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Notes on Hosta Tissue Culture

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Title: Notes on Hosta Tissue Culture


1
Notes on Hosta Tissue Culture
  • John Wessel
  • www.hostatissueculture.com

2
5 Distinct Phases Of Doing Tissue Culture
  • Preparation of the medium

3
Preparation of Medium
  • Murashige Skoog MS
  • NAH2PO4
  • KH2PO4
  • Agar
  • Sucrose
  • Benzyladenine BA
  • Naphtaleneacetic Acid NAA
  • NAOH

4
Preparation of Medium
  • Heat 1 liter of solution to 180 deg. F
  • Pour 25 ml into baby food jars
  • Place in pressure cooker

5
Sterilization
  • Hood Preparation
  • Self
  • Paper Plates
  • Instruments
  • Any thing that enters the hood

6
Laminar hood
7
Hosta Crown and Buds
8
5 Distinct Phases Of Doing Tissue Culture
  • Preparation of the medium
  • Preparation of the meristem
  • Initiation Stage I

9
Meristem Isolation
  • Remove dirt from crown
  • Cut offshoot from crown with a small portion of
    crown material
  • Wash off all dirt from offshoot
  • Peel layers from offshoot
  • Wash off again and remove any blemishes

10
Meristem Isolation
  • Use a scalpel to cut into bud
  • Remove all leaf tissue down to the meristem
  • Put it in a piece of sterile cheese cloth
  • Put in 70 ethyl alcohol for 20 sec.
  • Transfer to 10 bleach solution for 20 minutes
  • Rinse three separate times in sterile water

11
Meristem Explant
12
Initiation Stage I
  • Place explants in individual test tubes
  • Controls contamination

13
5 Distinct Phases Of Doing Tissue Culture
  • Preparation of the medium
  • Preparation of the meristem
  • Dividing the explants
  • Multiplication Stage II

14
Multiplication Stage II
  • Remove from Test Tubes and Divide
  • Put 10 to 15 explants in Baby food jars
  • Take about four to six weeks

15
Culture Room
16
5 Distinct Phases Of Doing Tissue Culture
  • Preparation of the medium
  • Preparation of the meristem
  • Dividing the explants
  • Rooting Stage III

17
Rooting Stage III
  • Transfer to a different medium
    No BA, .01-.05 NAA

18
5 Distinct Phases Of Doing Tissue Culture
  • Preparation of the medium
  • Preparation of the meristem
  • Dividing the explants
  • Rooting
  • Growing on or acclimation
  • Stage IV

19
Acclimation Stage IV
  • Place in 1206 trays 72 per tray
  • Cover with a plastic dome
  • Lights 24 hours per day
  • Move to outside after a couple weeks

20
Growth Room
21
Greenhouse
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