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. 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

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