Transcript Arabidopsis Handling

Best Practices
For researchers using
Biotech Support Facility & Rightmire Hall
Growth Chambers and Greenhouse
Introductions
 Gary Posey, Greenhouse Superintendent
 Day to Day at BSF & Rightmire
 posey.8 292-9241
 Joan Leonard, Program Manager
 Day to Day at Bio Sci Greenhouse
 Oversee renovations and implementation of
best practices at BSF & Rightmire
 leonard.4 292-7904
Why?
1. Reduce/ eliminate pest and disease
issues & improve plant health
2. Increase experiment success
3. Improve uniformity and repeatability
4. Abide by all regulations set by NIH,
APHIS, EPA, ODA and Federal laws
Planting & Growing “Do’s”
 DO use clean and sterile pots and trays
 DO label all pots/flats with researcher name -unlabeled plants will
be considered abandoned and will be disposed of.
 DO surface sterilize seed (10% bleach, 10 min.)
 DO follow order of entry to chambers/growth rooms/greenhouse
 DO water and fertilize properly
(use care not to overwater)
 DO wear dedicated lab coat for each growing space
 DO know how to ID thrips, fungus gnats and signs of INSV
 Do make sure all staff and students have read the best
practices and met with Gary for a training session.
Potting Room 100 BSF
 No seeds are permitted in the potting
area, room 100 BSF to prevent
contamination of media
 Fill pots in 100 BSF then plant seeds in
room 160A, the greenhouse or your lab
 For Arabidopsis, 3 scoops of media = 1
flat. Incorporate 2 TBL of osmocote for
each flat prepared.
Photo courtesy of Purdue University
Proper Watering
 Arabidopsis IS NOT AQUATIC!
 Overwatering: #1 cause of plant diseases, pest
problems and plant death! Too much-Too often.
 Overwatering weakens roots and allows pests and
diseases an opportunity
 Never leave plants in a tray of standing water. Soil will
reach saturation within 10 minutes—excess water in
trays after this will only weaken the plants.
 Green algae growing on soil surface is sure indication
of overwatering.
 Plants found standing in trays of water will have holes
poked into the trays!
Quantity




Watch nested flats: water can get “trapped”
Only water pots/cells that are dry instead of whole flat.
Know if plants need water—what is dry?
If bottom watering whole flat, add only enough water to
cover bottom of flat. This should get taken up within a
few minutes. If not, then plants were not thirsty.
 Pour off excess water after 10 minutes!
 Never leave standing in water longer than 30 minutes.
Water not used in 30 minutes will deprive roots of
oxygen.
Frequency
 Check daily BUT only water as needed!
 Water individuals when dry rather than watering all
plants in flat at same time
 Don’t follow a rigid watering schedule
 Avoid “watering insurance”
 This is overwatering to compensate for not checking on plants
daily.
 Heavy watering on Friday will make sure plants don’t need
any over weekend and possibly even until following Friday or
longer!
 No need to overwater to make it through the weekend. A wellwatered plant on Friday will be fine on Monday.
 Rarely have we seen underwatered plants! This is easily fixed
by adding water. Overwatering cannot be easily fixed.
Disposal “DO’s”
 DO cover all plants during transport and disposal
 DO promptly dispose of unnecessary plants in yellow
steam carts (no plastics, no stakes, etc)
 DO keep growing spaces and adjacent areas, and
potting room 100 clear of debris/dirty pots/dead plants
 DO report infestations of pests and diseases to
manager
 DO immediately discard all INSV infected plants
 DO make sure all lab staff are educated in
procedures and are following them. This is the
responsibility of the PI.
Handling “DON’Ts”
 DON’T leave chamber/rooms doors
hanging open
 DON’T move plants from one
chamber/room to another
 “DON’T put all your eggs in one
basket.” (Stagger germination dates and
location of valuable plants to increase
chances of success.)
Thrips ID
 Western Flower Thrips
Adults are less than 1/20th of an inch
(<2mm) long
Eggs are laid in epidermis on underside
of leaf
Only larval stages acquire INSV while
feeding
Pupal stages occur in soil
Adults spread INSV when feeding
Fungus Gnat ID
Larvae feed on plant roots
& spread diseases
Slows plant growth & can
kill seedlings
Thrive in wet soils
Best control is prevention
through proper watering—
do not overwater
Powdery Mildew
Common in crowded conditions
with poor air circulation, low
light and dampness—AKA a
growth chamber!
Spores are short-lived and
need moisture to germinate.
Sleeved plants are especially
susceptible—cut holes in
sleeves for better air flow.
Water carefully!
INSV in Arabidopsis
 Dead Leaves
INSV in Arabidopsis
 Yellow Mid-Rib
INSV in Arabidopsis
 Purple Leaves
INSV in Arabidopsis
 Random Death
INSV Handling
 Plants testing positive for INSV will be
immediately removed from growth spaces and
destroyed.
 No new plants will be permitted in an infected
space.
 Any plant to be removed from an infected
space must be bagged before removal.
 Anyone working in an INSV infected space is to
immediately exit the building when work is
completed- they may return the next day.
Beneficials
 N. cucumeris
Feeds on Thrips larvae
Beneficials
 Hypoaspis miles
Feeds on Thrips pupae and Fungus Gnat larvae
Beneficials
 Nematodes
feeds on Fungus Gnat larvae
Space Rotation
It is critical to break the thrips lifecycle by
enforcing a chamber clean out every 3 months.
 All chambers in 005 Rightmire and in Biotech Support
Facility are common shared space.
 Researchers requiring a chamber with special temp or
daylength for a specific project will need to pre-arrange
space with Gary.
 There will no longer be any continuous cycling in
chambers—all chambers will enter the rotation cycle.
Space Rotation Detail
1
2
Planting
Window
~6 weeks
Maturation Window
Planting
Window
~6-7 weeks
Clean Planting
out
Window
Maturation Window
Clean
out
• Growth spaces will have a 6 week window for planting. This
window may close sooner than 6 weeks IF space is full or INSV is
detected. (Check for available space BEFORE planning to plant!)
• Spaces will have posted on them the last date for new plants. No
new plants will be allowed in the space after the end date —even if
there is space available.
• No plants are permitted to move from one space to another.
• Once a space is empty, it will be cleaned by GH staff and heat
treated for 4 days, then reopened for planting.
Order of Entry
 Order of entry is posted on the dry erase boards in the
BSF and Rightmire 005. Information is updated weekly
based on actual pest counts.
 Users should note these daily as they go about their
work to make sure they are following the correct order
of entry.
 Users who have entered a space lower on the list
cannot enter or re-enter a space above it on the list
until the next day.
 Dedicated lab coats are required for all growth spaces.