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 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!