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Genetic Engineering. Topics. Concepts. Agrobacterium Gene gun Protoplasts Anti-sense technology. DNA code is universal Plants can be transformed with “foreign” DNA using one of several methods Antisense technology can eliminate a trait Successful genetic engineering
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Genetic Engineering Topics Concepts • Agrobacterium • Gene gun • Protoplasts • Anti-sense technology • DNA code is universal • Plants can be transformed • with “foreign” DNA using one • of several methods • Antisense technology can • eliminate a trait • Successful genetic engineering • depends on a plant’s offspring • carrying the new gene Terms • Cloning • Agrobacterium • Plant hormones • Callus
Damage done by Lepidoptera Corn borer Gypsy Moths Monarch Butterflies Cabbage Butterflies
Bacillus thuringiensis is an effective biocontrol agent Intact bacteria Spore Bt-toxin helios.bto.ed.ac.uk/ bto/microbes/bt.htm
Scientists have used genetic engineering to move the Bt-gene into plants
CLONING • Cutting and splicing genes • Generating an exact copy • Cells • Organism (i.e potato)
CLONING: 2 definitions • Cutting and splicing genes • Generating an exact copy - cells - organism (i.e. potato)
In nature, Agrobacterium carries a Tumor-inducing (Ti) plasmid Genomic DNA Ti-plasmid (tumor-inducing) Agrobacterium
The Ti-plasmid carries Transfer DNA (T-DNA) T-DNA Plant promoter Plant hormone genes Opine Synthesis genes
Agrobacterium delivers the Transfer DNA (T-DNA) to the plant cell Agrobacterium Plant Cell
The Transfer DNA (T-DNA) integrates into to the plant DNA Agrobacterium Plant Cell
The Transfer DNA (T-DNA) integrates into to the plant DNA Plant promoter Plant hormone and opine Synthesis genes Plant Cell
Agrobacterium forces plant to become a opine factory http://helios.bto.ed.ac.uk
In genetic engineering, scientists highjack the Ti-plasmid and clone the Bt-toxin gene into it Ti-plasmid Plant promoter Bt-toxin gene T-DNA
The Transfer DNA (T-DNA) integrates into to the plant DNA T-DNA + Bt-toxin gene Agrobacterium Plant Cell
Goal: To get your gene of interest into every cell of a plant If you want to sell your genetically modified seed – EVERY seed needs to have the “new” gene
3 ways to get your gene into every plant cell • Use Agrobacterium to transform the gametes of a plant, then grow a plant from the seeds after pollination. • Use Agrobacterium to transform plantprotoplasts, then regenerate plants from those cells. • Use gene gun to transform plantcells, then regenerate plants from those cells.
Genetically engineering plants: floral dip Flowers Dip flowers in a solution of Agrobacterium (with your gene plus herbicide- resistance gene)
Agrobacterium can move into pistil and specifically transform the ovules (gametes) Bent lab, U. of Wisconsin
Transforming plants: floral dip Flowers Dip flowers in a solution of Agrobacterium (with your gene plus herbicide- resistance gene) Seeds growing on herbicide: only seeds with herb.-resistance can grow Seed pods Bent lab, U. of Wisconsin
Only Seedlings with T-DNA integrated into their own DNA can survive in presence of herbicide EH, U. of Wisconsin Success Rate = 1/ 3000
Floral dip transformation has only been successful for a few plants (dicots)
3 ways to get your gene into every plant cell • Use Agrobacterium to transform the gametes of a plant, then grow a plant from the seeds after pollination. • Use Agrobacterium to transform plantprotoplasts, then regenerate plants from those cells. • Use gene gun to transform plantcells, then regenerate plants from those cells.
Piece of Potato Leaves J. Helgeson, UW-Madison
Protoplasts Released from Leaves (cell walls stripped) See 541-542 in Life
Transform protoplasts with Agrobacterium T-DNA (engineered withgene of interest plus herbicide-resistance gene) Agrobacterium cell
Grow protoplasts in a plant medium that contains: • Plant hormones (to stimulate cell division and differentiation) • Herbicide (to prevent non-engineered cells from growing)
Clumps of plant cells grow (called callus). Only cells with herbicide-resistance gene will grow.
New plant will grow from callus … and every cell will contain your gene of interest...
…including the gametes which will provide the gene to the next generation.
3 ways to get your gene into every plant cell • Use Agrobacterium to transform the gametes of a plant • Use Agrobacterium to transform plant protoplasts (stem cells) and regenrate plants from those cells • Use gene gun to transform plant cells and regenerate plants from those cells
Transform plant cells directly with gene gun (especially monocots) Plant cell Gold beads covered with DNA from your gene of interest + herbicide-resistance gene
The gene gun can be used to shoot callus. Then regenerate a plant from transformed callus tissue. (Grow it in a medium with plant hormones & herbicide.)
Steps in Genetic Engineering Step 1: Isolate your gene of interest. Step 2: Clone into a Ti-plasmid:your gene of interest plus an herbicide-resistance gene. Step 3: Transform your plant the altered Ti-plasmid. Step 4: Regenerate entire plant from transformed cells, callus, or seeds.
Softening is due to destruction of pectin Pectin: Cell rigidity PG Polygalacturonase
Softening is due to destruction of pectin Pectin: Cell rigidity PG Polygalacturonase
Antisense Technology: How to prevent PG enzyme from being made SENSE ANTISENSE P TATATACGAAGGCAC…… ATATATGCTTCCGTG…… TACGAAGGCAC..TATA ATGCTTCCGTG…ATAT P AUGCUUCCGUG UACGAAGGCAC… AUGCUUCCGUGUACGAAGGCAC Double stranded RNA cannot be translated because it binds with antisense mRNA.
Antisense Technology Tomato plants carrying the antisense PG gene showed 95% reduction in PG enzyme. These Flavr-Savr tomatoes ripened on the vine but stayed firm for a longer period of time.
Issues in Genetic Engineering What are the impacts of genetic engineering? • Biological or environmental • Socio-economic • Political or legislative