GE Crops - Techniques of Genetic Engineering Flashcards
What is transformation?
The introduction of a DNA sequence into a plant
Which two methods of transformation work for most plants?
- Biolistic transformation
2. Agrobacterium mediated transformation
Why do we need to be able to select for the plants that got the desired DNA sequence into the chromosome? What do we typically use as a selectable marker?
So we can tell which plants got the desired DNA sequence into the genome so that we can produce more of that plant. Typically use antibiotic resistance, put the seeds on a plate and only the ones that survive will have the desired DNA sequence
How does the DNA get into the plant cell when using biolistic transformation?
Purified DNA is coated onto tiny metal particles and blasted into the plant cells using a gene gun
How do we get the cells transformed with biolistic transformation to grow into new plants?
After selecting for the plants containing the transgene, cuttings are grown in tissue cultures and plant hormones are manipulated to trigger root and shoot formation until we have a new plant
What is Agrobacterium?
A soil bacterium that is a plant pathogen. It has evolved to genetically modify single plant cells to get them to produce opines and proliferate
What two sets of genes are contained in T-DNA of the native Ti plasmid? What do they do?
- Opine synthesis genes - makes the plant cells synthesize opines to feed the bacteria
- Oncogenes - makes the transformed cell proliferate
What is contained in the Ti plasmid normally in Agrobacterium?
The T-DNA region that contains the opine synthesis genes and oncogenes, and the left border and the right border
What are the left and right borders in the Ti plasmid of Agrobacterium? Why are they important for genetic engineering?
Critical regions on either side of the T-DNA that dictate what will be transferred to the plant. We can remove the opine synthesis genes and oncogenes and put whatever we want in there and we know it will get transferred to the plant cells
What are the two plasmids used in Agrobacterium mediated transformation?
Helper and vector
What is contained on the helper plasmid in Agrobacterium mediated transformation?
The virulence genes, everything needed to copy and transfer the T-DNA
What is contained on the vector plasmid in Agrobacterium mediated transformation?
The left border, right border, and the sequence of interest that comprises of a selectable marker and the transgene
What are the steps of Agrobacterium mediated transformation?
- Produce and clone the vector plasmid in E. coli to have the transgene
- Put the vector plasmid into Agrobacterium that already has the helper plasmid
- Dip the flowers of the plant into Agrobacterium
- Harvest the seeds and screen for transformed plants
- Plant the transformed plants back into the dirt
