Asceptic Germination of Chickpeas:Development of Transformation

1. Asceptic Germination of Chickpeas:Development of Transformation By Lindsay Stafford and Ken Poff

2. Introduction Agrobacterium Transformation: How is it performed and what is its significance? What other legumes have been transformed? How does Agrobacterium Transformation relate to dicots in general?

3. Objectives for Research Develop some background on chickpeas. Establish the reason for needed Agrobacterium Transformation study with chickpeas. Examine techniques and data gathered from the growth of chickpeas in culture. Use these data to develop a plan for other aseptic dicot culture and Agro. Trans. with plants such as: Sunflowers and Grapes.

4. Hypothesis Chickpeas will be easily grown in culture and these techniques will be useful for growing other dicots.

5. History of Chickpeas Native to the orient originating 7000-6000 BC. A high protein staple of the Mediterranean Basin(climate limitation) A legume that requires high temperatures during the summer months.

6. Methods Imbibition of seeds Steps and Applications Formulation of MS Medium Aseptic Culture Techniques Growth Inspection Application of Outside Knowledge

7. Results Assess Viability of Dried Chickpeas. Determination Successfully Cultured? Trial 1 Trial 2 Trial 3 Savable Tissue Direct Composite

8. Discussion What were the Pros and Cons of this experimentation? Pros Cons What was found overall? Overlook Do any of these gathered data support the original goals?

9. Conclusions and Future Ambitions Successes Goals and Accomplishments Failures Learning From Experience Where Could the Results of this Experiment Aide Future Research? Ambitions and Actions

10. Ken Poff Marcia Ewers The Class of Bot 499 Everyone Here Today

11. Results From First Trial These results were some of my first samples and they helped me to develop a better sense on what could be done for improvement. Back

12. Results From Second Trial These results were quite favorable and will they will be helpful in further research . Back

13. Results From Third Trial 12 Hours of Light 24 Hours of Light After One Week After One Week Back

14. Agrobacterium Transformation has been used on many plants over the years. In general, the technique is described as insertion of transferred DNA or T-DNA via the agrobacterium host. This can be done through a number of methods. Some of these methods include: the wounding of a plant or explant(i.e.: petiole, leaf or anther), whole plant vacuum procedure, or co-cultivation processes. There is a gene created modeling the one below that is used for the specific resistance that the scientist would desire in the resulting plant: The induction of this gene can be introduced into the plant through the methods mentioned before and infect the cells as illustrated below: Back

15. List of Legumes Subjected to Agrobacterium Transformation Research Dry Beans Fababeans Lentils Lupine Pigeon Pea Alfalfa Back Back

16. Dicots Dicotyledons are usually more susceptible to successful agrobacterium transformation because they are able to be transformed through the co-cultivation method. -Monocotyledons are not able to be used in this method This knowledge will be increasingly useful in the completion of agrobacterium transformation techniques. Back

17. Imbibition 1.) Soak Chickpeas in water over night for reviving them from a dried state. 2.) Rinse Chickpeas in 50% bleach solution. 3.) Remove embryo covering from Chickpeas. 4.) Multiple dissection efforts - Sliced in half - Sliced in half fully containing the embryo - Totally removing the embryo from starchy host. Back

18. 1.) Long sleeves must be dealt with. 2.) Hair must be out of the way. 3.) Wash hands and forearms with soap follow by 95% ethanol spray. 4.) Don't touch face or hair during procedures. 5.) Wipe cabinet with an ethanol cloth before and after use. 6.) Spray all non-sterile objects with ethanol. 7.) Always work with most critically aseptic items at the back of the hood. 8.) Flame tops of all things to be poured, probing utensils, and tongs. Back

19. Test determining life: - Reviving the chickpeas - Methods: The chickpeas were soaked over night and several dissection techniques were followed placing each section in a petri dish with a filter paper soaking in water. - Test to verify that they are alive. - Results: The chickpeas showed little or no response to the embryo being removed from the starch host and set to grow on filter paper. However, there was a fairly decent response to the halving effect of the chickpea grown on the filter paper as well as keeping the whole chickpea intact while simply removing the outer covering. Back

20. Pros The results of this experiment were conveniently done in a laboratory. Most of the results were favorable. Techniques were learned that could be used for building blocks in the future. Back

21. Cons Chickpeas were unresponsive by the third try. There was limited time to carry out the project to its full extent. Field research might have added to the experiment rather than relying on books. Back

22. Savable Data First Trial - Was interesting but basically used for testing the basic structure of the experiment. Second Trial - Was the most productive but errors occurred ~Break in experimentation Third Trial - Was not very successful Back

23. Overlook Production of tissue that could be grown further to produce substantial leaves, roots and shoots. Knowledge about techniques that work for chickpeas and could be similar with other dicots. Back

24. Goals and Accomplishments Learned techniques of chickpea cultivation will be useful for experimentation on other dicots. Tricks with Grapes and Sunflowers Chickpea culture is beneficial in research involving agrobacterium transformation. Back

25. Learning Experiments should be done with more time or added supplies to ensure efficient cultivation of chickpeas. More trials should be performed to add more variety to the plants that can then be taken to the next level of the experiment. Back

26. Ambitions and Actions Grow cultivated plants in magenta boxes so they can expand and produce more plant tissue. Harvest plant tissue for agrobacterium induction. Complete test for successfully transformed plants. Back