Transcript Research Focused Undergraduate Education

Plant and Mammalian Tissue Culture
Plant Culture Laboratory
- Start Flytrap or Rose Culture
Lab Goals
Become familiar with Plant Tissue Culture
Work aseptically with plant culture
Maintain cultures for an extended period of
time
• Choose Either Rose or Venus’ Flytrap to use for
Micropropagation
• Prepare cultures for stage I and II (rose) or stage II
and III (flytrap)
Create Cloned Rose or Flytrap Plants!
Contamination
What we are trying to avoid!
Venus’ Flytrap
 Grown in moist swamps of North and South
Carolina
 Soil is typically nitrogen poor so plants have
evolved to use nitrogen (amino acids) from “other”
organic matter
 Start with Stage II culture.
Image from wikipedia (Dionaea muscipula closing trap animation)
Venus’ Flytrap
 Prepare work area – sterilize work area with 70%
ethanol, UV light and work with airflow in hood.
Mop area in front and around work area with
10% bleach
Have beaker of 70% ethanol and flame handy
Wear lab coat and tie back hair
NO TALKING – can cause contamination!
Prepare tools for culturing
• Don’t forget, once sterile, avoid placing on non-sterile
surface. Use sterile culture plate!
Venus’ Flytrap
 Culturing
 Remove flytrap from tube with medium
 Place on sterile Petri dish
 Sterilize forceps and disecting tool in ethanol bath and
flame
 “Tease” culture apart into plantlets –
• Use teasing needles in each hand to separate plantlets
Venus’ Flytrap
 Culturing
 Transfer half of the plantlets into multiplication medium
and half into pretransplant medium
 Those plants in multiplication medium (stage II) should be
subcultured 3-4 weeks.
 Those in pretransplant medium (stage III) can be hardened
for transfer to soil in 4 to 6 weeks
Miniature Rose
Micropropagation
Starting with miniature rose plants,
culture into cloned transplant roses
Miniature Rose
Micropropagation
 Nodal Explant Preparation
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Sterilize prep area and scissors w 70% ethanol
Obtain single stem, transfer to workstation
Remove compound leaves
Cut nodes apart
Wash with running water
Node cutting
- Just material
within shaded
area
Miniature Rose
Micropropagation
 Prepare Explant– prepare single stem nodes, sterilize and
place in agar medium.
 Important note: keep nodes wet at all times!
 Stems will be cut from the mother plant
 Set up the work area as shown below.
forceps
4X
50%
EtOH
Transfer
Basket
Sterile
water
20%
bleach
Sterile
water
Sterile
Antiox
70%
EtOH
Transfer dish (Petri)
Sterile
water
Medium Tube
Miniature Rose
Micropropagation
Prepare the Explants
Wash the nodes in each wash to
sterilize the surface of the explant
Using sterilized forceps, place
node into initiation medium
Cap and wait 2-3 weeks –
culture in unsealed zip-lock bag
Miniature Rose
Micropropagation
 After 3 weeks young shoots and leaves
should start to grow.
 Growth hormones promote stem formation
but not root formation (stage II)
 After 6 weeks the culture can be divided
into new initiation medium or placed in
rooting medium
 7-10 days after rooting medium,
plantlets can be placed in soil!