Alfalfa Breeding, Genetics, and Cytogenetics

Ploidy Levels of in vitro Regenerated Alfalfa Plants from Salt Tolerant Cells
B. Rodriguez-Garay, A. Gutierrez-Mora and S. Perez-Perez..........................2

Agronomic and Physiological Effects Associated with Biparental Plastid Inheritance
in Alfalfa - R N. Keys, S. E. Smith, and W. T. Molin.............................3

Karyotype Analysis of Alfalfa Using C- and N-Banding Techniques
Gary R Bauchan and M. Azhs Hossain ...............................................4

Magnetic Resonance Imaging of Aluminum Toxicity in Alfalfa
T. A. Campbell and C. D. Foy .....................................................5

Cold-tolerant annual medics: Medicago rigidula and M. rigiduloides
R W. Groose, R A. Ballard, N. Charman and A. W. H. Lake...........................6

Breeding for Annual Cropping Systems Using North American and Arabian Non
dormant Germplasm - S. Ray Smith ...............................................7

Forage Yield, Dormancy and Disease Resistance Response to Two Cycles of Divergent
Selection for Root Architecture in Alfalfa - J. F. S. Lamb, D. A. Samac,
D. K. Barnes, and K. I. Henjum ...................................................8

Comparing Winterhardiness and Associated Traits using Identical Genotypes in Alfalfa
(And Mapping Genetic Regions Affecting these Traits)
D. J. Brouwer and T. C. Osborn....................................................9

Alfalfa Perenniality, Adaptation and Persistence - Edwin T. Bingham ...................10

Relationship of Alfalfa Genetics to Alfalfa Variety Testing - Thad H. Busbice..........11

Breeding for Grazing Tolerance in Alfalfa - J. H. Bouton ..............................12

Reduced Bloat Incidence in Grazing Trials of Alfalfa Selected for Low Initial Rate
of Digestion (LIRD) - B. Coulman, W. Majak, T. McAllister, B. Berg,
D. McCartney, K. -J. Cheng, J. Hall, and B. Goplen ...............................13

R.N. Keys, S.E. Smith, and W.T. Molin
Department of Plant Sciences, University of Arizona
Tucson, AZ 85721 USA

Research using chlorophyll deficient mutants, chloroplast DNA markers-, and ultrastructural
analysis of egg cells, embryos, and microgametophytes has shown that plastids are inherited
biparentally in alfalfa (1). Plastid inheritance in alfalfa also shows strong paternal bias.
Most progenies have only paternal plastids and paternal plastids predominate in progenies
with some maternal plastids. Positioning of maternal plastids towards the micropyle within
the egg cell has been shown to be at least partially responsible for this paternal bias.
Most maternal plastids are sequestered within the basal cell of the two-celled embryo and
therefore not inherited (2). Any adaptive significance of biparental inheritance of plastids
is not well understood and forms the basis of our research. Previous- research on
microgametophyte function has shown that the number of plastid nucleoids per generative cell
is unrelated to ploidy, mating system, life history strategy, or floral size (3). We have
also shown that this trait is negatively associated with fertility as a pollen parent (4).

The research described here deals with our first attempts to relate the mode of plastid inheritance to the growth and development of the sporophyte.?? Our hypothesis is that sporophyte performance may be influenced by an interaction between nuclear and plastid genes in response to either the source of plastids per se or the ratio of maternal paternal plastids present within a cell. Our approach is based on the fact that progenies from reciprocal crosses in alfalfa will not differ in their nuclear genetic constitutions, but -their plastids will be derived largely from the paternal parent in the cross (Table 1). We chose to concentrate first on traits associated with plastid function and generated three sets of reciprocal crosses using a single common genotype and three other nondormant genotypes. Forage yield was evaluated in the greenhouse in parents and S1 progenies from seven F1s from each reciprocal cross. Parents did not differ in forage yield, however in two of the three pairings, forage yield over six harvests differed significantly between reciprocals (+11.8 and +33.2) greater yield in higher yielding reciprocal). Significant differences were observed among some parents and reciprocal crosses in anatomical and chemical traits associated with plastid function (chlorophyll a, total chlorophyll, adaxial stomatal index and the length of the stomatal aperture). However, there were no apparent patterns in plastid-associated traits that could consistently explain differences in forage yield between reciprocals.? Analysis of photosynthetic activity and secondary pigment in parents and reciprocals is ongoing.

References
1. Mogensen, H. L. 1996. The hows and whys of cytoplasmic inheritance in seed plants. Am. J. Bot. 83:383-404.
2. Losoff Rusche, M., H.L. Mogensen, T. Zhu, and S.E. Smith. 1995. The zygote and proembryo of alfalfa: quantitative, three-dimensional analysis and implications for biparental plastid inheritance. Protoplasma 189:88-100.
3. Keys, R. N., S. E. Smith, H. L. Mogensen, and E. Small. 1995. Microgametophytic plastid nucleoids content and life history traits if tribe Trifolieae (Fabaceae). Plant Syst. Evol. 196:89-98.
4. Keys, R. N., S. E. Smith, and H. L. Mogensen. 1995. Variation in generative cell plastid nucleoids and male fertility in Medicago sativa. Sex. Pl. Reprod. 8:308-312.

Gary R. Bauchan and M. Azhar Hossain
USDA-ARS, Soybean & Alfalfa Research Lab. Beltsville, MD 20705

Chromosomes of two diploid (2n=2x=16) subspecies of Medicago sativa, ssp. caerulea and ssp. falcata, their hybrid and tetraploid (2n=4x=32) cultivated alfalfa (M. sativa, ssp. sativa) were studied. Seeds were scarified and germinated in petri dishes at room temperature of four accessions of ssp. caerulea, PI 212793, PI 212798, PI 243225, and PI 299046 plus cv. CADL; four accessions of ssp falcata, PI 262332, PI 464728, PI 467970, and UAG l806; and cv. 'Saranac' and PI 536539 of M. sativa ssp. sativa. Modified C- and N-banding techniques (1 ,2) and an image analysis system were used (3) Chromosome banding techniques rely on the differential staining of heterochromatin and euchromatin. The heterochromatin is tightly bound to the DNA and thus produces a characteristic band on the chromosome when the chromosomes are lightly denatured and stained with Giemsa stain. The image analysis system is an efficient method of obtaining quality images of alfalfa chromosomes due to its enhancement capability. Enhancement of the chromosomes by pseudocoloration and enlargement of the images enable the edges of the chromosomes and the heterochromatic bands to be distinguished for easy identification. The chromosomes of ssp. falcata have only centromeric bands and thus are not as useful for identifying individual chromosomes However, a multitude of bands were observed in both the C- and N-banding pattern of ssp. caerulea and ssp sativa enabling the precise identification of each of the eight sets of chromosomes and development of a karyotype. All of the chromosomes had centromeric bands, and in addition, all of the chromosomes have telomeric bands in their short arms. Except for chromosome 7, all of the chromosomes have interstitial bands on their short arms and chromosomes 1, 2 and 3 each have one prominent interstitial band on their long arms. The N-banding pattern of ssp caerulea is different from the banding pattern observed in C-banding. All of the chromosomes have a centromeric band with the exception of chromosome number 4. The telomeric bands occur on all the chromosomes except chromosome 2 and all of the chromosomes except chromosome 7 have interstitial bands on their short arms. Chromosomes 1, 2 and 4 each have two interstitial bands on their long arms and chromosome 3 only has a single interstitial band on the long arm The differences in banding patterns between the diploid subspecies makes it possible to identify hybrids between these subspecies and to distinguish chromosomes from each subspecies in hybrids cells. Using these techniques it will be possible to detect the introgression of chromosomal material between these species. Karyotypic analysis of tetraploid alfalfa revealed that alfalfa has four nearly identical sets of chromosomes based on their identical chromosome morphology and banding patterns, thus providing support that alfalfa is an autotetraploid
1. Hossain, M.A. 1985. An improved Giemsa C-banding technique. Bangladesh J Bot 14:37-40
2. Endo, T.R. and B.S. Gill. 1983. Identification of wheat chromosomes by N-banding. In: G.
Kimber, ed. Proceedings of the Sixth International Wheat Genetics Symposia. Kyoto, Japan,
pp 355-359.
3. Bauchan, G.R. and T.A. Campbell. 1994. Use of an image analysis system to karyotype
diploid alfalfa (Medicago sativa L.) J Hered 85:18-22.
Maqnetic Resonance Imaging of Aluminum Toxicity in Alfalfa

An estimated 40% of arable soils and 70% of non-arable soils of the world are acidic and, in many of these soils, Al toxicity is the primary growth limiting factor for plants. Excess exchangeable Al is especially undesirable in subsoils because it reduces rooting depth and branching and predisposes plants to injury by drought. In most soils, liming the plow layer does not neutralize phytotoxic Al in sub-surface layers and applying lime to subsoils is generally not economically feasible. Al-tolerant germplasm offers an alternative or supplemental solution to the problem, unfortunately, current protocols for developing Al-tolerant alfalfa populations are only marginally effective (1), and the possibility of using new technologies to develop more effective protocols is being investigated (2).

MRI(Magnetic Resonance Imaging) techniques are non-invasive and nondestructive, so that repeated images can be made using the same tissue without causing injury or interfering with growth(3). MRI is based on the fact that some nuclei, such as protons, have a magnetic moment which will align with a strong static magnetic field. A weak radio pulse of the proper frequency (based on the element and the magnetic field strength) will cause the net magnetic moment to rotate 90 deg;. When the pulse is removed, the moment loses energy and relaxes back to its previous position. Energy loss is differential and is based on the environment surrounding each proton, e.g., relaxation times (T) for protons within a tumor would be different from those within normal tissue. T2 represents loss due to inter nuclear interaction and is particularly sensitive to the chemical environment. Because relaxation times can be represented as specific pixel colors, T can be used to form images.

MRI was used to investigate Al uptake and movement in young alfalfa roots. Experimental design was a split-plot with three replications. Whole plots were 55 1 tanks of pH 4.5 modified Steinberg solution containing 0 or 111 micromol Al. Sub-plots were four plants of an Al tolerant or an Al sensitive clone. Longitudinal T2 images were made of sections of young alfalfa roots. While all T2 changes were not attributable to Al, relaxation times were markedly less with Al in the system, and more Al was noted in the epidermis than in the interior of the root. Tolerant clones often had less internal Al than did sensitive clones, and relaxation times with 0 Al were generally longer for the tolerant than for the sensitive clone, indicating, perhaps, differential chemical states. Differences noted may be useful for studying the basic mechanisms of Al tolerance and as a basis for selecting Al tolerant plants.

1. Campbell, T. A., Z. L. Xia, P. R. Jackson, and V. C. Baligar. 1994. Diallel analysis of tolerance to aluminum in alfalfa. Euphytica 72:157 162.

2. Campbell, T. A., P. R. Jackson, and Z. L. Xia. 1994. Effects of aluminum stress on alfalfa root proteins. J. Plant Nutrition 17:461-471.

3. Pfeffer, P. E., Shu-I Tu, W. V. Gerasimowicz, and J. R. Cavanaugh. 1986. In vivo 31p NMR studies of corn root tissue and its uptake of toxic metals. Plant Physiol. 80; 77-84.

R.W. GROOSE', R.A. BALLARD2, N. CHARMAN2 and A. W. H. LAKE2

'Department of Plant, Soil and Insect Sciences, University of Wyoming,
PO Box 3354, Laramie WY 82071 USA
South Australian Research and Development Institute,
Plant Research Centre, Waite Precinct, GPO 397, Adelaide SA 5001 AUSTRALIA

Medicago rigidula (L.) All. and M. rigiduloides E. Small are more winter hardy than other annual medics (see Krall et al. 1996 in this volume) and show potential for use as self-regenerating winter annual pastures ("green fallow") in rotation with winter wheat on the U.S. Western High Plains. These two species were recently differentiated from a single species, " M. rigidula," (reviewed by Heft and Groose, 1996 in this volume). M. rigidula and M. rigiduloides are, respectively, primarily European and Asian in their distribution. The objectives of this research were (1) to investigate Rhizobium specificity of these species and (2) to evaluate interspecific fertility between M. rigidula and M. rigiduloides as part of a plant breeding program to develop cold-tolerant annual medics with optimal adaptation to the wheat-fallow agroecosystem of the U.S. High Plains. To investigate Rhizobium requirements of these species five lines each of M. rigidula and M. rigiduloides were tested in a glasshouse experiment with nine replications where eight treatments were no nitrogen ("nil"), mineral nitrogen ("+N"), four Rhizobium meliloti strains (CC169B, WSM688, WSM826 and WSM1366) isolated in Europe and Australia and recommended for various cultivated annual Medicago spp. in South Australia, and two experimental Rhizobium meliloti strains (M18 and M49) isolated in Asia that were good nodulators of a M. rigiduloides line in a preliminary experiment. Surface-sterilized seed were germinated for 48 hours under sterile conditions on inverted Water Agar plates and transferred to sterilized 20 X 150 mm test tubes containing vermiculite moistened with N-free McKnight plant nutrient solution. Three-day-old seedlings were inoculated with five-day-old Rhizobium cultures. For the +N treatment 4 mL of a l9mM solution of NH4NO3 was added at 7 day intervals commencing 7 days after inoculation. Shoots were harvested 35 days after inoculation. Shoot biomass (dry weight) of plants grown on N-free media was used as an indicator of successful inoculation and N2 fixation. M. rigidula and M. rigiduloides differed markedly in Rhizobium specificity. Distinct within-species differences in Rhizobium specificity among medic lines were also evident. All five M. rigiduloides lines produced more shoot biomass with the Asian Rhizobium strains M18 and M49 than with the four Rhizobium strains isolated in Europe and Australia. None of the latter strains performed better than nil with any M. rigiduloides line. Conversely, Rhizobium strains CC169B and WSM826 were more effective than nil with all M. rigidula lines except for CC169B which was ineffective with one line. WSM688 and WSM1366 were effective in various combinations with the M. rigidula lines. Clearly, successful commercialization of M. rigidula and M. rigiduloides will require attention to the Rhizobium requirements of these species. Hybridization experiments involving a set of 20 lines of M. rigidula and M. rigiduloides support the taxonomic split between these species (Table 1). F1 plants of both intra- and interspecific crosses had reduced fertility compared to mid-parent means (all significantly different at P=.05) but in all cases interspecific F, plants exhibited a greater depression of fertility than did intraspecific crosses.
 

S. Ray Smith, Department of Plant Science, Univ. of Manitoba
Winnipeg, Manitoba, Canada R3T-2N2

There is a growing interest in using forage legumes in annual cropping systems in North America. Although a number of species have been proposed, most seem to have significant limitations. Non-dormant alfalfa cultivars have been proposed as an option for annual systems, and the cultivar 'Nitro' was released in the late 1980's as a non-dormant with high nitrogen fixation potential. The limitation with Nitro and other North American non-dormants is that they do not always winterkill, forcing producers to rely on tillage or herbicides before planting the subsequent year annual crop. Alfalfa germplasm from the Arabian pennisula and North Africa may offer a better alternative for annual systems because they show rapid regrowth after cutting, high forage production in the fall and rarely fail to winterkill in the north central United States. These germplasms are limited though by susceptibility to many of the alfalfa diseases and insects common in North America and because of their lack of frost tolerance. The objectives of this research project were two fold: 1) To combine the desirable growth attributes of Arabian alfalfa germplasm with the disease and insect resistance of North American cultivars; and 2) To determine the most efficient selection methodology to combine these characteristics. The base populations consisted of the Arabian germplasms Hejazi, Quassimi and Hudeiba and the North American non-dormant cultivars Valley, Rio, P5683, GT13R, P5715 and Florida 77. The breeding methodology consisted of three cycles of selection. CYCLE 1 - Superior genotypes were selected from all cultivars and germplasms (in a space planted nursery) for improved fall growth and frost tolerance (-4 deg C) during early October 1993. The Arabian germplasms and the non-dormant cultivars were maintained as separate populations and reciprocal crosses were made between these populations during the winter of 1993/94 (pollen parent was random). The crossed seed was then bulked by maternal cultivar or germplasm resulting in 9 distinct populations. CYCLE 2 - Cycle 1 seed and the original cultivars and germplasms were planted in the field in a space plant nursery in the spring of 1994. The superior genotypes for fall growth and frost tolerance were selected from all cycle 1 populations, cultivars and germplasms in early October. Reciprocal backcross populations were developed as follows: Between each of the Arabian cycle 1 selections and their original germplasm (pollen parent random); and between each of the non-dormant cultivar selections and their original cultivar (pollen parent random). Each Arabian cycle 1 selection and each non-dormant cultivar selection were also intercrossed to produce 2 RPS (recurrent phenotypic selection) populations. Twenty distinct populations were developed. CYCLE 3 - Seed was planted from all cycle 2 populations in the field in a space planted nursery in the spring of 1995. The superior genotypes for fall growth and frost tolerance were selected from all populations in early October. Random pollination was conducted within the Arabian and non dormant cultivar populations during the winter of 1995/96 and seed was bulked to produce the following six populations. 1) Arabian O x S - AO x AS(AO x 790); 2) Arabian S x O - AS(AO x 790) x AO; 3) Arabian RPS; 4) ND79 0 x S - 790 x 795 (790 x AO); 5) ND79 S x O - 79S(790 x AO) x 790; 6) ND79 RPS. (Note: O = original cultivar or germplasm; S = selected cultivar or germplasm.) These populations will be tested in field tests in 1997 to determine the degree to which the desirable characteristics of fall growth, frost tolerance, disease and insect resistance, and winterkill have been combined.

Fertilizer N is the single most expensive input in most crop productions systems and has been implicated in declining ground water quality due to nitrate contamination. Improvements in N cycling and efficient use of symbiotically-fixed N can have a marked effects on the economic and environmental impact of agricultural systems. A major limitation to better management of symbiotically-fixed N from alfalfa is the lack of varieties with specific characteristics that influence the N cycle. Characteristics of interest would include root morphology, root elongation rate, nitrogen uptake and biological nitrogen fixation. Johnson (1992) showed that modern alfalfa varieties (released after 1980) had little variability for root morphological traits. Most alfalfa varieties were tap rooted with a few secondary roots and a small amount of fibrous roots. Older varieties (released before 1970) and Plant Introductions showed a large amount of variability for the number of secondary roots, the amount of fibrous root mass, and taproot diameter. The objectives of this study were (1) to evaluate experimental alfalfa germplasms that have undergone two cycles of divergent selection for the number of secondary roots, amount of fibrous root mass and (2) determine the impact of selection for root traits on root and forage yield, dormancy, and disease resistance.

The experimental germplasm MWNC (dormancy=3) underwent two cycles of selection for no or few (LF, low fibrous) vs. many (HF, high fibrous) fibrous roots and one cycle of selection for few (TAP) vs. many (BRH) secondary roots. Selected and unselected populations were established in two experiments at both Becker, and Rosemount, MN in May 1994. The experimental design was eight replications of a randomized complete block with a split plot arrangement of the treatments, with fertilizer rates (0 kg N ha-1 and 200 kg N ha-1) as whole plots and alfalfa populations as subplots. One experiment at each location was dug in October 1994 and evaluated for taproot diameter (measured in mm), number of secondary roots (scored, 1= few, 5= many), and amount of fibrous roots (scored, 1= few, 5=many). The remaining experiment at each location was harvested for forage yield four time in 1995. In October 1995 these experiments were dug and the plants were evaluated for root traits in the same manner as described above.

MWNC-HFC2BRH had significantly more fibrous and secondary roots than MWNC-LFc2TAP and the unselected parent population. MWNC-LFc2TAP was not significantly different from the unselected parent population for fibrous or secondary root score. MWNC-HFc2BRH had the greatest forage yield. Fibrous root and secondary root scores were positively correlated with root and forage yield. Disease resistance and dormancy rating were unaffected by selection for root traits. Results indicate that (1) two cycles of divergent selection for root morphological traits produced populations that were significantly different in root architecture, (2) high fibrous branched populations demonstrated a yield advantage, and (3) selection for populations differing in root architecture did not change dormancy or disease resistance.

References:

Johnson, L.D. 1992. Morphology and genetics of root types in alfalfa. Ph.D. Thesis. University of Minnesota.

Comparing Winterhardiness and Associated Traits using Identical Genotypes in
Alfalfa (And Mapping Genetic Regions Affecting these Traits)

D. J. Brouwer and T. C. Osborn
Department of Agronomy, University of Wisconsin-Madison
1575 Linden Dr., Madison, WI 53706

Reliable trait data for mapping experiments requires replicated trials. Clonal propogation can be used; however, clonal propagules have altered root and crown morphology. A study was conducted to compare seedlings and cuttings for winterhardiness, fall dormancy, and freezing tolerance and to evaluate germplasm for a mapping population. The 15 genotypes used covered a broad range of dormancy. The field trial was RCB with six replications and 12 plants per replication. Every plant was clonally propagated and the treatments were arraigned in a factorial design - seedlings versus cuttings (SC) as one factor and genotypes (G) the other one. Contractile growth was measured as the unifoliate internode length on 6 week old seedlings, fall dormancy as vertical regrowth on Nov 7, 1994, and freezing tolerance by freezing a 1 cm piece of the root at -8C for two hours and measuring the electrical conductivity of the leachate (Sulc et al., 1991). The SC*G interaction was not significant for all traits. Contractile growth, freezing tolerance and fall dormancy were correlated to winterhardiness by r= 0.69, 0.66, and 0.92 respectively. Our results suggest that cuttings can be used to replicate genotypes in alfalfa winterhardiness evaluations.

A mapping population was created using two parents selected from the clonal propagule experiment. The Blazer XL 17 (B17) parent was very dormant, winterhardy, and freezing tolerant. The Peruvian 13 (P13) parent was non-dormant, non-winterhardy, and freezing sensitive. The mapping populations consist of 101 backcross progeny to B17 and 108 backcross progeny to P13. In the first year field trial, significant differences were found between the two backcrosses for fall dormancy (21.0 cm vs. 26.5 cm), freezing tolerance (54 umoh/gram fw vs. 77 umoh/gram fw) and winter injury (1.8 vs 1.0 on a 1-5 scale). More than 100 RFLP probes are being screened, most of them previously mapped in diploid alfalfa. These markers will be mapped following Wu et al., 1992. Twenty two markers have been scored on the BC populations with most (16/22) of these detecting multiple alleles segregating from the F1. Regression analysis of marker genotype on phenotype will be used to detect genetic factors affecting each trait. This will be especially powerful when two alleles inherited from one parent can be followed in the backcross other parent (13/22 markers so far).

References

1. Sulc, R. M., Albrecht, K. A., Palta, J. P. , and Duke, S. H. 1991. Leakage of intracellular substances from alfalfa roots at various subfreezing temperatures. Crop Science 33:1575-1578.
2. Wu, K. K., Sorrels, M. E., Tew, T. L., Moore, P. H., Tanksley, S. D. 1992. The detection and estimation of linkage in polyploids using single dose restriction fragments. Theor. Appl. Genet. 83:294-300.

Edwin T. Bingham
Department of Agronomy-University of Wisconsin
Madison, Wisconsin 53706 U.S.A.

The ability of perennial alfalfa to live more than two years is dependent on its adaptation and persistence (3,4). These traits are interrelated; they have known and unknown biological components, and they are genetically complex. Selection for persistence maximizes all components of survival. Moreover, selection for persistence is a proven practice in alfalfa. Alfalfa breeding experiments indicate that persistence has its own genetic base. The genetic base is not necessarily the same for all materials with equivalent persistence, and there are many interactions of survival traits. The genetic base is not fixed in most materials and requires selection to maintain it. Population genetic theory and empirical results indicate that selection for persistence should be at or near the end of the breeding exercise. This minimizes shifts due to genetic drift, and negative genetic linkages and associations with other traits under selection.
High levels of multiple pest resistance do not necessarily ensure persistence. This was determined in a study of 30 ycars of Wisconsin alfalfa variety trials (2), and when a study of alfalfa varieties from three eras (1) was evaluated for five years. Thus, the benefits of pest resistance are best obtained in persistent alfalfa. Selection for persistence lengthens the breeding cycle; however, if a new cycle is started each year, there is only one lag phase after which a new product can be selected each year.
Research strategies on persistence include: 1) identifying the types of disease resistance in plants that have persisted for five years or more; 2) monitoring shifts in persistence over sexual generations of selection for other traits; 3) gaining a basic understanding of persistence in fall dormant vs. relatively non-fall dormant genotypes; and 4) identifying chromosome blocks with measurable genetic effects on persistence. The goals are to breed alfalfa that lives up to its potential, and to understand how it does it.
References

1. I Holland, J. B. and E. T. Bingham. 1994. Genetic improvement for yield and fertility of alfalfa
cultivars representing different eras of breeding. Crop Sci. 34:953-957.

2. Ipson, R. B. 1991. Thirty years at Wisconsin Alfalfa Variety Trials. M.S. Thesis, Univ. of
Wisconsin-Madison.

3. Lowe, C. C., V. L. Marble and M. D. Rumbaugh. 1972. Adaptation, varieties, and usage. In
Alfalfa Science and Technology. ASA Monograph No. 15. P. 391-414.

4. Tysdal, H. M. and H. L. Westover. 1937. Alfalfa Improvement. Yearbook of Agriculture.
USDA. P. 1122-1153.

Relationship of Alfalfa Genetics to Alfalfa Vanety Testing
Thad H. Busbice, Great Plains Research Company, Inc.
3624 Kildaire Farms Road, Apex, NC 27502

Early plant breeders noticed that alfalfa behaved differently from other crops. Three unusual breeding patterns were observed: 1. F2 progeny rows appeared surprisingly uniform. Alfalfa did not show the wide segregation observed in F2 progenies of corn, peas, and other familiar crops. 2. Mild inbreeding resulted in a dramatic loss of vigor. 3. Experimental varieties, which showed large yield advantages, performed disappointingly mediocre when they reached the farmer. When it was understood that alfalfa was autotetraploid, it was possible to explore how autotetraploid genetics might influence alfalfa's somewhat strange behavior. As one might have suspected, the breeding behavior, that appeared strange at first, fit autotetraploid genetics perfectly. To understand autotetraploid breeding behavior, we must first understand the diploid gamete. When the gamete is diploid, as with alfalfa, defective genes are masked by sound ones, and defects are passed from generation to generation. A genetic load of deleterious genes builds in the genome of an autotetraploid species such as alfalfa. The result of the genetic load is dramatic inbreeding depression. The surprising uniformity that is observed in F2 progeny rows is also the result of the diploid gamete. Because genes are distributed in couplets, segregation and recombination is restricted. Segregation is restricted to an even greater degree in alfalfa varieties. Not all genotypes can occur in early generations of seed increase, and several generations are required for all segregates to appear. For example, in a 6 parent variety over 17,000 distinct genotypes are formed at a locus in the third generation, while the first generation is relatively uniform. Plant to plant variation is limited in the early generations of seed increase. What does all this have to do with variety testing? Alfalfa varieties change with each generation of seed increase. The greatest change comes in the Syn 2, and the variety stabilizes in the Syn 4. Equilibrium is reached asymptotically. Early generations may differ dramatically from later generations. The Syn 1 and Syn 2 are the breeder and foundation seed generations, respectively, and often are tested under experimental designations. It is the Syn 3 and Syn 4, which represent the third and fourth generations of seed increase, respectively, that are sold to farmers as planting seed. Based on autotetraploid genetics and our experience with alfalfa breeding we know that the Syn 1 and Syn 2 generations are: 1. More uniform than the commercial variety. 2. Higher yielding than the commercial variety. Ramzy Gurgis (1) in 1976 demonstrated how vigor is lost with each generation of seed increase. He compared selected synthetics with the unselected Cherokee variety in 18 replications. The best F1 hybrid yielded 150% of the check. The Syn 1 generation yielded 132% of the check, the Syn 2 generation yielded 116% of the check and the predicted Syn 3 generation yielded 109% of the check variety Cherokee. The yield reduction that we observe as we go from the Syn 1 to the Syn 3 is due to slight inbreeding, and to changes in the genotypic composition of the variety. What does all this have to do with variety testing? It means that many of our variety trials and the data they have produced are mostly worthless in comparing commercial varieties, because the data are confounded with generation of seed increase and seed source. What is required to produce valid data? 1. You must sample commercial varieties from commercial seed sources. This way you will test the generation that the farmer is buying. 2. Draw you own seed samples. Independent sampling is essential to unbiased testing. 3. Never change an experimental designation to a variety name or commercial designation. The experimental is likely to be the Syn 1 or Syn 2 generation. Early generation data, when presented to the fanner, is misleading and deceptive. All characters influenced by heterosis or genotypic structure such as yield, plant height, and persistence are confounded by the generation of seed increase. For these traits commercial varieties must be compared using commercial seed samples.
References:
1. Busbice, T. H. and Ramzy Y. Gurgis. 1976. Evaluating Parents and Predicting Performance of Synthetic Alfalfa Varieties. USDA, ARS-S-130. June 1976.

J. H. Bouton, Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602

Our approach at the University of Georgia to test and develop grazing persistent alfalfa cultivars has been to use the grazing animal during screening. It is important to remember that stand losses occur under grazing for a variety of reasons with defoliation and crown damage from hoof action being the two main stresses. Insuring both stresses occur is essential for a successful screen. Recently, a standard test for alfalfa cultivar grazing tolerance was proposed for inclusion in the NAAIC Standard Tests to Characterize Alfalfa Cultivars manual (Unpublished, J.H. Bouton and S. Ray Smith, Jr., 1996). The procedures described in this standard test rely on overgrazing and are not meant to be used as grazing recommendations for producers. The described protocol and the data for each check have been used to differentiate cultivar grazing tolerance in over 25 location years of testing. More importantly, the cultivar identified as a grazing tolerant check, Alfagraze, has also shown excellent stand survival for livestock producers over a range of grazing pressures, systems, and environments.
The main points of this standard tests are based on published procedures (1,2) and can be summarized as follows: Land preparation, liming, fertilization, and pest control for establishment and maintenance are the same as those used for any alfalfa yield or performance trial. All entries to be tested, plus Alfagraze, the grazing tolerant check and Apollo, the intolerant check, are sown at the normal planting date for alfalfa in replicated sward plots within a grazing paddock. Approximately 7-10 days after an initial hay harvest or graze down, initial stand measurements are made. Fourteen to 21 days after initial stands are determined, the test is exposed to intensive, continuous stocking by beef cattle (entire paddock area constantly grazed to a stubble height of 5 to 7.5 cm). Once checks are significantly different from each other (p<0.05) and within their acceptable ranges for final stands and/or survival % (e.g. Final plant counts/Initial plant counts X 100), data are analyzed and each tested entry is compared to the checks. An entry must, at a minimum, show significantly (p<0.05) better final stands and/or survival % than the intolerant check and be no different from the tolerant check in order to claim grazing tolerance.
Results obtained thus far confirm that this standard procedure is effective when screening for grazing tolerance and that selection and intermating of suriviving plants within intolerant cultivars results in germplasms with greatly improved grazing tolerance (2).
References
1. Bouton, J.H., S.R. Smith, C.S. Hoveland, and M.A. McCann. 1993. Development of grazing tolerant alfalfa cultivars. p. 416-418. Proc. XVII International Grassland Congress, 8-11 February 1993. Palmerston North, New Zealand.

2. Smith, S.R., Jr., and J.H. Bouton. 1993. Selection within alfalfa cultivars for persistence under continuous stocking. Crop Sci. 33:1321-1328.

Reduced Bloat Incidence in Grazing Trials of Alfalfa Selected for Low Initial Rate of Digestion (LIRD)

B. Coulman, W. Majak, T. McAllister, B. Berg, D. McCartney, K.-J. Cheng, J. Hall, and B. Goplen
Saskatoon Research Centre
Agriculture and Agri-Food Canada
107 Science Place
Saskatoon, Saskatchewan S7N 0X2 Canada

Bloat-safe legumes have a lower initial rate of digestion than bloat-causing legumes such as alfalfa (1). This suggests that selection of alfalfa for slower initial digestion would reduce its bloating potential. Such a selection program was initiated at the Saskatoon Research Centre in 1979 and four cycles of recurrent selection were carried out. Individual genotypes were evaluated by a 4-hoLIr nylon bag digestion in the rumen and those with the lowest digestion were intercrossed for the next selection cycle. The cycle-3 and -4 low initial rate of digestion (LIRD) populations had vegetative stage initial digestions approximately 85% of those of unselected cv. Beaver. At more mature growth stages, initial rates of digestion were not different. The LIRD populations had thicker epidermal and mesophyll cell walls than unselected populations (2).
LIRD cycle-3 was assessed for its bloating potential in feedlot and grazing trials between 1990 and 1992 (3). These trials involved a daily 6 h feeding period followed by 18 h without feed. This interrupted feeding system provoked a high incidence of bloat and there was, at best, a slight trend to a reduced bloat incidence with LIRD cycle-3 as compared to unselected cv. Beaver.
More recent trials involved 24 h grazing of LIRD cycle-4 and cv. Beaver pastures at three locations (Kamloops, Lethbridge and Melfort) in 1994 and 1995. Four fistulated steers were placed on each pasture for 2-3 week periods. Approximately half-way through each grazing period the animals were crossed-over (i.e. those on LIRD were moved to Beaver and visa versa). Of the eight grazing periods over the locations, five showed significantly (p=0.05) reduced incidences of bloat for LIRD-4. For the other three periods, there was little or no bloat seen, and consequently no significant differences between the two pastures. Over the five significant grazing periods, the reduction in bloat on LIRD-4 as comparcd to cv. Beaver ranged from 40 - 88% with a mean reduction of 62%. The differences in bloat incidence could not be explained by maturity, as there were no differences in growth stage between the two lines during the grazing periods. LIRD-4 was slightly higher than cv. Beaver in NDF, but did not differ in ADF, lignin and crude protein during the vegetative stage of growth. In other experiments at the mid bloom stage of growth, LIRD-4 was found to be higher than cv. Beaver in ADF, NDF and lignin likely due to longer
stems and a lower percentage of leaf.

These grazing trials will be continued at all three locations in 1996. If there is still a positive reduction in bloat incidence in LIRD-4, we are planning to release it as a cultivar in 1997. In initial forage yield trials, LIRD-4 has

produced from 95-110% percent as much dry matter as cv. Beaver.

References

Howarth, R.E., B.P. Goplen, S.A. Brandt, and K.-J. Cheng. 1982. Disruption of leaf tissues by rumen microorganisms: An approach to breeding bloat-safe legumes. Crop Sci. 22: 564-568.

Goplen, B.P. R.E. Howarth, and G.L. Lees. 1993. Selection of alfalfa for a lower initial rate of digestion and corresponding changes in epidermal and mesophyll cell wall thickness. Can. J. Plant Sci. 73: 111-122.

Hall, J. W., W. Majak, D. G. Stout, K.-J. Cheng, B. P. Goplen, and R. E. Howarth. 1994. Bloat in cattle fed alfalfa selected for a low initial rate of digestion. Can. J. Anim. Sci. 74: 451-456.