BIO 599 Graduate Independent Study
Karen Beitler
Advisor: Dr. Edwin Wong
Fall 2006

Nature of the project;
Students will be examining the effect of amendments (pH) on Rhizobium-treated legumes in sterile potting soil. Students will grow pea plants in sterile soil that has been treated with a symbiotic nitrogen-fixing bacteria; Rhizobium. Plants will be watered with different pH solutions to determine the effects on the nitrogen-fixing Rhizobium and overall plant grow. The affects of acid rain on this symbiotic relationship will be examined as well as the best pH for Rhizobium fixation. Possible further study may include the identification of the best species of Rhizobium for enhancing plant growth.

The Project involves the nitrogen-fixing bacteria rhizobium and their cohorts the legumes - peas. This unit intertwines biotech and environmental science. Students will be growing a specific type of legume, snow pea, in sterile potting soil. We will seed the soil with Rhizobium and then treat the soil with different amendments - change pH and alkalinity of the water we use to feed them. We are looking for optimal growth of the plant using specific species of rhizobium, specifically if the rhizobium attached and has the nitrogen fixing gene. We will continue standard measurements in the green house through the winter and hope to transfer offspring to an outdoor garden in the spring. We will use PCR to amplify and identify the nif (nitrogen-fixing gene) and DNA fingerprinting to identify species of Rhizobium. We hope to discover a species of Rhizobium that fixes nitrogen for optimal growth in a specific soil amendment. Ultimately, a second experiment will then be to seed the optimal rhizobium species, under the right conditions, into different lawn plants and grasses and see if we can get a symbiotic relationship going with them. If the experiments work as I hope, then my students can spend some time writing to lawn care companies in the state asking for reduction of fertilizers on lawns by making suggestion of plants that will fix nitrogen and reduce cost too! The big picture will be to reduce the number of run-off nitrates from fertilizers into our waterways!

Methods used in the project

  • Grow pea plants to flowering; examine roots for nodules; quantify nodules
  • Use PCR and DNA fingerprinting to show presence of Rhizobium species by presence of their nitrogen-fixing gene(s);
  • (optional) Use PCR and DNA sequencing to determine species of Rhizobium present;.
  • Determine which soil amendment is ideal for plant growth with Rhizobium

Materials
Pots (3˛pots?) and Trays to hold and label by amendment
Sterile potting soil
Rhizobium inoculant
Pea seeds
Watering cans & water
Thermometer
Fertilizer
3 amendments ­ student choice (pH paper, acids, bases???amt of Rhizobium, fertilizer)
Constants for all; light in the greenhouse, temperature, soil, seed, pots, trays
PCR thermocycler
Primers & PCR kit
Micropippetor
Microcentrifuge
Agarose gel Electrophoesis chamber
Electrophoresis buffer
DNA stain

Criteria to evaluate the student's performance
Report & poster for WISTR in Spring 2007

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