CSB351Y1 Lecture Notes - Lecture 16: Agrobacterium Tumefaciens, Ti Plasmid, Genetically Modified Crops
26 May 2018
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Lecture 16: Biotechnology I
Transgenic animals
• Prion-free cattle
• Transgenic salmon – grow much more quickly with less food
• Transgenic pigs for xenotransplantation (prevent donor rejection) – not enough organs for transplantation to
use
• Biopharmaceutical and therapeutic proteins produced in milk (insulin, monoclonal antibodies)
• Egg and nucleus hybrid between two animals grow then embryo implanted in surrogate mother
• Dolly genetically identical to mammary cell donor
- Problem – old chromosomes (short)
Transgenic plants can be generated from a diverse array of plant tissues
• Undifferentiated carrot cell turned into differentiated by adding hormones to mature mass into real plant
Methods used for plant transformation
• Agrobacterium tumefaciens – all over the place, bacteria in soil
• Biolistic delivery with Gene Gun
• Other:
- PEF (polymer that keeps molecule stable) mediated transformation of protoplasts
- Elctroporation of protoplasts (tricky to use)
◼ Protoplasts are plant cells with cell wall removed (by cellulose)
• Agro is the causal agent of crown gall disease (tumors) in dicotyledonous plants
- Rod shaped bacteria
Generation of transgenic plant via Agrobacterium-mediated transformation
1. Ti plasmid has tDNA and restriction site
2. We add gene of interest in tDNA and make recombinant Ti plasmid and transform them into Agrobacterium to
infect plants
3. Get Ti plasmid to incorporate tDNA into genome of the plant
4. Grow plant with new trait
1. Foreign gene (gene of interest) with antibiotic resistance gene (marker) is incorporated into Agrobacterium
2. Piece of leaf on dish with bacterial suspension (gene transfer)
3. Callus is undifferentiated, add antibiotics (only plant cells with foreign plasmid antibiotic resistance should grow
4. Use hormones to start growing plants that have roots and shoots → potato transgenic plant
Ti (tumor inducing) plasmid
• Agrobacterium contains tumor-inducing plasmid (virulence (vir) genes and transferred DNA (tDNA) region)
• Genes of interest inserted into the tDNA
• WT Ti plasmid has ORFs – Opine, Auxin and Cytokinin which help Agrobacterium form tumor
- Are knocked out when making transgenic plants and replaced with gene of interest
- Cytokinin and auxin – phytohormones cause plant cells to divide uncontrollably
- Opine – AA derivative required for growth of bacteria
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Transgenic plants can be generated from a diverse array of plant tissues: undifferentiated carrot cell turned into differentiated by adding hormones to mature mass into real plant. Methods used for plant transformation: agrobacterium tumefaciens all over the place, bacteria in soil, biolistic delivery with gene gun, other: Pef (polymer that keeps molecule stable) mediated transformation of protoplasts. Protoplasts are plant cells with cell wall removed (by cellulose: agro is the causal agent of crown gall disease (tumors) in dicotyledonous plants. Ti (tumor inducing) plasmid: agrobacterium contains tumor-inducing plasmid (virulence (vir) genes and transferred dna (tdna) region, genes of interest inserted into the tdna, wt ti plasmid has orfs opine, auxin and cytokinin which help agrobacterium form tumor. Are knocked out when making transgenic plants and replaced with gene of interest. Cytokinin and auxin phytohormones cause plant cells to divide uncontrollably. Opine aa derivative required for growth of bacteria.
