In February 2003, researchers from the Plant Science Research Unit/ARS-USDA, U.S. Dairy Forage Research Center/ARS-USDA, The Samuel Roberts Noble Foundation, Inc., and Forage Genetics International met in Ardmore, Oklahoma to create an alliance for the purpose of improving characteristics of alfalfa, such as yield, nutritional content, and digestibility.

“This consortium represents a unique union of government, private non-profit, and for-profit scientists,” said Rick Dixon, director of the Plant Biology Division of The Samuel Roberts Noble Foundation. “However, our focus is on the end product an improved alfalfa plant. The beneficiaries of this effort will be alfalfa producers, cattle and dairy operations, and consumers.”

The Consortium for Alfalfa Improvement (CAI) will regularly convene to identify key research projects, prioritize research efforts, coordinate scientific resources, and develop potential collaborations, both within CAI and external to CAI. CAI members will utilize the varying resources and expertise of the respective members to maximize the effectiveness and efficiency of each research initiative. “The participating institutions will share their research and seek to publish their findings in the spirit of academic research,” said Mark McCaslin, president of
Forage Genetics, Inc.

Alfalfa and alfalfa hay is the principal feed stock for dairy cows but is also an important food source for horses, beef cattle, and sheep. Alfalfa contains between 15-22% crude protein as well as other important vitamins and minerals. This high protein content directly impacts milk, beef, and wool production. There are approximately 23 million acres of alfalfa cut for hay in the United States annually. This represents the fourth most widely grown crop in the United States behind only corn, wheat, and soybeans. Alfalfa hay production is estimated to generate more
than $7 billion annually. Other forms of alfalfa, including alfalfa meal and cubes, are exported to other countries from the United States with an annual value in excess of $45 million.

Alfalfa, as a legume, is capable of “fixing” nitrogen claiming atmospheric nitrogen and returning it to the soil. While crops such as wheat and corn require substantial quantities of nitrogen fertilizers, alfalfa often requires no external nitrogen fertilizer. Specifically, alfalfa is capable of fixing from 120 to 500 lbs of nitrogen per acre annually. It is estimated that alfalfa in the United States fixes approximately 2-2.5 million tons of nitrogen year. When planted in rotation with cereal crops, alfalfa can significantly reduce the requirements for additional nitrogen fertilizer in the rotated, non-alfalfa crops. Alfalfa further evidences significant value in preventing nitrate leaching; reclaiming phosphates, nitrogen, and other contaminates from the soil; and mitigating or preventing erosion.

Despite the widespread use and recognized value of alfalfa, this forage maintains certain traits and susceptibilities that could be corrected through science. The first two CAI research initiatives will focus on cell wall digestibility, for example, through lignin reduction, and improving efficiency of protein utilization, areas in which the various parties have already developed patented technologies. “This pooling of intellectual resources represents an exciting development in the effort to improve this important agricultural and economic crop using applied biotechnologies and conventional plant breeding,” said Joe Bouton, acting head of the Forage Biotechnology Group of The Samuel Roberts Noble Foundation.

Update on Biotech Traits for Improved Forage Quality

In 2003 Forage Genetics, the U.S. Dairy Forage Research Center (USDFRC) and the Noble Foundation launched the Consortium for Alfalfa Improvement (CAI) to examine the potential use of genetic engineering/genomics for improving alfalfa forage quality. The CAI is currently focusing on two traits: improved fiber digestibility and improved efficiency of protein utilization.

Improved Fiber Digestibility

Rationale – Forages are the primary source of fiber in ruminant diets. A minimum amount of dietary fiber is required for normal rumen function, animal health and milk fat content. Fiber is the least digestible part of most dairy diets, and has the lowest energy content. Increasing fiber digestibility will increase feed intake and energy content of the diet and decrease the amount of excreted undigested fiber (manure solids).

Approach – Lignin is a phenolic compound found in most plant secondary cell walls. Lignin is indigestible and cross- links with cellulose to decrease cellulose digestibility. Lignin content increases, and cell wall digestibility decreases as alfalfa plants mature. Genetic engineering can be used to “knock-out” genes coding for one or more of the several enzymes the plants use to make lignin. Brown midrib corn is an example of a natural mutation that caused a “knock-out” of one of the lignin biosynthetic enzymes.

The Noble Foundation has created transgenic alfalfa plants with independent knock-outs of most of the plant enzymes required for lignin synthesis. In FGI field tests (2002-2004) several of these transgenic plants have demonstrated a significant reduction in lignin content and an increase in cell wall digestibility >10%. This improvement can be compared to conventional breeding where over 15 years selection has resulted in a 2-3%
increase in cell wall digestibility.

Next steps – To complete proof-of-concept testing, the CAI partners will begin comprehensive field tests to evaluate agronomic/forage quality of elite transgenic alfalfa plants in 2005. Several different alfalfa promoters and biotech methods for gene knockout are now also being compared at FGI. Based on these results, we anticipate commercial transgenic plants will be produced in 2005 followed immediately with integration of the new trait into elite germplasm, product development and the generation of data required for Federal deregulation of the trait.

Potential economic impact – The USDFRC estimates that a 10% increase in fiber digestibility could result in $200M annual increase in milk and beef production in the U.S., and a 200M ton annual decrease in production of manure solids.

Improved efficiency of protein utilization

Rationale - Although alfalfa has a high protein content, alfalfa protein is rapidly degraded in the rumen, and inefficiently utilized by dairy cows. As a result even high alfalfa diets often require protein supplements when fed to high producing dairy cows. Inefficient utilization of alfalfa protein also results in increased losses of nitrogen to the environment, potentially affecting water quality. In the making alfalfa haylage there is an extended period of time for post-harvest protein breakdown, often resulting in high nonprotein nitrogen (NPN) content of typical alfalfa haylage. This significantly further decreases efficiency of protein utilization, compared to alfalfa hay. Decreasing protein degradation in the rumen and in the making of alfalfa silage would decrease the need for supplemental feed protein and the decrease the loss of nitrogen to the environment in a dairy system.

Approaches – Red clover haylage has significantly lower NPN content than alfalfa haylage. The USDFRC has identified a gene in red clover (PPO) that is responsible for a compound that significant ly reduces post- harvest protein degradation. This gene has now been expressed in transgenic “PPO alfalfa”. The USDFRC has shown that in the presence of a required substrate, PPO alfalfa has much reduced post- harvest protein degradation.

Tannins are phenolic compounds found in many plants. Tannins generally bind with proteins, decreasing rate and extent of protein degradation. Forage legumes (e.g. birdsfoot trefoil) that produce tannins in leaves or stems have increased stability of protein in the rumen, resulting in more intact protein by-passing the rumen and going directly to the cow’s stomach. This is often referred to as by-pass protein, or rumen undegraded protein (RUP). For example alfalfa protein in hay is generally about 20% RUP vs. >30% RUP in birdsfoot trefoil. Alfalfa produces tannins in seed coats and in the tips of glandular hairs, but not in leaves and stems. Genetic engineering offers tools that can be used to modify tannin expression in alfalfa, producing transgenic plants that express tannin in leaves and stems. The Noble Foundation and other FGI collaborators have identified several candidate genes that may be useful in producing transgenic “tannin alfalfa”.

Next steps – New “commercial” transgenic PPO alfalfa plants will be produced in 2005. Various methods for supplying the required PPO substrate will be compared and analyzed. In 2005 FGI and the Noble Foundation will be comparing several different genes that regulate the tannin pathway and/or modify tannin expression in alfalfa. These genes will be evaluated singly and in combination with one another.

Potential economic importance – USDFRC whole- farm dairy models predict that tannin alfalfa would decrease protein supplementation by 60%, decrease on-farm nitrogen losses by 25%, and increase farm income by 12%. Worldwide bloat losses are currently estimated to be > $200M/year.