The Use of Membrane Rafts in Somatic Embryogenesis

The Use of Membrane Rafts in Somatic Embryogenesis:
Results of 12 Selected Lines of Norway spruce (Picea abies, L. Karst.)

The following application note is based on a short publication^¹ and the figures from the lecture by Martin Vagner et al.^² at a symposium devoted to the use of liquid systems for in vitro plant culture.

1 Martin Vagner, Zuzana Vondrakova, Lucie Kurnstyrova and Jana Opatrna

Institute of Experimental Botany AS CR, Rozvojova 135, 165 02 Prague 6, Czech Republic,

Email: vagner@ueb.cas.cz

2 1^st Int. Symp. “Liquid Systems for in vitro Mass Propagation of Plants”, As, Norway, May 29^th –June 2^nd, 2002.

Semi-Solid vs. Liquid Suspension Culture in Somatic Embryogenesis

Liquid culture is preferred for cost saving and automation of production techniques. However, semi-solid media are predominantly used in the majority of protocols for conifer somatic embryogenesis because of the difficulty in adapting the growth conditions in liquid suspension culture to the embryo requirements. Examples of the types of problems encountered may be seen in Figure 1, which compares the morphology of somatic embryos grown on semi-solid (agar) medium versus those grown in liquid medium.

**Figure 1.**

C	D
Figure 1. A. C111 Embryos proliferated on Agar. B. C111 from Suspension Culture. C. Agar Matured Embryo. D. Suspension Culture Matured Embryo. Cells grown in suspension culture are clearly shorter and thicker than those from agar, while the matured embryos do not develop cotyledons, and have problems with their epidermal layer. They also suffer from hyperhydricity, as evidenced by the fact that their water content is 86-90%, versus the 70-75% for normal embryos.

LifeRafts as a Solution

LifeRaft has a number of advantages over solid media and suspension culture, which may be seen in Figure 2. As with solid medium, it keeps the embryos well aerated at the surface. Hyperhydricity is avoided due to a minimum amount of contact with liquid medium held in the 25 micron membrane pores.

Embryo production is easily automated, as they can be moved from each stage of development by simply lifting the raft out of the old medium and placing it in a vessel containing the new medium. Alternatively, opening the lid, suctioning out the old medium and introducing new medium can exchange the medium.

While the study only used the Magenta sized rafts & vessels, large scale pilot or commercial production can be carried out more economically in the LifeGuard vessels using 90 x 90mm rafts.

Figure 2. A. 25 micron membrane LifeRaft and float. B. Top view of embryos on LifeRaft. C. Side view of embryos on raft and float.

A.		B.
C.

Results of Culturing on LifeRaft vs. Agar

Table 1. Comparison of 25μm membrane LifeRaft System to Solid Medium:

	LifeRaft	Solid Medium
Embryo Morphology	Normal	Normal
# of Developed Embryos per gram of FW culture	7 of 12 cell lines show greater #’s of mature SE	Lower than Liferaft, greater than suspension
Time to embryo maturation	Average of 6 days shorter time	Longer than raft, same as suspension
Germination Frequency	Statistically identical for 9 lines, significantly greater for 3 cell lines
Laboratory Time Required	Much faster	More involved than raft or suspension culture

6 Week Profileration on 25 μm raft
F2 Generation from Microtubers

3 Week Dessication

Germinated Embryo

Martin Vagner, Zuzana Vondrakova, Lucie Kurnstyrova and Jana Opatrna

Institute of Experimental Botany AS CR, Rozvojova 135, 165 02 Prague 6, Czech Republic,

Email: vagner@ueb.cas.cz

1^st Int. Symp. “Liquid Systems for in vitro Mass Propagation of Plants”, As, Norway, May 29^th –June 2^nd, 2002.

Literature|Links|

LifeRafts as a Solution

Results of Culturing on LifeRaft vs. Agar

Germination Frequency