General Cycad Somatic Embryogenesis Protocol
Most of our experiments in the past have focused on species in
the genera Zamia and Ceratozamia. While work on
these genera is still continuing, we have now expanded our
efforts to include: Cycas, Dioon, Encephalartos,
Lepidozamia, Macrozamia and Microcycas.
Note: All photographs were taken by Richard Litz.
I. Protocol
II. Media
III. References
IV. Links to Some Cycad Sites
I. Protocol
 Young, succulent, non-expanded pinnae are
removed from the rachis. These are
surface-sterilized in 5-20% commercial bleach to which a
few drops of Tween® 20 have been added. The exact
concentration of bleach and Tween® used varies with
species and stage of development.
An additional immersion in 70-95% ethanol can be added,
especially for extremely hirsute species.
After surface-sterilization, the material is rinsed three
times in sterile, deionized water and blotted on sterile
paper toweling.
Pinnae can be plated to induction media entire or sliced
into segments.
Cultures are maintained in the dark and, generally,
transferred monthly.
|
 |
| Cycad somatic embryos proliferate and develop
well, but cultures are generally not synchronized. |
 |
The
embryos may be monocotyledonous or dicotyledonous, or the shoot
apex may be enclosed in a coleoptile-like sheath.
After
root germination, the somatic embryos are transferred
individually to test tubes containing a medium with activated
charcoal.
Shoot
germination occurs in this medium.
II. Media
Unless otherwise specified, all media are solidified with Gel-Gro™ at 2 g/L. All media are adjusted to pH 5.8 before
autoclaving.
Basal Induction Medium
This medium consists of Gamborg B5 major salts with MS minor
salts and organics, glutamine (400 mg/L) and sucrose (20-60 g/L).
Arginine and asparagine (each at 100 mg/L) may also be added.
For Ceratozamia mexicana var. Robusta, the optimal
growth regulator combination for callus induction was found to be
kinetin (4.7 µ) and 2,4-D (4.5 µ); the best combination for C.
hildae was determined to be kinetin (1.2-4.6 µ) and 2,4-D
(4.5 µ).
Maturation Medium I
This medium is composed of a modified Gamborg B5 major salts
formulation that contains no ammonium nitrate, MS minor salts and
vitamins, glutamine (400 mg/L) and sucrose (20-60 g/L).
III. References
Chavez, VM, Litz, RE, Moon, PA and Norstog, K (1992) Somatic
embryogenesis from leaf callus of mature plants of the Gymnosperm
Ceratozamia mexicana var. Robusta (Miq.)Dyer (Cycadales).
In Vitro Cell. Dev. Biol. 28P: 59-63.
Chavez, V, Litz, RE, Vovides, A and Norstog, K (1994) Somatic
embryogenesis from leaves of a mature plant of Ceratozamia
euryphyllidia (Cycadales, Gymnospermae), an endangered
species. VIIIth International Congress of Plant Tissue and Cell
Culture, 12-17 June, 1994, Firenze. abstract S11-22.
Litz, RE, Moon, PA and Chavez, VM (1995) Somatic embryogenesis
from leaf callus derived from mature trees of the cycad Ceratozamia
hildae Gynmospermae. Plant Cell, Tissue and Organ Culture 40:
25-31.
Litz, RE, Chavez, VM and Moon, PA (1995) Somatic Embryogenesis
in the Cycadales. IN: Jain, S, Gupta, P and Newton, R (eds.)
Somatic Embryogenesis in Woody Plants, vol. 3. pp. 1-15. Kluwer
Academic Publishers, Dordrecht.
IV. Links to Some Cycad Sites
The Cycad Society
The
Cycad Pages
The Virtual
Cycad Encyclopedia
Gymnosperm Database
Home Page
Palm and Cycad
Societies of Australia
Introduction
to the Cycads (UC-Berkeley)
Yahoo Groups -- home of the Cycad
Listserv
go
back to the Plant Tissue Culture at TREC Page
If you have any questions or comments about cycad tissue culture,
please send them to Pamela
Moon.
