Agricultural crops have been bred for many different traits to increase the yield, strength, or pest resistance. It is no secret that high-throughput screening methods are essential for viable plant breeding programs. Techniques such as PCR and marker assisted selection (MAS) have been at the forefront of plant breeding since the mid-90s. Some traits are not as easy to select for by MAS because the genes that control these
traits may not be known or no markers are linked to the genes of interest. In these cases, other selection methods must be used such as multiple trait selection (Falconer and Mackay 1996). Because maize is used primarily as livestock feed, many traits are selected for the ability to improve the diets of these livestock.
One example of improving methionine in corn and soybeans is described by Wright and Orman (1995). They used a microbe to catabolize the methionine and measured the cell density of the culture to determine the amount of methionine in the substrate. This microbial method was much less expensive than the accepted chemical method, and the authors showed that the microbial method was highly correlated to the chemical method. This is an example of a bioassay because they were using a microbe instead of a chemical to quantify the substrate. This microbial assay suggests a measure of bioavailability, not chemical availability, which was important to their study when discussing the utilization of substrates in livestock. A modified version of this method was also used by Darrigues et al. (2005) to evaluate methionine in maize. The authors suggested that the methods used to do their high-throughput screening may not be as accurate as accepted chemical methods, but their methods are much less expensive, repeatable, and can produce results where the samples can be ranked. These are some of the more important features of screening methods in plant breeding programs.
Plant cell wall digestibility is not well-suited to MAS because, as of yet, it is poorly characterized genetically. Simultaneous saccharification and fermentation (SSF) is an accepted method to determine the digestibility of the cell wall in order to create ethanol, but requires a week’s time and special, expensive equipment. Other high- throughput screening methods that are precise and reliable need to be developed in order
to make progress with this trait in breeding programs. One of these methods recently
developed is described by Weimer et al. (2005). The authors describe in vitro gas
production as a high-throughput method to evaluate feedstocks for ethanol production
potential. The method basically consists of an in vitro digestion of untreated, ground
feedstock by anaerobic ruminant microorganisms in sealed serum bottles. During the fermentation, the gas pressure is recorded and converted to the amount of gas produced by the reaction. The amount of gas produced correlates to the amount of ethanol that would be produced by the feedstock. This method gets around having to measure the ethanol directly by HPLC or GC as in the SSF procedure. This method can be used to evaluate up to 64 samples in a single replication and takes 24 or 96 hours to complete, depending on what parameters are used. There are two main advantages of this particular assay over SSF: 1) this method does not require any sterilization due to the high
inoculum levels added, and 2) this method is more sensitive than SSF due to the measurement of gas production rather than ethanol directly. The main disadvantage of this assay is that is uses rumen microflora to conduct the digestion rather than an enzyme cocktail and yeast which are easier to store, distribute, and collect. Both SSF and the method described by Weimer et al. (2005) are bioassays, which model the process of producing ethanol from yeast better than chemical methods, but they still require a lot of time, are run in rather large vessels, and each sample must be read one at a time at the end of the reaction. The method presented in Chapter 2 called simultaneous
saccharification and catabolism (SSC) improves upon each of these weaknesses while also increasing the number of samples that can be evaluated in one batch. SSC uses a
microbial biosensor in order to evaluate the availability of sugars in corn stover, a lignocellulosic feedstock.
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