Post on 13-Jan-2016
Plant and Mammalian Tissue Culture
Plant Culture Laboratory
- Start Flytrap or Rose Culture
Lab Goals
Become familiar with Plant Tissue CultureWork aseptically with plant cultureMaintain 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% bleachHave beaker of 70% ethanol and flame handyWear lab coat and tie back hairNO 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 mediumPlace on sterile Petri dishSterilize 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 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.
50% EtOH
Sterile water
20% bleach
Sterile water
SterileAntiox
70% EtOH
forceps
Transfer dish (Petri)Transfer Basket
4X
Sterile water
Medium Tube
Miniature Rose Micropropagation
Prepare the ExplantsWash 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!