SPECIAL TOPICS SYMPOSIUM

Research on Transgenic Alfalfa as a Bioreactor for the Production of Industrial Enzymes
and Pharmaceuticals - Edwin T. Bingham and Sandra Austin.............................68

Root Physiology and Alfalfa Persistence: Myths, New Paradigms, and Future
Explorations - J. J. Volenec, B. C. Joern, S. M. Cunningham and A. Ourry ............69

Breeding for Improved Forage Quality in Alfalfa: An Animal Nutritionist's View-
H. G. Jung.....................................................................................70

Grazing Alfalfa in Argentina - E. H. Hijano and N. A. Romero..............................71


Research on Transgenic Alfalfa as a Bioreactor
for the Production of Industrial Enzymes and Pharmaceuticals

Edwin T. Bingham and Sandra Austin
Department of Agronomy and Plant Biotechnology Laboratory
University of Wisconsin, Madison, WI 53706 U.S.A.

Most industrial enzymes currently are produced from native or recombinant microorganisms by large-scale fermentation. Prior to the advent of molecular genetics, production was limited to twenty or so enzymes that could be produced cheaply in large amounts. The use of genetic engineering to clone the genes of non-abundant enzymes promises to expand the use of enzymes in industrial applications. Research on using alfalfa to produce certain enzymes is underway. Machinery and methods to squeeze protein-rich juice from alfalfa have been developed.

Transgenic alfalfa expressing two proteins, manganese-dependent lignin peroxidase (Mn-P) from Phanerochaete chrysosporium and alpha-amylase from Bacillus licheniformis, have been produced and field tested (1). Other enzymes whose production may be economically feasible in alfalfa include lactase, to be used to convert the large amounts of lactose present in cheese whey to glucose and galactose, and to reduce the lactose content of whole milk; cellulases and hemicellulases for use in biomass conversion or to be used as additives to silage; papain and other plant-derived proteases; and lysozyme, a bacteriostat that obviously cannot be produced readily by bacterial fermentation. Production of pharmaceuticals in transgenic alfalfa also appears to have potential. Currently, two companies are testing the feasibility of producing antibodies in transgenic alfalfa.

Breeding strategies which will provide for maximum expression of the desired protein, product protection, and control of reproduction need to be considered (2). The homozygous tetraploid condition, in which the integrated gene is raised from one to four copies, needs to be studied. Indeed, each independent transformation event may be on a different chromosome or different chromosome arm. Independent transformants can be combined by breeding and multiplied by four to produce a very high copy number.

Production of certain growth hormones or pharmaceuticals may require complete containment. A method to provide complete containment of transgenic germplasm in alfalfa by using a recessive flowerless mutant has been developed. Genetic manipulation is done using normal plants, heterozygous for the mutant. This sterile mutant co-segregates in breeding populations with any transgene not linked to the mutant. The selected sterile transformed plants can be propagated from shoot cuttings to produce a high-value product.

References

Austin, S., Bingham, E.T., Mathews, D., Shahan, M., Will, J. and Burgess, R.R. (1995)
Production and field performance of transgenic alfalfa expressing alpha-amylase and
manganese dependent lignin peroxidase. Euphytica. 85:318-393.
Micalef, M.C., Austin S., and Bingham E.T. (1995) Improvement of transgenic alfalfa by
backcrossing. InVitro-Plant. 31:187-192.


Root Physiology and Alfalfa Persistence: Myths, New Paradigms, and Future Explorations

J.J. Volenec', B.C. Joern', S.M. Cunningham', and A. Ourry2
1Department of Agronomy, Purdue University, West Lafayette, IN 47907-1150 and 2INRA. Physiologie et
Biochimie Vegetales, Institut de Recherche en Biologie Appliquee, Universite, 14032, Caen, Cedex, France.

No other facet of alfalfa physiology has been studied more extensively than the role of root carbohydrates in alfalfa performance. The concept that carbohydrate reserves control shoot regrowth and persistence has been so widely accepted that few other aspects of alfalfa root physiology have been explored. However, many studies where root carbohydrate reserves are positively correlated with persistence and shoot regrowth contain significant confounding because they include mis-management treatments (frequent harvesting, late fall harvests, pests,...) that, in addition to reducing root carbohydrate reserves, alters numerous physiological processes. This makes it impossible to unequivocally assign plant responses solely to differences in root carbohydrate reserves. We have used genetic approaches to further evaluate the role of root carbohydrate reserves in alfalfa regrowth and persistence. We have found no correlation between root carbohydrate concentrations and genetic differences in shoot regrowth after defoliation (Volenec, 1985). Furthermore, when we evaluated genotypes differing up to four-fold in root carbohydrate concentrations, we found that these lines had similar shoot regrowth rates after harvest (Habben and Volenec, 1990). Additional work indicated that multiple defoliations that essentially exhaust carbohydrate reserves in roots of low-starch lines did not reduce herbage regrowth rates (Boyce and Volenec, 1992). Because our research showed no relationship between root carbohydrate reserves and genetic variation in alfalfa regrowth and winterhardiness, we examined other physiological factors that may influence alfalfa growth and persistence. This has led us to our current interest in alfalfa root N pools.
Our results to date show the following: I) Using 15N as a tracer we have been able to quantify N transfer from roots and crowns to shoots after
harvest. Results of these studies show extensive, rapid turnover of N and subsequent transfer to growing
shoots (Kim et al., 1991, Barber et al., 1996). These results show unequivocally that substantial taproot
N is mobilized to shoots during regrowth. 2) Which root N pools serve as this source of N? Analysis of root N pools revealed that the buffer-soluble
N fraction is nearly equally split between buffer-soluble protein N and low molecular vvt. N (amino acids,
small peptides, nitrate, etc.,). Alfalfa taproots contain three proteins we refer to as vegetative storage
proteins (VSP), that comprise approximately 20% of the soluble protein pool, which are preferentially
degraded during shoot regrowth (Hendershot and Volenec, 1993). 3) In contrast to carbohydrate reserves, root N reserve levels are closely associated with alfalfa shoot
regrowth rate after harvest (Ourry et al., 1994).

References

Barber, L.D., B.C. Joern, J.J. Volenec, and S.M. Cunningham. 1996. Effects of supplemental nitrogen on alfalfa regrowth and
taproot nitrogen mobilization. Crop Sci. (in press).
Boyce. P.J., and J.J. Volenec. 1992. Taproot carbohydrate concentrations and stress tolerance of contrasting alfalfa genotypes.
Crop Sci. 32:757-761.
Habben, J.E., and J.J. Volenec. 1990. Starch grain distribution in taproots of defoliated Medicago sativa L. Plant Physiol.
94: 1056-1061.
Hendershot, K.L., and J.J. Volenec. 1993. Taproot nitrogen accumulation and use in overwintering alfalfa. J. Plant Physiol.
141 :68-74.
Kim, T.H., A. Ournv, J. Boucaud, and G. Lemaire. 1991. Changes in source-sink relationship for nitrogen regrowth of lucerne
(Medicago sativa L.) following removal of shoots. Aust. J. Plant Physiol. 18:593-602.
Ourry, A., T.H. Kim. and J. Boucaud. 1994. Nitrogen reserve mobilization during regrowth of Medicago sativa L.
Relationships between availability and regrowth yield. Plant Physiol. 105:831-837.
Volenec, J.J. 1985. Leaf area expansion and shoot elongation of diverse alfalfa germplasms. Crop Sci. 25:822-827.

 

Breeding for Improved Forage Ouality in Alfalfa: An Animal Nutritionist's View

H. G. Jung
USDA-Agricultural Research Service, Plant Science Research Unit and US Dairy Forage
Research Center Cluster, St. Paul, MN

Alfalfa is the primary perennial forage crop used in US dairy production. Two trends within the dairy industry have contributed to the reduction in alfalfa acreage. First, dairy cow numbers have been declining since 1950. However, total milk production has increased during this time because of the second major trend, a doubling of milk production per cow during the last 30 years. The increased milk production by America's dairy cows places a negative pressure on alfalfa production because of the inability of alfalfa to adequately meet the nutrient requirements of elite cows. Grains and other high energy feeds represent an ever increasing proportion of dairy cow diets to meet the nutrient demands of the high-producing cow. A major challenge to the alfalfa breeding industry will be to improve forage quality of alfalfa simply to continue current levels of alfalfa usage by dairy cattle.

Alfalfa contains too much protein, too little energy, and too much fiber to meet the needs of a high-producing dairy cow. Specifically, the protein in alfalfa is too rapidly degraded by rumen bacteria to allow efficient re-cycling of the nitrogen into microbial protein. A major goal of ration formulation for dairy cows is to match protein and energy substrate degradation rates to allow maximal production of microbial protein. Variation in alfalfa germplasm has been identified for protein degradability, with the Medicago falcata germplasm being less rapidly degraded than M. sativa. However, even M. falcata has rates of protein degradation exceeding 1 6%/h. This compares to fiber degradation rates of less than 9%/h and even corn starch is only degraded at 8 to 10%/h. So selection for decreased protein degradation rates appears to be a limited option. Introduction of tannins via biotechnology to reduce ruminal protein degradation has been suggested as an alternative approach. While low to moderate concentrations of tannins can improve animal performance by limiting ruminal protein degradation, higher levels of tannins actually over-protect protein and restrict animal performance. Energy and fiber are intimately related. Fiber is needed in dairy cow rations for maintenance of rumen health and a favorable fermentation profile for milk component production. However, fiber is fermented more slowly and less completely than starch and other carbohydrates, so fiber limits energy availability. Alfalfa fiber is more quickly digested than fiber in grasses, but the extent of fiber digestion is lower in alfalfa. A major goal of alfalfa breeders should be to increase the potentially digestible fiber content of the crop. Fiber concentration might also be reduced in alfalfa stems. Alfalfa does not store starch in its vegetative tissues so increasing this energy source would be difficult. The simple sugars in alfalfa leaves are present at low concentration, but represent a very rapidly fermentable energy source. Pectin, a polysaccharide in the cell wall, is a major constituent of alfalfa leaves and has fermentation rates equal to alfalfa's protein. Selection for this component would increase both energy content and contribute to more favorable fermentation profiles. Genetic variation for pectin concentration appears to exist in alfalfa germplasm.

E. H. Hijano and N. A. Romero INTA - Argentina

In Argentina, there are approximately one million hectares of pure alfalfa stands and 4 million hectares of pastures with alfalfa as the main component. It is estimated that between 75 and 80% of the alfalfa crop is grazed. Twenty five years ago, continuous grazing was a common practice among farmers. As a consequence, wide-crowned regional ecotypes developed with dormancy grades 3 to 5. These ecotypes were severily damaged in the early 70 s by pea aphid (Acyrthosiphon pisum). The introduction of aphid resistant, -non dormant cultivars- and the results of a cooperative research program between FAO and INTA, dramatically change the overall picture of alfalfa in Argentina. An impressive number of grazing trials were established at several INTA stations to determine the most convenient grazing system in terms of beef and milk production, recovery periods, grazing time, stocking rate, etc. In these trials, a major effort was devoted to maintain pasture production and stand persistence. In mixtured pastures, alfalfa is usually combined with bromegrass (Bromus sp.), orchardgrass (Dactylis glomerata), tall fescue (Festuca arundinacea) or wheatgrass (Elytngia sp.). These pastures facilitate weed control, reduce bloat, allow to maintain the level of production when alfalfa is not growing under optimum conditions, and improve the recovery of soil structure. Sixty five to 75% of the milk produced in Argentina is obtained by grazing alfalfa. In the major dairy areas of the country the use of non-dormant alfalfa cultivars have had a major impact in the farmer s economy: more stable milk production during the different months of the year and lower cost of the milk due to a significant reduction of the more expensive annual winter grasses. Rotational grazing js -by far- the most common alfalfa management system used in the country. Six to 12 days of grazing is common for beef cattle wheres for dairy herds these periods are reduced to 1 to 5 days. A 6-paddock subdivision of the field (system 7 x 35) is preferred for beef production whereas the 36-paddock (1 x 35) is commonly used for dairy herds. In all cases, a resting period of 35 to 42 days is used to recover the crop and to time up grazing with the alfalfa growth. Mixtures of alfalfa and perennial grasses are quite common but management has to be carefully applied if the proportion of the different species are able to be maintained. In general, the best cultivars under cutting are also the best under rotational grazing. When this type of grazing management is used, stand persistence will depend on alfalfa genotype.