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Week 9 Handout A03: Amanda Rajapaksa

arajapak@ucsd.edu 

OH: M 3-4@CLICS /by appt.

Agrobacterium Review:

 Agrobacterium tumefaciens is a free-living soil bacteria that infects plants and induces theformation of ___gall tumors (undifferentiated cells)___. The Agro bacteria create a protein

bridge to the plant cell and transfers DNA to the nucleus where the T-DNA integrates.

T-DNA transforms the plant cells via oncogenes ( __tmr__ and __tms__). These genes, as wellas the __opine__ biosynthesis genes, are found on the T-DNA section of the Ti plasmid. All the

genes on the T-DNA are eukaryotic, so they can only be expressed in eukaryotic cells.

Q: What does tms code for? And tmr? (How many steps does each take?)

-  Tms: Auxin, 2

-  Tmr: Cytokinin, 1

Q: Fill in the chart!

GENOTYPE HORMONESPRODUCED

TUMOR?

WT Agro tmr+ tms+ Auxin, cytokinin Gall (Callus in TC)

Single mutants: tmr+ tms - Cytokinin Shooty tumor

tmr- tms+ Auxin Rooty tumor

Double mutants: tmr- tms- No hormones None

Q: You have a mutant Agro that can only create a rooty tumor. What hormone is it creating?

What would you need to add in order for it to create a callus?

-  It is creating auxin; need to add cytokinin

Similar results can be observed in tissue culture by varying the levels of hormones applied to

plant cells in culture. Shoot or root growth can be induced by varying the levels of hormone

applied.

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Plant Regeneration:

Q: What are the steps in regenerating a plant from a single leaf?

-  1. Grind up leaf to produce fragments

-  2. Treat fragments with hydrolases to digest the cell walls

o  Result = protoplasts (individual cells without cell walls)-  3. Maintain cells in culture with nutrients and osmoticom (isotonic solution)

o  Over time, protoplasts will regenerate and rebuild its cell walls

-  4. Place cells on an agar plate and add auxin and cytokinin

o  Cells divide and produce clumps of tissue –  callus 

  Undifferentiated growth

-  5. Transfer callus to a new plate without auxin (only cytokinin)

o  Shoots form off of callus-  6. Transfer shoot cutting to a new agar plate with only auxin (no cytokinin)

o  Roots form!

-  7. FINAL RESULT = new intact plant (shoot and root)

Plant Transformation:

By introducing new genes into T-DNA on Agro, and then using Agro to transfer the genes into

the plant genome, plant biologists are able to produce transgenic plants. If the new gene wassimply spliced into the T-DNA region to produce a chimeric Ti plasmid, the cells infected with

the new Agro would form __transformed callus___.

Q: Can the transformed callus be used to generate an intact plant? Why or why not?

-  No, the tumor cells produce an overabundance of hormones; therefore, skewing the

hormone levels will not result in shoot/root growth

Modified T-DNA:

By eliminating ___oncogenes and opine genes___, plant biologists were able to create a

modified T-DNA that could be used in plant transformations. With this, plant biologists are ableto use hormones and generate an __intact transgenic plant__ from the transformed cells. Every

cell in the transgenic seedling will have the new gene.

Q: How are the transformed cells separated from the non-transformed ones?

-  Selectable markers are used in the modified T-DNA 

o  this allows you to select for those cells which are transformed  

o  usually an antibiotic resistant gene, such as a gene that confers resistance to the

drug: Kanamycin 

Binary Plasmids:

Because the Ti plasmid is so large, it is difficult to get the genes into the plant cell. To increase

the efficiency of transformations, plant biologists began using the binary plasmid system.

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Q: What are the two plasmids that are used in the binary plasmid system?

o  T-DNA plasmid (without vir genes)

  Plasmid that contains T-DNA, multiple cloning sequence, selectablemarker, etc

o  Deleted Ti plasmid

  Plasmid without T-DNA

  Does contain vir genes

  vir genes from the Ti-plasmid mobilize the T-DNA-  Both plasmids are needed

-  Vir genes don’t need to have the LB and RB on the same plasmid to work  

Things to focus on for this week’s lectures: 

-  Green Revolution

o  Who was involved?

o  Why was this so important?

o  How did they accomplish the Green Revolution?

  New plants, new farming techniques, etc

o  What were the advantages of this? The disadvantages?

-  Industrial Agriculture

o  What does it rely on?

o  What are the advantages? The disadvantages?

-  Video: Harvest of Fearo  What are some of the examples used in the film?