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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Seed Science and Technology

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.

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