# Presentation: 860396

## Genetic Engineering in Alfalfa A tool for improving forage quality and nutrient management on dairy farms

**Genetic Engineering in Alfalfa**** *****A tool for improving forage quality and nutrient management on dairy farms***

## Why Alfalfa?

- High quality forage crop
- High protein content, high fiber digestibility
- High forage yield, long-lived perennial legume
- Rotational benefits
- Nitrogen credit (~100 lb N)
- Improved yield of succeeding corn crop (~20 bu/A)
- Break in disease/insect/weed pest cycle
- Improved soil tilth

## Why Alfalfa?

- Environmental benefits
- Decreased soil erosion on sloping land
- Low pesticide use
- Lessens need for N fertilizer on succeeding crop

## Why Plant Genetic Engineering

- Introduce traits not available in                          alfalfa or alfalfa relatives.
- Roundup Ready (bacterial gene)
- Improved protein bypass (corn gene)
- Phytase (fungal gene)
- Knock out existing genes that negatively affect crop performance/crop quality
- Improved digestibility (lignin knockout)

## Genetic engineering for improved forage quality in alfalfa

- Desired improvements in alfalfa quality
- Better source of fiber
- Improved fiber digestibility
- Higher energy content in forage
- Higher milk production potential
- Decreased manure generation
- Better source of protein
- Improved efficiency of protein utilization
- Increased rumen bypass
- Improved protein stability in silage
- Higher milk production potential
- Decreased N excretion

## Alfalfa Fiber Quality Impact on dairy production

- Alfalfa is a primary source of fiber in most dairy diets
- Lignin cross-linking with cell wall polysaccharides limits both rate and extent of fiber fermentation in rumen
- Impact of rate and extent of fiber digestibility
- Energy content of forage (energy is common limiting factor in diet for high producing dairy cows)
- Non-digestible fiber is primary manure solids component

## Engineering the lignin biosynthetic pathway in alfalfa

**Down **
**regulation**

## Genetic engineering for improved forage quality in alfalfa

- Altered lignin content/composition in alfalfa
- Low lignin transgenic alfalfa produced based on “knockouts” of enzymes involved in lignin biosynthesis.

|  | COMT
pkat/mg | CCOMT pkat/mg | Klason Lignin % | S/G 
ratio |
| --- | --- | --- | --- | --- |
| Control | 6.55 | 23.77 | 17.91 | .47 |
| COMT- | 1.24 | 22.26 | 12.46 | .04 |
| CCOMT- | 14.39 | 0.78 | 14.58 | 1.05 |
| Dual- | 0.78 | 5.59 | 14.72 | .23 |

- Dixon et. al., 2000

## Effect of gene knockout on in vivo digestibility in alfalfa

- Guo, et. al. 2001

- Altered lignin content and composition resulted in increased rate and extent of rumen digestion

## Potential benefits of transgenic alfalfa with increased cell wall digestibility

- Increased energy from forage
- Dietary fiber is required for rumen health, increased digestibility of this fiber will result in more energy for milk/beef production.
- Increased milk/beef production potential
- USDFRC estimates that a 10% increase in fiber digestibility would result in an annual $350 million increase in milk/beef production.
- Decreased generation of manure
- USDFRC estimates that a 10% increase in fiber digestibility = 2.8 million tons decrease in manure solids produced each year.

## Alfalfa Protein Quality Impact on dairy production

- Alfalfa is a primary source of protein in most dairy diets
- Alfalfa protein is rapidly degraded in the rumen (low RUP)
- This rapid degradation of alfalfa protein limits the efficiency of protein utilization in ruminant animals
- Inefficient utilization of alfalfa protein requires the feeding of  supplemental protein with high RUP to maximize milk production.
- Inefficient utilization of alfalfa protein also results in increased N losses to soil/water/air.

## Strategies for improving efficiency of alfalfa protein utilization in the rumen

- Transgenic alfalfa expressing a novel protein (high RUP)
- Novel leaf storage proteins (LSP) can be designed that largely bypass rumen fermentation, yet be fully digested in the gut.
- Such novel LSPs have now been expressed in alfalfa
- Transgenic alfalfa with leaf tannins
- Tannins bind with plant proteins, slowing rate of rumen degredation
- Forage species with leaf tannins:
- Have higher RUP than those species without leaf tannins
- Do not cause bloat when grazed

## Impact of improving efficiency of alfalfa protein utilization in the rumen

- Transgenic alfalfa expressing a novel protein (high RUP)
- Decrease/eliminate requirement for supplemental feed protein
- Decrease in $ and N input required
- Transgenic alfalfa with leaf tannins
- Decrease/eliminate requirement for supplemental feed protein
- Decrease in $ and N input required
- Decreased rate of protein fermentation = more efficient use of alfalfa protein (decreased N content of excretia).
- Decreased N losses likely

## Post Harvest Proteolysis in Alfalfa Impact on dairy production

- Post harvest degradation of alfalfa protein begins at mowing/conditioning and continues until limited by moisture (hay) or pH (haylage).
- Post harvest proteolysis results in increased non-protein nitrogen (NPN) in the forage.  NPN is less efficiently utilized in the rumen than intact protein.
- Nitrogen in alfalfa hay is typically 20% NPN
- Nitrogen in alfalfa silage is typically 60-75% NPN
- Trend is for more alfalfa silage, less alfalfa hay

## Post Harvest Proteolysis in Alfalfa Impact on dairy production

- Increased NPN decreases the efficiency of protein utilization in ruminants
- Inefficient utilization of alfalfa protein requires the feeding of  supplemental protein with high RUP to maximize milk production.
- Inefficient utilization of alfalfa protein also results in the excretion of excess rumen NH3, leading to increased N losses to the environment.

- Typical NPN
- content of silage

## Strategies for decreasing post-harvest proteolysis in alfalfa silage

- Some compounds bind with alfalfa protein to decrease rate of post-harvest proteolysis.  Transgenic alfalfa will be produced that contain these compounds.
- Tannins – altered expression of genes for alfalfa tannin biosynthesis
- Polyphenol oxidase (PPO) – gene isolated from red clover (USDA)
- Potential “knockout” of key plant enzymes important in post harvest proteolysis.
- PPO
- Protease inhibitors

## Impact of improving efficiency of alfalfa protein utilization in the rumen

- Transgenic alfalfa with decreased post-harvest proteolysis
- Much higher protein content of alfalfa silage
- Increased N use efficiency of alfalfa silage
- Decrease requirement for supplemental feed protein
- Decrease in $ and N input required
- More efficient use of alfalfa protein (decreased N content of excretia).
- Decreased N losses to air/soil/water

## Genetic engineering for improved forage quality in alfalfa

- Genetic engineering for improved forage quality in alfalfa should offer added value for alfalfa growers and dairy producers.
- Increased cell wall digestibility = more energy from alfalfa = increased milk production.
- Increased alfalfa protein stability in the rumen and silo will increase efficiency of N utilization
- Decreased cost of protein supplementation
- Current cost ~$.70/cow/day

## Genetic engineering for improved forage quality in alfalfa

- Genetic engineering for improved forage quality in alfalfa should offer new tools to for better nutrient management on dairy farms.
- Increased cell wall digestibility=decreased manure production
- Increased alfalfa protein stability in the rumen and silo will increase efficiency of N utilization
- Decreased N losses to water/air/soil
- Decreased N input from protein supplement

## A multidisciplinary collaboration of public and private scientists

- Dairy Nutrition (USDFRC)
- Biochemistry (Noble Foundation and USDFRC)
- Molecular/cell biology (Noble, FGI and DowAgro)
- Agronomy
- Plant breeding (FGI)