Lum:Michael P.

Plant-Microbe Research

I am currently working on multiple projects in the Lum Lab.

• One project is to identify the nodulating bacteria associated with Lupinus chamissonis and to determine the role that heavy metals (particularly zinc) play in the symbiosis. This is of importance because of the high levels of heavy metals, such as zinc and copper, found in the Ballona wetlands, (adjacent to Loyola Marymount University) where L. chamissonis grows.
• Another project is to characterize the nodulation patterns of Caesalpinoid legumes, which are typically understudied.

Protocols

Seed sterilization

Make sure all materials are sterile

L. chamissonis

1. Scarify seeds on sand paper
2. Sterilize seeds with 20% bleach for 5 minutes, agitating constantly
3. Remove bleach
4. Rinse seeds with water for 10 minutes, agitating constantly
5. Remove water
6. Repeat steps 4 & 5 a total of 6 times

Chamaechrista

1. Scarify seeds on sand paper
2. Sterilize seeds with 70% ethanol for 5 minutes
3. Remove ethanol
4. Sterilize seeds with 50% Bleach for 30 minutes
5. Remove bleach
6. Rinse seeds with water for 10 minutes, agitating constantly
7. Remove water with a pipette
8. Repeat steps 6 & 7 a total of 6 times

Leucena

1. Scarify seeds on sand paper
2. Sterilize seeds with 70% ethanol for 5 minutes
3. Remove ethanol using a pipette
4. Sterilize seeds with 50% bleach for 30 minutes
5. Remove bleach
6. Rinse seeds with water for 10 minutes, agitating constantly
7. Remove water with a pipette
8. Repeat steps 6 & 7 a total of 6 times

Cowpea

1. Sterilize seeds with 70% ethanol for 5 minutes
2. Remove ethanol
3. Sterilize seeds with 50% bleach for 30 minutes
4. Remove bleach
5. Rinse seeds with water for 10 minutes, agitating constantly
6. Remove water
7. Repeat steps 5 & 6 a total of 6 times

Albizia

1. Scarify seeds on sand paper
2. Sterilize with sulfuric acid for 20 minutes (handle acid with extreme precaution)
3. Remove acid
4. Rinse seeds with water for 10 minutes, agitating constantly
5. Remove water
6. Repeat steps 4 & 5 a total of 6 times

Gleditsia

1. Scarify seeds using a razor blade to cut through the seed coat
2. Sterilize with sulfuric acid for 20 minutes (handle acid with extreme precaution)
3. Remove acid
4. Rinse seeds with water for 10 minutes, agitating constantly
5. Remove water
6. Repeat steps 4 & 5 a total of 6 times

PCR for nodule bacteria

• 12.5 µL 2x Phusion Mix
• 0.25 µL 16S-27F Primer
• 0.25 µL 16S-1424R Primer
• 4 µL DNA
• 3 µL Ultra-pure H20

SCASM/SCCUR 2010 Poster

Poster