Plantbio - Week9handout-2003

Week 9 Handout

A03: Amanda Rajapaksa [email protected] OH: M 3-4@CLICS /by appt.

Agrobacterium Review: Agrobacterium tumefaciens is a free-living soil bacteria that infects plants and induces the formation 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 well as 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

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Tmr: Cytokinin, 1

Q: Fill in the chart! GENOTYPE WT Agro Single mutants: Double mutants:

tmr+ tms+ tmr+ tms tmr- tms+ tmr- tms-

HORMONES PRODUCED Auxin, cytokinin Cytokinin Auxin No hormones

TUMOR? Gall (Callus in TC) Shooty tumor Rooty tumor 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.

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

1. Grind up leaf to produce fragments

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2. Treat fragments with hydrolases to digest the cell walls o Result = protoplasts (individual cells without cell walls)

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3. Maintain cells in culture with nutrients and osmoticom (isotonic solution) o Over time, protoplasts will regenerate and rebuild its cell walls

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4. Place cells on an agar plate and add auxin and cytokinin o Cells divide and produce clumps of tissue – callus 

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Undifferentiated growth

5. Transfer callus to a new plate without auxin (only cytokinin) o Shoots form off of callus

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6. Transfer shoot cutting to a new agar plate with only auxin (no cytokinin) o Roots form!

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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 was simply 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 able to 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. 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, selectable marker, etc

o Deleted Ti plasmid 

Plasmid without T-DNA

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Does contain vir genes •

vir genes from the Ti-plasmid mobilize the T-DNA

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Both plasmids are needed

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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?

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Industrial Agriculture o What does it rely on? o What are the advantages? The disadvantages?

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Video: Harvest of Fear o What are some of the examples used in the film?