Notes on Hosta Tissue Culture

John Wessel

www.hostatissueculture.com

5 Distinct Phases Of Doing Tissue Culture

• Preparation of the medium

Preparation of Medium

• 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

• 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

• 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

• 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