Notes on Hosta Tissue Culture
John Wessel
www.hostatissueculture.com
5 Distinct Phases Of Doing
Tissue Culture
• Preparation of the medium
Preparation of Medium
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Murashige & Skoog MS
NAH2PO4
KH2PO4
Agar
Sucrose
Benzyladenine BA
Naphtaleneacetic Acid NAA
NAOH
Preparation of Medium
• Heat 1 liter of solution to 180 deg. F
• Pour 25 ml into baby food jars
• Place in pressure cooker
Sterilization
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Hood Preparation
Self
Paper Plates
Instruments
Any thing that enters the hood
Laminar hood
Hosta Crown and Buds
5 Distinct Phases Of Doing
Tissue Culture
•Preparation of the medium
•Preparation of the meristem
Initiation Stage I
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
Meristem Isolation
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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
Meristem Explant
Initiation Stage I
• Place explants in individual test tubes
• Controls contamination
5 Distinct Phases Of Doing
Tissue Culture
•Preparation of the medium
•Preparation of the meristem
•Dividing the explants
Multiplication Stage II
Multiplication Stage II
• Remove from Test Tubes and Divide
• Put 10 to 15 explants in Baby food jars
• Take about four to six weeks
Culture Room
5 Distinct Phases Of Doing
Tissue Culture
•Preparation of the medium
•Preparation of the meristem
•Dividing the explants
•Rooting Stage III
Rooting Stage III
• Transfer to a different medium:
No BA, .01-.05 NAA
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
Acclimation Stage IV
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Place in 1206 trays 72 per tray
Cover with a plastic dome
Lights 24 hours per day
Move to outside after a couple weeks
Growth Room
Greenhouse