Seed Science and Technology

LABORATORY EXCERCISE
Encapsulation

Introduction
Encapsulation is the process where a sample tissue is enveloped in a synthetic bead which
acts as a protective layer to the particular tissue. This is widely used in novel
cryopreservation techniques especially in vitrification techniques as the encapsulated plant
specimens prove to germinate well after thawing. The correct methodology of bead
formation should be learnt in order to have the best viable samples.

Objectives
1. To provide simulation for student to perform encapsulation of seeds or other explants.
2. To expose students the importance of encapsulation.
3. To evaluate the effects of various concentrations of sodium alginate and the effects of
polymerisation time (duration).

Materials
Beakers; 1%, 2%, and 3% sodium alginate (NaC6H7O6); 100mM calcium chloride (CaCl2);
Pasteur pipettes; petri dishes; sterile distilled water.

Methodology
Effects of various concentrations of sodium alginate on characteristics of the beads
1. A beaker was filled with 1% sodium alginate.
2. By using Pasteur pipette, at least ten drops of 1% sodium alginate were transferred into
the beaker containing calcium chloride.
3. Slow but constant agitation was done by shaking the beaker for several minutes, to
ensure complete polymerization of beads.
4. Calcium chloride was discarded and beads were transferred into a petri dish filled with
sterile distilled water for ten minutes to stop polymerization process and avoid
desiccation. The beads formed were observed visually.
5. Step 2-4 was repeated for 2% and 3% sodium alginate.
6. Results were recorded.

Relationship between duration of polymerisation and the characteristics of the beads

1. A beaker was filled with 3% sodium alginate.
2. By using Pasteur pipette, at least ten drops of 3% sodium alginate were transferred into
the beaker containing calcium chloride.
3. Slow but constant agitation was done by shaking the beaker for several minutes, to
ensure complete polymerization of beads.
4. Calcium chloride was discarded and beads were transferred into a petri dish filled with
sterile distilled water to stop polymerization process and avoid desiccation.
5. The beads formed were observed visually after 2, 10, 20, and 30 minutes in sterile
distilled water.
6. Results were recorded.

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Results and Discussion

Two tests were studied in this experiment. The first experiment emphasized on the different
concentrations of sodium alginate used in encapsulation bead formation and the second
experiment focused on duration of sodium alginate forming into beads of calcium alginate
(polymerization time).

In the first test, three different concentrations of sodium alginate were used: 1%, 2% and 3%.
Petri dishes with beads made of 1% and 2% sodium alginate shown in Figure A. For 1% of
sodium alginate, the beads formed were not uniformly shaped as it was spherical with a tail.
But the clarity of the beads formed was crystal clear. The beads are easily broken as it is
fragile, hence the elasticity is minimal. As for the 2% of sodium alginate used, the beads
exhibit a more uniform shape; which is spherical and the beads are crystal clear as well. The
beads are not easily broken thus as it is firm and the degree of elasticity showed by the
beads are very elastic and bouncy. 3% of sodium alginate was perfectly sphere, with clear
beads. But the beads were relatively hard and possessed good elasticity.

Figure A: Beads of 1% and 2% sodium alginate

The second test tested on the characteristics of beads formed based on the exposure period
of sodium alginate in calcium chloride. Polymerization time were set to be the parameter in
different periods: 2 minutes, 10 minutes, 20 minutes, and 30 minutes. All the beads formed
were perfectly spherical. As for the clarity, the beads exposed for 2 minutes were all crystal
clear (Figure B); 10 minutes exposure showed normal clearness (Figure C) but as the time
increased to 20 minutes, the beads exhibit turbidity. And for prolonged exposure to 30
minutes, the beads were very turbid (Figure D). The beads formed at the exposure of 2
minutes were too soft and the beads formed at 10 minutes exposure was just soft. For
extended period of 20 minutes, the beads were firm and further prolonging the period to 30
minutes revealed that the beads formed were too hard. The degree of elasticity increased as
the duration of exposure was increased.

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Figure B: Beads formed after 2 mins exposure in calcium cloride

Figure C: Beads formed after 10 mins exposure in calcium cloride

Figure D: Beads formed after 30 mins exposure in calcium cloride

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When encapsulating plant explant for long term conservation through cryopreservation, the
size of the beads (depends on tip size of Pasteur pipette) should be taken into consideration
as well. Large beads causes suffocation to explants that will be cultured as the aeration of
carbon dioxide and oxygen will be limited leading to death of explants. Hard beads simulate
the characteristics of thick seed coat and this will be a barrier towards germination and
growth of explants, thus dormancy occurs. Soft beads, in the other hand, are too fragile and
constantly break. This will not provide a proper encapsulation to the explants and the
explants are prone to environmental damages.

The time taken for formation of beads is closely related to the clarity of beads. When a bead
is turbid, light penetration through the bead will be insufficient for the explants. Thus, light
requiring species for germination may not germinate well.

Conclusion
From this experiment, we become aware and understood about how to perform
encapsulation of seeds or other explants, the effects of various concentration of sodium
alginate, and the effects of polymerization time (duration).

References
Kuhtrieber, W.M., Lanza, R.P., and Chick, W.L. 1999. Cell Encapsulation Technology and
Therapeutic. Birkhauser Publishing, New York, USA.