INTRODUCTION TO PLANT BREEDING

AGRONOMY 815 / COURSE NOTES

P. STEPHEN BAENZIGER, 338 Keim Hall, 472-1538

DEPARTMENT OF AGRONOMY / UNIVERSITY OF NEBRASKA

GENETIC ENGINEERING

In general, genetic engineering refers to the isolation of a gene from one organism and then the introduction of the gene into another organism.

How are genes isolated? If a protein is known, then a portion of the DNA sequence can be estimated from the amino acid sequence and used as a probe (probe -- a small piece of DNA or RNA which will hybridize with a larger segment). The larger segment is made by isolating DNA and then cutting it with "restriction enzymes" -- enzymes that only cut at very discrete regions. If the probe is radioactive, the large segment can be identified using X-ray film to identify its position on a gel. Also specialized films or columns will separate single stranded and double stranded DNA. Once the large segment is identified, it can be sequenced or cut with different restriction enzymes to determine which part of the DNA is the functional gene.

If no protein is known, the gene may be identified by transposon mutagenesis (a transposable element of known DNA structure randomly inserts itself in the genome causing mutations at the insertion site). By isolating the transposable element, the accompanying gene can be identified. This allows genes, where only the phenotype is known, to be isolated.

If a gene is isolated in one species, its DNA can often be used to isolate the gene in a different species, (heterologous probe).

How will the gene be regulated? With more genes being isolated and studied, more information is being developed on the regulatory sequences. Example: chlorophyll proteins are often regulated by light. Hence, any gene that has their regulatory sequences will be light regulated. Seed storage proteins are only expressed in seed, hence their regulation will be specific to seeds. Similarly there are flower specific, etc. regulatory sequences. For strong expression sequences, often the regulatory sequences from plant pests (viruses) are used. The gene will be expressed in all plant tissues throughout the lifetime of the plant.

Sometimes it is beneficial for plant breeders to reduce the effect of a gene. Transformation only adds something. How do you take a gene away -- anti-sense DNA. By making a copy of the non-template DNA, can transform plants with the DNA that makes the wrong strand of mRNA. The correct and incorrect strands of mRNA interact and the effect of the gene is greatly reduced because there is less "effective" mRNA.

How is the isolated gene with its proper regulatory sequence introduced into a new organism? Usually, by the use of Agrobacterium tumefacians, a naturally occurring genetic engineer. DNA can also be taken up by direct DNA uptake (soaking), or other mechanical means (particle-gun, electroporation, osmotic shock, etc.).

How will the transformed cell or plant be identified in population of cells or plants? In addition to the regulatory sequence and the gene of interest, there is usually added a gene that is a selectable marker (resistance to an antibiotic, etc.). The transformed plants survive the "challenge," while the untransformed cells and plants die. Selectable markers are very important in genetic engineering because they allow the finding of transformed cells and plants which are rare events.

So far, plant expression of foreign genes has been relatively stable (they are not lost in propagation) and behave in a Mendeliam manner. Where they are inserted can change their level of expression. A rule of thumb is that 20 insertions must occur (i.e., 20 lines must be transformed) before a suitable insertion with proper gene expression is identified.

The power of this technique is that it greatly increases the amount of genetic variation that is available for plant breeding. The biosphere becomes a genetic resource. It also can make linkage work for the plant breeder because the inserted DNA is usually small (hence, no crossovers) but may contain a few genes which, due to no crossovers, are tightly linked.