Asceptic Germination of Chickpeas:Development of Transformation

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Asceptic Germination of ChickpeasDevelopment of
Transformation

• By Lindsay Stafford
• and Ken Poff

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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?

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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.

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Hypothesis

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

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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.

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Methods

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

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Results

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

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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?

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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

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

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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

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Results From Second Trial

• These results were quite favorable and will they
  will be helpful in further research .
• Back

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Results From Third Trial

• 12 Hours of Light 24 Hours of Light
• After One Week After One Week
• Back

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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

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List of Legumes Subjected to Agrobacterium
Transformation Research

• Dry Beans
• Fababeans
• Lentils
• Lupine
• Pigeon Pea
• Alfalfa
• Back
• Back

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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

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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

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• 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

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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

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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

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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

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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

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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

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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

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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

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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