leaving
counter-clockwise.
the autoclave is very hot.
Remove the pan of soil. Make sure the Keep all body parts
autoclave tape shows “Autoclaved”. If it does away from the steam.
not, tell the maintenance staff that the autoclave
is not working properly.
Let the pan of soil sit out for at least one hour before planting to allow it to cool
down.
Fill out the autoclave log in the autoclave room. Be sure to write down the time
the autoclave was started and when it was finished.
Timer Dial
Mode
Selector
Dial
Pressure
Gauge
Figure 7: Autoclave settings
Start Dial
Chapter 2: Planting
13
Sterilizing seeds
The seeds are sterilized using a chemical method. This can be done while the soil is
cooling off to save time. Sterilizing removes any fungus spores and bacteria that could
affect growth.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Transfer about 100 seeds to a clean 1.5 mL
microcentrifuge tube. 100 seeds will just
barely cover the bottom of the tube (Figure
8 shows about 100 seeds).
Add 1 mL of 95% ethanol to the tube using
a micropipette (Figure 9). See Appendix
A: Using Micropipettes for tips on using
micropipettes.
Close the lid and throw out the
Figure 8: Seeds for sterilization
micropipette tip.
Gently mix the contents for 3 minutes.
Use the micropipette with a clean tip to remove the ethanol. Be careful to leave
the seeds in the tube.
Add 1 mL of a 20% bleach/0.1% Tween-20 solution using a clean tip to the seeds.
For instructions on making solutions, see Appendix B: Sterilization Solutions.
Close the lid and gently mix the contents for 3 minutes.
Use the micropipette with a clean tip to remove the bleach solution, leaving the
seeds in the tube.
Add 1 mL ddH2O (sterile MilliQ water) to the seeds using a clean tip.
Close the lid and shake three times.
Remove the water using the
micropipette with a clean tip,
leaving the seeds in the tube.
Repeat steps 9-11 three more times,
using a new micropipette tip each
time.
Add 1 mL ddH2O to the seeds. The
seeds can be kept in this state for up
to 24 hours before planting.
Figure 9: Sterilizing seeds
14
Care of Arabidopsis
Preparing flats
Before the seeds can be planted, the ARASYSTEM flats must be prepared. This involves
adding soil to the ARABASKETS, watering the soil and adding the fertilizer. The
completed flat is shown in Figure 10.
1.
2.
3.
4.
5.
6.
7.
8.
Place the ARAFLAT into the ARATRAY.
Label the flat on a piece of tape with the name of the Arabidopsis seed line and
the date.
Open the pan of autoclaved soil.
Fill the ARABASKETS 3/4 full with the soil and place them in the ARATRAY
pots.
Wet the soil with deionized water. The soil should Tip – While watering
the soil, dump water
be damp to the touch, but not muddy.
from tray before it
Add one fertilizer pellet to each ARABASKET.
gets too full.
Wet the soil again with deionized water.
Label a white plant marker. The marker should include:
Name of seed line (ex: WT Col)
Lineage of seeds
(ex: From: 1/12/05 seeds)
Date Planted (ex: 3/15/05)
Figure 10: Prepared flat
Chapter 2: Planting
15
Planting Seeds
The seeds can be planted immediately after the flats are ready. If the soil is allowed to
dry out, the plants will not receive the proper amount of water and will grow poorly.
Planting the seeds is the most difficult part of planting Arabidopsis. The seeds are too
small to plant by hand, so they mush be planted using the micropipette. If you are not
confident with the micropipette, practice moving wild-type seeds around before moving
on to other seed lines. See Appendix A: Using Micropipettes for more help.
Tip – In some instances, the seeds must be cold treated before they can be
planted. To cold treat seeds, place the sterilized seeds (in water) in the
refrigerator for two days. This helps ensure all of the seeds will germinate at
the same time.
1.
2.
3.
4.
5.
6.
7.
Use the micropipette to transfer all of the seeds to a clean 50 mL beaker.
Add diH2O to fill the beaker (Figure 11). This makes it easier to get out one seed
at a time.
Use the micropipette to transfer one seed
to each basket in the flat. The seed is Tip – It is okay for two or three
simply squirted onto the soil (Figure 12). seeds to end up in each pot.
However, any more than that
Add water to the beaker as necessary.
will have a negative effect on
Cover the finished flat with plastic wrap.
growth.
Secure the plastic wrap with binder clips.
Pour out all water in the ARATRAY.
Take the finished flat to the growth room.
Figure 11: Seeds ready to be
planted
Figure 12: Planting seeds
Chapter 3: Growing Arabidopsis
Chapter 3: Growing
19
Chapter 3: Growing Arabidopsis
After planting, Arabidopsis will fully mature in 6 to 8 weeks. During this time, the plants
need additional care to ensure proper growth. They must be watered regularly, supported
while growing and kept free of pests.
Watering
The plants must be watered regularly. A consistent watering schedule produces healthier
plants and produces more seeds. Proper watering also helps reduce algae and fungus
growth on the plants.
When the flats are first planted, the soil holds the moisture. The seeds are on top of the
soil, so the water must come up from the bottom. If the flats were watered from the top,
the seeds would mix into the soil and could not grow as well. The ARATRAYS are filled
with water, providing a reservoir of water to keep the plants healthy.
As the water is used or evaporated from the top of the flats, more water is pulled up from
the tray reservoir, a process called capillary action. If this capillary column is broken by
allowing the tray to run dry, fresh water will no longer move up to the surface of the soil.
The plants will begin to dry out, stopping growth and seed production.
When the flats are first brought to the growth room, remove the plastic wrap and place
the flat on a rack. Fill the tray about half full with deionized water from the large plastic
jug. There is a diH2O tap in the sink room next to the growth room to refill the jug.
The flats need to be watered every other day. The water level should be kept between 1/4
and 1/2 full (about 10 mm full). The trays can be filled more if they will not be watered
for two or three days, such as over a weekend. However, this could result in rapid growth
of green algae on the soil.
Water the flats until the plants begin turning
brown and the siliques start to open, generally 6
to 8 weeks after planting. This is the start of plant
senescence. Once this begins, the plants are
allowed to dry out (Figure 13).
Figure 13: Dried Plants
20
Care of Arabidopsis
Growth Support
Arabidopsis plants can grow over one foot tall. The individual plants must be supported
or they will fall over. This could contaminate other plants on the same flat or on other
flats. In a research setting, the individual plants must be kept separate to ensure the seeds
from each plant are the correct seeds.
The ARASYSTEM used in the Center for Plant Lipid Research contains special
equipment to support and isolate each plant. The ARACONS in the set consist of a long
plastic tube and a cap that holds it up. The cap catches any seeds that fall off of the plant,
and the tube keeps the plant upright.
Place the caps on each pot soon after the plants start bolting (see page 5 for a description
of growth stages). This is typically after about 3 weeks of growth. Place the tubes in the
caps over the plant. The plants can then grow up through the cap and tubes, as shown in
Figure 14.
Figure 14: 6 week old plants
Chapter 3: Growing
21
Pest Control
The growing Arabidopsis plants must be kept free of pests to ensure proper growth and
seed production. The most common pests encountered in the growth rooms are thrips
(Figure 15). These small flying insects eat the leaves of the plants. If left untreated,
entire flats of Arabidopsis can be ruined.
Figure 15: Thrips larvae
The best way to control thrips is to use preventative measures. Always be on the lookout
for any flying insect in the growth room. Early detection and prevent much of the
damage to the plants. The following steps help keep thrips from becoming established in
the growth room.
1.
2.
3.
Never transfer flats of plants from one growth room to another.
Hang several pieces of flypaper in the growth room.
Replace old flypaper every month.
Sometimes, thrips become established on the plants despite these
precautions. Once again, prompt treatment will minimize Caution: Use a
respiratory mask
damage to the plants. If thrips larvae are on the leaves, spray all
and wear gloves
of the flats in the growth room with Diazinon or Malathion before handling
insecticide. These solutions are kept in either the sink room or any pesticide.
the tobacco growth room. Liberally spray the plants, even the
ones with tubes. This treatment kills all insects that the spray can reach. These
insecticides have very little impact on the plants, so as long as the infestation is caught
early, the plants should be safe.
To kill any remaining thrips, use a slow release insecticide. Add one small scoop of
Marathon 1% granules to the tray of each flat. The insecticide is then drawn up into the
plant with the water.
Chapter 4: Harvesting Arabidopsis
Chapter 4: Harvesting
25
Chapter 4: Harvesting Arabidopsis
The flats of Arabidopsis are grown until they begin to senesce. At this point in their life
cycle, the seed pods open to release the seeds. Watering stops and the plants begin to
dry. Once the plants have completely dried out, collect the plants and harvest the seeds.
These new seeds can then be used for various experiments or to grow more flats of seeds.
Collecting Plants
Before the seeds can be harvested, the plants must be cut down. The plants are stored in
a -80°C freezer to kill any insects or fungus on the plants. Each seed line is harvested
separately in a new 8x11 envelope and weighed. These weights can be used to determine
how well the plants grew.
Ideally, the harvesting should be done
outside of the growth rooms to prevent cross
pollination of other plants. Unfortunately,
there is usually no place to move the flats.
You must be extremely careful to not stir up
plant materials while harvesting.
1.
2.
3.
4.
5.
6.
7.
Weigh a new, empty 8x11 envelope.
Record the empty weight on the
envelope.
Fold the bottom corners up to keep Figure 16: Plant material in envelope
seeds from falling out of the
envelope.
For each pot in the flat, lift the cap
slightly and cut the plant as close to
the soil as possible.
Lift the cap and tube out of the soil.
The plant should stay in the tube.
Dump the tube into the envelope.
Press the plant to the bottom.
Repeat for all plants in each seed
line.
Press all of the plants to the bottom
Figure 17: Finished envelope
of the envelope (Figure 16).
26
Care of Arabidopsis
8.
9.
10.
11.
Fold the envelope in half and close the top.
Label the envelope with the name of the seed line, the date of harvesting (DOH),
and the number of plants harvested (Figure 17).
Weigh the full envelope. Record the weight on the envelope. The plant dry mass
is the final weight minus the empty weight.
Place the envelope in the Arabidopsis door of the -80°C freezer for three days
(Figure 18).
Figure 18: Arabidopsis freezer
Harvesting Seeds
After the plants have been frozen, the seeds can be harvested. Harvesting the seeds
involves separating the plant material from the seeds.
1.
2.
3.
4.
Let the envelope sit out for 20 minutes
before harvesting.
Open the envelope and crush the plants
by hand.
Remove the large plant stems (Figure
19). Only seeds and siliques will be left
in the envelope.
Spread the large sheet of paper over the
table (Figure 20).
Figure 19: Crushed plant stems
Chapter 4: Harvesting
Figure 20: Seed harvesting setup
5.
6.
27
Figure 21: Straining seeds
Pour the seeds through the strainer onto a piece of paper (Figure 21). Tap the
side of the strainer to make the seeds fall through the holes.
Pour the strained seeds back through the strainer onto another piece of paper.
Repeat as many times as necessary to remove the small white plant matter
(Figure 22).
Tip – It is impossible to remove all plant material from the seed. The seeds
are clean when seeds make up 99% of the vial.
7.
8.
9.
Pour the cleaned seeds into a small glass screw top vial.
Label the vial with the name of the seed line, the date of harvest (DOH) and the
date the parent seeds were harvested (Figure 23).
Cap the vial and place in the seed desiccator.
Figure 22: Cleaned seeds
Figure 23: Finished seeds
28
Care of Arabidopsis
Storing Seeds
Once the seeds are harvested, they are kept in a seed desiccator (Figure 24). The screw
cap vials are good for short term storage of the seeds. All of the seeds in the desiccators
are constantly being rotated and used for research.
For long term storage, the seeds must be kept cold. Place the open vials in the desiccator
for 5 days to remove any excess moisture. The vials can then be tightly closed and
placed in the -20°C freezer. These seeds can be stored for several years with minimal
loss of viability.
Figure 24: Seed desiccator
Chapter 5: Cleaning Arabidopsis Materials
Chapter 5: Cleaning
31
Chapter 5: Cleaning Arabidopsis Materials
Once the seeds are harvested, clean the planting equipment. While this step seems
simple, there are a few tips that speed up the process. Following these steps can also
prevent contamination of future flats of Arabidopsis.
Disposing of Plant Tissue
The first step is to get rid of the Arabidopsis plants. Usually, plant material that has been
genetically modified must be autoclaved before it can be thrown out. This is to protect
the naturally occurring varieties of the plants. However, Arabidopsis cannot grow in
North Texas. The hot, dry summers quickly kill all plants. This means that the modified
plant material does not need to be autoclaved before it is thrown away.
The material removed from the seeds during
harvesting can be immediately thrown out with
the normal trash. Dispose of the soil by dumping
each basket into the trash (Figure 25). The dry
soil is very powdery, so it can irritate the nose and
lungs. A filter mask will keep out most of the
dirt.
Figure 25: Disposal of soil
Cleaning Planting Equipment
One of the advantages of the ARASYSTEM is that it is reusable. Cleaning the planting
equipment, while not difficult, can be very time consuming. After washing, the
equipment needs to dry at least 24 hours.
The foil pan used for the soil is simply rinsed out with water and left to dry. Use the
aluminum foil that covers the soil over again if it does not have any rips or holes in it.
Wash the ARATRAY with running water and a brush. Remove all roots and dirt. There
may be a film on the edge of the tray where algae started to grow. Set the tray upside
down to dry.
32
Care of Arabidopsis
Wash the ARAFLAT with running water and the large black brush. Clean each pot
separately. Make sure there are no roots clogging the drain holes in the pots. Clean the
bottom of the tray as well, removing all dirt between the pots. Remove all tape from the
tray.
Wash the remaining pieces of the ARASYSTEM in a solution of water with three capfuls
of bleach. The baskets and caps are easily washed in a large bin using a small white
brush. Rinse the bleach water off after washing. Stack the caps and baskets on a layer of
newspaper to dry overnight.
Wash the tubes in a tub with the large black brush. Place the tubes upright in the water
and run the brush down the tube two or three times to remove any plant material. Rinse
the tubes in running water. Often, the tubes will come apart. Simply rinse the flat tube
and put it back together. Stack the tubes horizontally on newspaper to dry (Figure 26).
Once the equipment is dry, it needs to be
stored. The flats and trays can be stacked
together and kept near the worktable. Store
the baskets in a box above the worktable.
Place the caps in one of the two
ARASYSTEM boxes above the cabinets.
The tubes remain assembled and are kept in
any box large enough to hold them. These
boxes are usually kept either above the
cabinets or below the workbench.
Figure 26: Cleaned tubes
Works Cited
Works Cited
35
Works cited
Martínez-Zapater, José M. and Julio Salinas, eds. Arabidopsis Protocols. New Jersey: Humana
Press, 1998.
TAIR About Arabidopsis. 2 January 2003. TAIR. 20 March 2005
<http://www.arabidopsis.org/info/aboutarabidopsis.jsp>.
Photo Credits
Photo Credits
39
Photo Credits
Cover
Background: http://www.nottingham.ac.uk/bennett-lab/targetsite/targetmeetings.htm
Top left: http://www.rothamsted.bbsrc.ac.uk/corporate/Strategy.html
Top right: http://www.uni-kl.de/FB-Biologie/AG-Neuhaus/Publikationen/
Lower left: http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookflowersII.html
Chapter 1
Figure 1: http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookflowersII.html
Figure 2: http://www.expasy.org/sprot/ppap/
Page 6, Step 1: http://www.rothamsted.bbsrc.ac.uk/corporate/Strategy.html
Chapter 2
Figure 5: http://www.arabidopsis.com/main/cat/arasystem/!as01.html
Chapter 3
Figure 15: http://www.uidaho.edu/so-id/entomology/BerryInsects.htm
Appendix A
Figure 28: http://www.fao.org/DOCREP/005/AC802E/ac802e05.htm
Glossary
Glossary
43
Glossary
chromosome – a unit of the genome containing many genes; the DNA in the chromosome is
double stranded and surrounded by different proteins.
desiccator – a sealed jar with a water absorbent material used to remove all water vapor from a
sample.
DNA – deoxyribonucleic acid; the genetic material of an organism. An organism’s traits can be
determined by the sequence of its DNA. DNA is the building block of all multicellular
organisms.
ecotype – a subgroup of a species with distinct characteristics.
gene – a segment of DNA that encodes a protein or other gene product.
genome – the entire sequence of an organism’s DNA; the sequence determines the traits of the
organism.
germination – the emergence of the plant embryo from the seed.
mutant – a change in the DNA from the wild-type form to produce a different trait.
pollination – transfer of pollen from the male portion of a flower to the female portion. In crosspollination, pollen is transferred from plants with different ecotypes.
radicle – the growing tip of the new root of the plant embryo.
senescence – programmed cell death, in which the plant cells stop carrying the basic functions of
life. This step is necessary to open the seed pods and allow the seeds to be dispersed.
transformation – the addition of foreign DNA into an organism to change the normal traits.
Appendix A: Using Micropipettes
Appendix A: Using Micropipettes
47
Appendix A: Using Micropipettes
Micropipettes are used to transfer small amounts of liquid. The micropipettes used at the
Center are calibrated for different liquid volumes and are very accurate when used
properly.
Setting up the Micropipette
The first step in using a micropipette is to choose the correct
size. Micropipettes come in sizes ranging from a maximum
capacity of 10 µL to a maximum capacity of 1000 µL (1 mL).
For sterilizing seeds, the 1000 µL micropipette is large enough
to handle all fluid transfers. These micropipettes are labeled
either “P 1000” or “200 – 1000,” depending on the brand.
Once an appropriate pipette is chosen, the volume must be set.
For the 1000 µL micropipette, the setting should read 1000 in
the volume window (Figure 27). To change the setting, move
the black ring counter clockwise to unlock the dial and turn the
knob at the end. Once set, lock the dial by turning the ring
clockwise.
Using the Micropipette
Figure 27: 1000 µL
settings
Once the micropipette is setup properly, it is ready
for use. Refer to Figure 28 for details of stop
positions.
1.
2.
3.
Place a tip on the micropipette. Tips are
kept in labeled boxes and are usually
already sterile. For the 1000 µL pipette, use
the blue 1000 µL tips.
Press the plunger from the rest position to
the first stop position using your thumb.
Insert the tip into the fluid to be transferred.
Figure 28: Micropipette stop
positions
48
Care of Arabidopsis
4.
5.
6.
7.
8.
Slowly release the plunger, drawing up the fluid. For planting seeds, make sure to
pick up only one seed.
Move the tip to the new container or soil pot.
Slowly press down on the plunger to expel the liquid. Press down all the way to
the second stop.
Release the plunger.
Eject the tip. Throw away the tip.
Tip – The same tip can be used to plant all of the seeds of the same type.
Use a clean tip for each cleaning step.
Appendix B: Sterilization Solutions
Appendix B: Sterilization Solutions
51
Appendix B: Sterilization Solutions
70% Ethanol Solution
1.
2.
3.
Add 40 mL of 95% ethanol to a labeled 50 mL conical tube.
Add 10 mL milliQ water.
Screw on cap and shake vigorously for 1 minute.
This solution can be kept on a shelf if the tube is tightly sealed.
20% Bleach / 0.1% Tween-20
Tween-20 is a powerful detergent used to remove any oils from the seeds. Tween-20 is a
very thick fluid, so pipette slowly to make sure you get all of the detergent.
1.
2.
3.
4.
Add 10 mL household bleach to clean, labeled 50 mL conical tube.
Add 50 µL Tween-20 to tube using a micropipette.
Add 40 mL milliQ water.
Gently shake for 2 minutes.
This solution can be kept for up to one month. After that, the Tween-20 begins to
precipitate out of the solution.
Sterile milliQ Water
1.
2.
3.
4.
5.
6.
7.
Add 60 mL milliQ water to clean, labeled screw top jar.
Barely screw on the lid. If the lid is too tight, the jar could explode in the
autoclave.
Add a piece of autoclave tape to the top of the lid.
Autoclave for 20 minutes under the “Slow Exhaust” setting.
Remove the water from the autoclave using autoclave gloves.
Allow the water to cool to the touch.
Tightly screw on the lid.
The water can be kept indefinitely as long as it is only opened under sterile conditions in
the clean room.