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