Plant and Mammalian Tissue Culture Plant Culture Laboratory - Start Flytrap or Rose Culture.

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!