Summary - The Backcross Breeding Process

Backcrossing is necessary because the lines that lend themselves well to tissue culture and transformation are typically older, low yielding lines. Plant breeders use backcrossing to transfer the transgene from these older lines into elite, high yielding lines.

The steps of backcross breeding are:
1. Mate a transgenic inbred with an elite inbred. (F1 generation). All progeny will have 1 copy of the transgene and 50% elite genes.
2. Harvest the progeny and mate them back to the elite inbred (BC1 generation). Half of the progeny will have 1 copy of the transgene. All will have 75% elite genes.
3. Select the 50% progeny with the transgene and mate back to the elite inbred. Again, half of the progeny will have 1 copy of the transgene. All progeny will have 87.25% elite genes.
4. Continue selecting out progeny with the transgene and mating back to the elite inbred until the desired percentage of elite genes is reached (usually 98+%). This will take about 5-7 generations.

Screening for transgenic plants Selectable markers - Adding a second gene with an easily detectable trait (often herbicide or antibiotic resistance) during transformation. Usually, the selectable marker inserts at the same location as the trait of interest. Plants are sprayed with the herbicide, and lines that did not receive the transgenes during transformation will die. Utilizing a selectable marker is the quickest most efficient method to screen a large number of lines. ELISA - Tissue samples are taken from a plant a kit is used to test for the presence of the Bt protein molecule encoded by the transgene. Testing time is approximately 5 minutes. Need to know the specific ’event’ being tested for and how that impacts the time of year tissue is sampled, and which tissue can be sampled. PCR - A laboratory test that detects the presence of the transgene (DNA) itself. This method is time consuming and expensive and best used when only a small number of lines need to be tested.

Yield drag is the negative effect on yield potential associated with crop plants that have a specific gene or trait. It can be caused by:
1. The transgene inserting into a gene important for plant growth and yield disrupting its expression.
2. A drain in the limited pool of amino acids caused by having to produce large quantities of new proteins. This limits the amount of amino acids available for the production of other proteins important to plant growth and yield.

Testing for yield drag is done by comparing the yield potential of the backcrossed transgenic line to that of the original elite line (isoline).

Yield lag is a difference in yield potential between a transgenic line and the newest elite lines. The difference is because there is no selection for increased yield during the 3-5 years of backcross breeding while non-transgenic lines have had selection for improved yield potential every year.

Testing for yield lag is done by comparing the yield potential of the backcrossed transgenic line to that of the newest elite inbreds.

Gene stacking combines desired traits into one line. There are two ways gene stacking is accomplished:
1. Transferring two or more genes into the cell nucleus during transformation. The use of a selectable marker in addition to the gene of interest would be considered gene stacking.
2. Mating two plants which each contain a desirable trait to give offspring with both traits. This is the easiest and fastest method of gene stacking.