Work of the plant breeder part 1: Measuring Phenotypes:

Visual selection with the human eye:

Life is relatively easy when a plant breeder can use their eyes to select plants that possess the desired traits as they are growing in the field. Grain color is the obvious example of this type of trait. One of Dr. Baenziger’s wheat breeding goals is to develop wheat lines that have a white kernel color instead of the red kernel types predominantly grown as bread wheat in the United States. Selection for this trait is straightforward; keep the whites, throw out the reds. Other seed traits such are starch composition or protein levels can relate to the way the kernel looks. However, selection based on visual inspection may be less reliable to the untrained eye. Alternative selection methods need to be used.

“Visual” selection with a machine

Reflection of wavelengths of light that our eyes cannot detect can reveal variation in grain characteristics. Near infrared light reflecting from either whole or ground up grain can be measured to determine the relative amount of protein or oil in the seed sample. The machine that does this is a Near Infrared Reflectance (NIR) machine and it can be programmed to read a sample, calculate a value based on some previous calibration and provide a percent protein or oil value. The test is performed on a family of seeds from the same parent or family of parents. This measurement is typically done in a testing lab and not in the field but it provides the breeder with a relatively easy way to measure these traits. For Dr. Baenziger’s wheat program, protein levels are important determinants of bread making quality. This trait is typically measured with the NIR on hundreds of samples each season.

Fig. 2: White wheat (left) and red wheat (right)

Fig. 3 Grain samples and an NIR machine.

Fig. 4 Grain samples

X-ray based measurements

Most of us have personally experienced an X-ray based assessment of tooth or bone quality. Chemists have found that ground and prepared samples of grain can be subjected to X-rays and the reflectance of this radiation will relate to levels of elements such as phosphorus or sulfur. Dr. Russell’s has uncovered aspects of the genetic control of seed phosphorus levels in corn using this technique. The procedure is more time consuming than the NIR analysis and thus more expensive, but a reliable measurement of this seed trait can be obtained on sample numbers needed in a breeding program without breaking the plant breeders budget.

Measurements based on chemical reactions.

The work of chemists who enjoyed test tubes and reagents has provided Dr. Baltensperger’s program with a test for measuring an important grain quality trait in proso millet. A chemical reaction to identify “waxy” grains in proso millet was developed first for more common crops such as corn or wheat. The same test works well on proso millet because these crops are making the same kinds of starches. Typically grains make both branched starch (amylopectin) and unbranched starch (amylose) as they store carbohydrate for energy the seed will need to germinate. Waxy types make mostly branched amylopectin. If iodine is added to a solution of starch it will react with the amylopectin and produce a dark, purplish color. If the grain has a higher level of amylose starch, the reaction produces a reddish color. The staining assay can be done in a sample well or right on the kernel in crop species with large enough seeds (Fig. 4).

Fig. 5 Simplified carbohydrate pathway in seeds

Fig. 6 Amylose starch stains red in the non-waxy wheat kernels (top row). Amylopectin stains purple in the waxy wheat (bottom row).

Measurements based on electrophoresis

In some situations, the plant breeder knows the gene or protein that is critical in determining the grain quality trait. By using the correct isolation method for the protein or PCR to make copies of the gene, they can then visualize the plants with the desired trait by examining their protein or DNA banding patterns. This is shown in Fig. 5. Granule bound starch synthase (GBSS) is the enzymethat allows seeds to make unbranched starch. Non-waxy plants would have one or both of the GBSS bands in the gel. Waxy plants lack this band of protein. When the plant breeder can use DNA methods to detect the gene encoding the critical proteins, they are able to assay any tissue in the plant to identify the desired genotypes for the grain trait of interest. This would provide advantages to the plant breeder if they want to verify a plant’s genotype before it has made seed. (Can you think of why this would help the plant breeder save time?)

Fig. 7. Protein gel of granule bound starch synthase (GBSS) extracted from durum wheat starch granules. Waxy wheat lacks this enzyme in the seed and no bands of this protein are present (lanes 7 & 8). Non-waxy durum wheat will have either two copies of this enzyme (lanes 2,3,9-12) or one of those copies (lanes 1,4,5,6,13).

More complex assays

Dr. Pedersen has an interest in a more complex aspect of grain carbohydrate levels in the sorghum lines he works with. The amount of energy an animal can extract from the grain is the critical trait. The ultimate test involves feeding trials with cattle, pigs or chickens. Obviously, Dr. Pedersen needed an assay that would provide a reasonable prediction of the feeding test and be easy enough to do to hundreds of lines in his breeding program each year. By collaborating with animal nutritionists, they devised a test tube assay that treated the sorghum grain and measured levels of energy-producing carbohydrates the animal would have available. Now, this assay is a component of their selection process. Assays to predict bread-making attributes of wheat have provided Dr. Baenzigers program with similar tools to making “early generation” selections; when breeders have small amounts of seeds from lots of families and they need to decide what to keep.