Alfalfa Germplasm Research Articles

A Comparison of Hydroponics, Soil, and Root Staining Methods for Evaluation of Aluminum Tolerance in Medicago truncatula (Barrel Medic) Germplasm

ABSTRACTAluminum toxicity and soil acidity are major constraints in alfalfa (Medicago sativa subsp. sativa) production in the world. Despite intense research efforts, neither an effective screening procedure nor an Al‐tolerant alfalfa germplasm is available. This dictates the need for identifying a new source of Al‐tolerant genes in the closely related species M. truncatula (barrel medic). Our objectives were to compare three Al tolerance screening methods: (i) a seedling‐based hydroponics method, (ii) a soil‐based plant method, and (iii) an Al‐stressed seedling‐based lumogallion root staining method in 32 M. truncatula accessions. The soil system compared the genotypes for dry root and shoot weights in unlimed soil and for relative weights. The lumogallion root staining of Al‐stressed seedlings compared the genotypes for fluorescence intensity of Al‐bound lumogallion within the root tips. In the hydroponics system, the genotypes were compared for root elongation and relative growth. The three methods were different from each other, with altered rankings for genotypes across the methods. The soil assay demonstrated a higher capacity for discriminating Al response among genotypes with a higher reproducibility. Most of the genotypes that were Al‐tolerant in soil were also Al‐tolerant using the hydroponics and root staining methods. The results suggested that a combination of soil‐based and hydroponics screening might be essential to identify Al‐tolerant genotypes possessing multiple Al tolerance mechanisms.

Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms

Fifteen alfalfa populations consisting of six public cultivars and nine historically recognized sources of alfalfa germplasm in North American cultivars were examined using sequence related amplified polymorphisms (SRAPs). Three bulk DNA samples from each population were evaluated with fourteen different SRAP primer pairs. This resulted in 249 different amplicons, of which over 90% were polymorphic. A dendrogram from the analysis suggests that the public cultivars are quite diverse from all the historical sources of germplasm. The highest mean genetic similarity among the nine original sources of Medicago germplasm was 0.85 between PI 536535 (Peruvian) and 536536 (Indian), while the lowest (0.47) was between PI 560333 (M. falcata) and 536539 (African). The highest mean genetic similarity among the nine original sources of Medicago germplasm and the public alfalfa cultivars was 0.78 between PI 536532 (Ladak) and Vernal, while the lowest (0.59) was between PI 536539 (African) and Oneida. Relationships based on SRAP analysis appear to generally concur with expected relationships based on fall dormancy. This report demonstrates that SRAPs are a promising marker system for detecting polymorphisms in alfalfa.

Tolerance of Australian lucerne (Medicago sativa) germplasm to grazing by sheep

The sheep grazing tolerance of a diverse range of lucerne germplasm (Medicago sativa subspp. sativa, falcata and caerulea) was investigated at Roseworthy in South Australia. Lucerne entries were established on a sandy loam soil in 1998 and managed with rotational grazing management for the first 12 months. Continuous grazing by sheep in the following year reduced the plant density of each entry as measured by plant frequency estimates by 2–98%. Lucerne was allowed to recover, then grazed continuously for another 12 months. There was great diversity in tolerance to sheep grazing among the entries tested, with final plant frequency ranging from 0–13% for highly winter-active entries, 7–23% (at least 7–23 plants/m2) for winter-active entries and 11–40% for winter dormant entries. A grazing tolerance index of commercial cultivars was determined by comparing their plant frequency decline under continuous grazing to that under rotational grazing in an adjacent experiment. Australian-bred highly winter-active cultivars displayed a greater level of tolerance than those developed overseas. The superior performance of several breeders’ lines in the continuously grazed trial indicates there is further scope for improvement with selection and breeding.

Germplasm Variability and Environmental Effects on Stem Cellulose and Lignin Concentrations in Alfalfa*

AbstractStem cellulose and lignin concentrations are major determining factors of alfalfa (Medicago sativa ssp. sativa L.) forage quality. Only limited information is available on the genetic variability and the influence of environmental effects on these two stem‐quality traits. Our objectives were to: evaluate the variability for stem cellulose and lignin concentrations in modern alfalfa germplasms across several harvests; observe the environmental stability of these two quality traits in 32 alfalfa clones selected high or low for either stem cellulose or lignin concentration; and examine the relationships between these two stem‐quality traits and leaf and stem crude protein (CP). Fifty alfalfa entries (cultivars and experimental populations) were established May 1993, and sampled for stem acid‐detergent lignin (ADL), stem acid‐detergent cellulose (ADC), and stem CP on Sep 1993, June and Aug 1994. Clones were vegetatively propagated from individual plants selected for extremes in stem ADL and ADC and transplanted into blocks at two locations in May 1994 and sampled for quality analysis in September 1994. For all samples, leaves were hand‐separated from stems and stem ADL, ADC, CP and leaf CP concentration were determined using near infrared reflectance spectroscopy. Entry differences for stem ADL and ADC were detected only at the June 1994 harvest date in the cultivar study. Spearman's ranked correlations over years in the clonal study demonstrated greater environmental stability for stem ADC (r = 0.70, P ≥ 0.01) than for stem ADL (r = 0.54, P # 0.05). Environmental effects had an impact on both traits, but stem ADC showed greater potential for improving forage quality. Simple correlations showed that decreasing stem ADL or ADC would have minimal effect on leaf CP and may increase stem CP.

Effects of alfalfa germplasm and stage of maturity on digestive process and productive response of dairy cows fed alfalfa hay-based diets

The effects on the digestive process and the productive performances of dairy cows fed diets containing alfalfa hay from different germplasms and maturity were assessed in the present study. Three different lots of first-cut alfalfa hay were used in the study: the improved variety “Boreal”, harvested at two consecutive maturity stages (early flowering and full flowering) and the ecotype “Vogherese”, at full flowering. Cutting the plant at an earlier stage of maturity improved hay quality in comparison with the more mature forages (crude protein: 21.4 vs 16.5% DM; P<0.01; NDF: 42.2 vs 53.5% DM; P<0.01). The comparison between improved variety and ecotype carried out at the same maturity showed a higher lignin content for the latter (8.6 vs 8.2% DM; P<0.01). Three isocaloric, isonitrogenous and isofibrous diets for lactating cows were formulated using an equal amount of corn silage and the maximum inclusion of one of the tested hays as forage base. The better quality of the early cut hay made it possible to increase its inclusion in the diet up to 44% of total dietary DM, while the two more mature hays covered only 36% of total DM of the respective diets. According to a 3 x 3 Latin square design, the diets were fed to 3 Italian Brown cows (initial average days in milk 121 ± 24 and milk yield 20 kg ± 1.3) fitted with ruminal cannula in 3 consecutive periods of 28 d each. Alfalfa stage of maturity and germplasm did not affect dietary DM intake (average 16 kg/d). Degradability parameters of dietary DM, calculated by in situ nylon bags technique, showed similar kinetics of rumen disappearance for all diets. No differences were noticed in the ruminal rate of passage of the solid phase among diets, while the liquid phase showed a slower rate of passage for the early flowering hay diet. Consistent with the degradation process, the stage of maturity or the alfalfa germplasm did not affect the rumen fluid data or the in vivo digestibility coefficients of the diets. Milk yield did not show any change due to either alfalfa maturity or cultivar, while milk protein was lowered when cows received the early cut hay diet. This result was likely due to an excess of rumen degradable dietary protein which could have been limited by replacing part of the dietary protein sources (soybean meal) with others more resistant to the ruminal degradation.

Improving Winter Hardiness in Nondormant Alfalfa Germplasm

Alfalfa (Medicago sativa L.) adapted to the midwestern USA typically becomes dormant in autumn to survive winter stress. Nondormant alfalfa germplasm produces higher biomass yield during autumn but usually dies during the winter. The objective of this study was to test the hypothesis that recurrent selection could decrease winter injury in nondormant alfalfa populations. Between 1997 and 2000, three cycles of selection were conducted at Ames, IA, in four nondormant cultivars (5939, GT13R+, CUF101, and Magna 8) by recurrently selecting the most vigorous plants in the spring following establishment. In May 2001, the original cultivars and the three cycles of selection (C0, C1, C2, and C3) of each cultivar were transplanted with six check cultivars in the field at Ames, IA, and West Lafayette, IN. Biomass yield was measured in September and November 2001 at both locations. In November 2001, half of the plants in each plot were removed and roots were analyzed for total sugar, starch, and protein concentrations. The remaining plants were scored for winter injury in April 2002. Strong negative linear trends were observed across cycles for both winter injury and autumn plant height. In contrast, forage biomass yields showed little change. Root total sugar concentration in November increased with cycle of selection, but root starch, protein, and amino acid concentrations remained constant. Little change was noted among cycles for forage nutritive value. Selection based solely on winter injury can improve winter hardiness of nondormant populations. A concomitant decrease in autumn plant height may not affect autumn yield.

Heterosis in a Broad Range of Alfalfa Germplasm

Crosses between Medicago sativa L. subsp. sativa and subsp. falcata (L.) Arcang. produce progeny exhibiting heterosis for biomass yield. The purpose of this study was to determine which subsp. falcata germplasm produced the best hybrids in testcrosses with elite subsp. sativa material to guide future breeding efforts. Over 100 falcata genotypes from 40 populations were test crossed to four elite sativa populations. Testcross progeny and parental clones were grown for 2 yr in two locations and harvested three times per year to determine biomass yield. A broad range of testcross performance was observed, with mean heterosis values approximately zero. The highest yielding sativa–falcata hybrids were derived from European falcata germplasm. North American semi-improved falcata germplasm performed well in hybrid testcrosses. Preselection of parental falcata genotypes for autumn growth was associated with higher yielding testcross progeny. Positive heterosis was seen during the first harvest, but negative heterosis was often observed during second and, to a smaller extent, third harvests. Superior sativa–falcata hybrids were observed that showed good biomass yield and heterosis during all three harvests. Parental yield was least predictive of hybrid progeny yield during first harvest (h2 = 0.12). Heritability increased during second and third harvest to 0.31 and 0.33, respectively. Expected genetic gain per selection cycle is greater from progeny testing compared with simple recurrent phenotypic selection.

Diallel Analysis of Carbon Isotope Discrimination and Its Association with Forage Yield among Nine Historically Recognized Alfalfa Germplasms

Designing effective breeding strategies to integrate selection for carbon isotope discrimination (A), as a means to improve alfalfa (Medicago saliva L.) water-use efficiency (WUE), will depend upon the association between Δ and forage yield. The current study was conducted to determine responses in Δ affiliated with significant yield responses that were previously observed among 36 diallel hybrids of nine alfalfa germplasms (commonly referred to as African, Chilean, Flemish, Indian, Ladak, M. falcata, M. varia, Peruvian, and Turkistan). The influence of additive and nonadditive gene action on Δ, and the behavior of A across harvests was also determined. Forage yield and A of the germplasms and their half-diallel hybrids were evaluated near Las Cruces, NM, during May, June, and July in each of 2 yr in seeded plots that were flood irrigated every 14 d. Values for A of hybrids did not significantly exceed those of the most extreme parents in any harvest. Variation for A among crosses was attributed primarily to general combining ability (GCA) effects. The magnitude of parental per se and GCA effects, and their ability to predict hybrid A varied substantially between the first harvest and later harvests. Variety effects for Δ in Ladak, M. varia, and M. falcata became increasingly negative as the growing season progressed, while those for African, Chilean, Flemish, Indian, Peruvian, and Turkistan increased. Results suggest that fundamental differences in stomatal conductance exist between subspecies sativa and falcata.

Comparative Chromosome Banding Studies of Nondormant Alfalfa Germplasm

A cytogenetic investigation was conducted on four historically putatively distinct nondormant alfalfa germplasm sources, African, Chilean, Peruvian, and Indian tetraploid alfalfa [Medicago sativa ssp. sativa (L.) L. &amp; L.; 2n = 4x = 32]. C‐banding, image analysis, and cluster analysis was used to test the hypothesis that chromosome structure differed among the four nondormant alfalfa populations. Cytogenetic analyses revealed polymorphisms for heterochromatic DNA in the number and location of constitutive heterochromatic DNA both within and among genotypes. However, this variability did not prevent recognition of homologous chromosomes. Karyotypes of Peruvian and Indian populations were developed. The reference African population was used to compare the karyotypes of Peruvian and Indian populations as well as the previously published Chilean population. In general, the number of heterochromatic DNA bands was similar for the African, Chilean, and Peruvian populations; however, the Indian population had significantly fewer heterochromatic bands than the other three. Cluster analysis based on all eight chromosomes yielded no clear separation of the nondormant alfalfa populations possibly because of the lack of chromosomal rearrangements, similar genetic backgrounds of the initial introductions, intercrossing of the different sources, genetic drift during maintenance, and/or common genetic backgrounds of the original parental germplasm sources.

Population Density and Harvest Maturity Effects on Leaf and Stem Yield in Alfalfa

A system has been proposed using alfalfa (Medicago sativa L.) as a biofuel feedstock, where the stems would be processed to produce energy and the leaves used as a livestock feed. Our objectives were to evaluate the effects and interactions of environment, population density, and harvest maturity on leaf and stem yield of alfalfa germplasms differing in fall dormancy and leaf/stem ratio. Four alfalfa germplasms established at four population densities (450, 180, 50, and 16 plants m−2) were harvested at the early bud and green pod maturity stages and evaluated in three environments for leaf and stem yield. All main effects and several two‐way interactions influenced leaf and stem yield (P &gt; 0.01). The population density × harvest maturity interaction had the greatest impact on yield. Leaf and stem yield per unit area increased as population density increased from 16 to 450 plants m−2 at the early bud stage. In contrast, leaf and stem yield increased as population density increased from 16 to 180 plants m−2, but decreased dramatically at 450 plants m−2 at the green pod maturity stage. Delaying harvest until the green pod maturity stage and decreasing population density to 180 plant m−2 maximized both leaf and stem yield in all four alfalfa germplasms studied. Decreasing population density to 180 plants m−2, and harvesting twice per season at a later maturity stage would be a effective management strategy for maximizing yield in an alfalfa biomass energy or biofuel production system.

Raffinose and Stachyose Accumulation, Galactinol Synthase Expression, and Winter Injury of Contrasting Alfalfa Germplasms

Large differences in winter hardiness exist among alfalfa (Medicago saliva L.) cultivars, but the physiological and molecular bases for these differences are not understood. Our objective was to determine how raffinose family oligosaccharide (RFO) accumulation and steady state mRNA levels for galactinol synthase (GaS) in roots relate to genetic variation in alfalfa winter survival. A GaS cDNA was isolated that possesses over 70% identity with GaS clones from other plant species. Induction of GaS transcripts in crowns of winter hardy alfalfa cultivars occurred within 8 h of exposure to 2°C, and was intensified by exposing plants to -2°C for 2 wk. Galactinol synthase transcripts increased in November in crown and root tissues of winter hardy alfalfa plants. This increase was accompanied by large increases in root RFO concentrations between October and December. A close positive association between RFO accumulation in roots in December and genetic differences in winter survival was observed in these alfalfa populations. Although roots and crowns of nondormant alfalfa cultivars accumulated both GaS transcripts and RFO, accumulation was delayed until December and these cultivars did not survive winter. Understanding the mechanisms regulating GaS gene expression and subsequent RFO accumulation in roots and crowns provides opportunity to genetically improve alfalfa winter hardiness.

AFLP-based assessment of genetic diversity among nine alfalfa germplasms using bulk DNA templates.

Improving commercial utilization of perennial Medicago collections requires developing approaches that can rapidly and accurately characterize genetic diversity among large numbers of populations. This study evaluated the potential of using amplified fragment length polymorphism (AFLP) DNA markers, in combination with DNA bulking over multiple genotypes, as a strategy for high-throughput characterization of genetic distances (D) among alfalfa (Medicago sativa L.) accessions. Bulked DNA templates from 30 genotypes within each of nine well-recognized germplasms (African, Chilean, Flemish, Indian, Ladak, Medicago sativa subsp. falcata, Medicago sativa subsp. varia, Peruvian, and Turkistan) were evaluated using 34 primer combinations. A total of 3754 fragments were identified, of which 1541 were polymorphic. The number of polymorphic fragments detected per primer combination ranged from 20 to 85. Pairwise D estimates among the nine germplasms ranged from 0.52 to 1.46 with M. sativa subsp. falcata being the most genetically dissimilar. Unweighted pair-group method arithmetic average (UPGMA) analysis of the marker data produced two main clusters, (i) M. sativa subsp. sativa and M. sativa subsp. varia, and (ii) M. sativa subsp. falcata. Cluster-analysis results and D estimates among the Chilean, Peruvian, Flemish, and M. sativa subsp. varia germplasms supported the hypothesis that Peruvian was more similar to original Spanish introductions into Central and South America than Chilean. Hierarchical arrangement of the nine germplasms was supported by their respective geographic, subspecific, and intersubspecific hybrid origins. Subsets of as few as seven highly informative primer pairs were identified that produced comparable D estimates and similar heirarchical arrangements compared with the complete dataset. The results indicate that use of primer-pair subsets for AFLP analysis of bulk DNA templates could serve as a high-throughput system for accurately characterizing genetic diversity among large numbers of alfalfa populations.

Population Density and Harvest Maturity Effects on Leaf and Stem Yield in Alfalfa

A system has been proposed using alfalfa (Medicago sativa L.) as a biofuel feedstock, where the stems would be processed to produce energy and the leaves used as a livestock feed. Our objectives were to evaluate the effects and interactions of environment, population density, and harvest maturity on leaf and stem yield of alfalfa germplasms differing in fall dormancy and leaf/stem ratio. Four alfalfa germplasms established at four population densities (450, 180, 50, and 16 plants m−2) were harvested at the early bud and green pod maturity stages and evaluated in three environments for leaf and stem yield. All main effects and several two-way interactions influenced leaf and stem yield (P > 0.01). The population density × harvest maturity interaction had the greatest impact on yield. Leaf and stem yield per unit area increased as population density increased from 16 to 450 plants m−2 at the early bud stage. In contrast, leaf and stem yield increased as population density increased from 16 to 180 plants m−2, but decreased dramatically at 450 plants m−2 at the green pod maturity stage. Delaying harvest until the green pod maturity stage and decreasing population density to 180 plant m−2 maximized both leaf and stem yield in all four alfalfa germplasms studied. Decreasing population density to 180 plants m−2, and harvesting twice per season at a later maturity stage would be a effective management strategy for maximizing yield in an alfalfa biomass energy or biofuel production system.

Physiological assessment and path coefficient analysis to improve evaluation of alfalfa autotoxicity.

Reseeding of alfalfa is affected until autotoxic chemicals break down or are dispersed, often requiring a year or more. Bioassays of seed germination and early seedling growth, on agar medium in petri dishes, were conducted to evaluate autotoxic responses of 20 alfalfa germplasms to water-soluble extracts of alfalfa leaf tissue. Root length, 120 hr after placing imbibed seed on agar, was more sensitive to the autotoxin(s) than was hypocotyl length, germination speed, and final germination percentage. Path coefficient analyses showed variation in root length had 7-17 times more effect than variation in hypocotyl length in determining autotoxic effects on total seedling length. Although variations in seed size and germination rate were negatively associated (P < 0.05) with final root length, the autotoxin had little effect on these factors relative to that on root length. Germplasms in the control differed (P < 0.05) in root length, requiring tolerance to be evaluated as percent of control. Germplasms, as percent of control, differed significantly (P < 0.05) at extract concentrations of 1.0 and 4.0 g l(-1), but the range and LSD were more favorable for selection at 1.0 g l(-1). Root length is appropriate for genetic assessments of tolerance to the autotoxin when expressed as percent of control.

Raffinose and Stachyose Accumulation, Galactinol Synthase Expression, and Winter Injury of Contrasting Alfalfa Germplasms

Large differences in winter hardiness exist among alfalfa (Medicago sativa L.) cultivars, but the physiological and molecular bases for these differences are not understood. Our objective was to determine how raffinose family oligosaccharide (RFO) accumulation and steady state mRNA levels for galactinol synthase (GaS) in roots relate to genetic variation in alfalfa winter survival. A GaS cDNA was isolated that possesses over 70% identity with GaS clones from other plant species. Induction of GaS transcripts in crowns of winter hardy alfalfa cultivars occurred within 8 h of exposure to 2°C, and was intensified by exposing plants to −2°C for 2 wk. Galactinol synthase transcripts increased in November in crown and root tissues of winter hardy alfalfa plants. This increase was accompanied by large increases in root RFO concentrations between October and December. A close positive association between RFO accumulation in roots in December and genetic differences in winter survival was observed in these alfalfa populations. Although roots and crowns of nondormant alfalfa cultivars accumulated both GaS transcripts and RFO, accumulation was delayed until December and these cultivars did not survive winter. Understanding the mechanisms regulating GaS gene expression and subsequent RFO accumulation in roots and crowns provides opportunity to genetically improve alfalfa winter hardiness.

First Report of Uromyces striatus on White Sweetclover in Kansas.

Rust was observed on leaves and stems of Melilotus alba Medik. plants in Riley County, Kansas, in November 1999. Rust reoccurred during the summers of 2000 and 2001. Uredinia were orange to reddish brown and predominately hypophyllous. Infected leaves wilted and abscised prematurely. Urediniospores were one-celled, globoid or broadly ellipsoid, and measured 20 to 25 × 18 to 22 μm. Walls were 1.0 to 1.2 μm thick, echinulate, and with three to five (mostly four) pores. Pores were equatorial, or nearly so, in approximately one half of the spores examined and scattered in the other half. Telia occurred in the fall, were sparse, amphigenous, exposed, and much darker than uredinia. Teliospores were single-celled, ovoid, subglobose or ellipsoidal, and measured 18 to 25 × 15 to 21 μm. Wall thickness was 1.3 to 2 μm. Walls had ridges approximately 1 μm wide, 2 μm apart, and arranged in a longitudinal pattern as shown in photographs of Uromyces striatus (2). Pedicels were hyaline, fragile, and short. Pathogenicity tests were conducted in growth chambers (4) with monouredinial isolate K-SC-R1 from M. alba. Pots containing plants of 80 USDA Plant Introduction (PI) core accessions representing 18 Melilotus spp. were evaluated. Total accessions tested per species were: 22 M. alba, 2 M. altissimus, 3 M. dentata, 2 M. elegans, 1 M. hirsutus, 4 M. indica, 2 M. infestus, 2 M. italica, 25 M. officinalis, 2 M. polonicus, 2 M. segetalis, 2 M. siculus, 2 M. speciosus, 2 M. spicatus, 1 M. suaveolens,2 M. sulcatus, 2 M. tauricus, and 2 M. wolgicus. Various amounts of rust developed on plants of 79 accessions; no plants of M. indica PI 234674 exhibited signs of rust. A host range study of an isolate of U. striatus from alfalfa (4) included these 18 Melilotus species; 10 were hosts. Therefore, the reactions of Medicago accessions to isolate K-SC-R1 were evaluated. These included nine alfalfa germ plasm sources representing the diversity in North American alfalfas and the cvs. Saranac and Moapa 69, which are commonly used susceptible controls in alfalfa rust evaluations. No rust resulted on any entry of Medicago sativa subsp. sativa. A few scattered, small, open uredinia occurred on plants of Medicago sativa subsp. falcata ('WISFAL' PI 560533). Of 11 Medicago lupulina PI accessions inoculated, rust resulted only on a few plants of PI 269926. All M. alba plants included as susceptible controls in all tests became heavily rusted. The causal fungus fits U. striatus Schroet. as described by uredinologists who disregard urediniospore pore position in this species. For example, illustrations of U. striatus urediniospores by Savulescu (3) show scattered pores, however, descriptions of U. striatus Schroet. in North America (1,2) specify equatorial pores. To my knowledge, this is the first report of scattered pores in U. striatus urediniospores in the United States. However, an examination of urediniospores from heavily rusted alfalfa plants collected in 2001 near Manhattan, KS and from isolate KR1 (4) also revealed urediniospores with variously distributed pores. U. striatus was reported on M. alba in Florida in 2001.

Chromosomal Polymorphism as Detected by C‐Banding Patterns in Chilean Alfalfa Germplasm 1

A cytogenetic investigation was conducted on the tetraploid alfalfa [Medicago sativa subsp. sativa (L.) L. & L.] Chilean germplasm source PI 536534 using the combined techniques of C-banding and image analysis. Cluster and multiple correspondence analyses were utilized to compare the C-banding patterns of the Chilean germplasm source and the previously published African germplasm source. Cytogenetic analyses revealed polymorphisms for heterochromatic DNA in the 19 plants observed in detail. Abundant variability in the number, intensity, and location of constitutive heterochromatic DNA was noted; however, this variability was not sufficient to preclude recognition of homologous chromosomes. Five out of the 50 plants studied were aneuploids (2n = 4x + 1 = 33 or 2n = 4x − 1 = 31) because of the presence or absence of a chromosome with a satellite. The Chilean karyotype resembled the reference tetraploid African alfalfa karyotype; however, a reduction in the total amount of heterochromatic DNA was observed. Cluster analysis and multiple correspondence analysis based on all eight alfalfa genome chromosomes yielded no clear separation of Chilean and African germplasms. However, the analysis of C-banding patterns of Homolog 1 of Chromosome 8 in Chilean and African germplasms was effective in separating the two germplasm sources with the exception of two individuals from each germplasm source which clustered together.

Production of a Biodegradable Plastic Polymer, Poly‐β‐Hydroxybutyrate, in Transgenic Alfalfa

Alfalfa (Medicago sativa L.) is primarily used as a source of forage and animal feed. Improving the economic value of alfalfa could be achieved by developing new uses of this perennial crop. To investigate the potential of alfalfa as a source of industrial materials, we employed a genetic transformation approach to produce a biodegradable plastic, poly‐β‐hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three genes from Ralstonia eutropha (formerly Alcaligenes eutrophus) encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrate‐co‐hydroxyvalerate, or PHB/V) (bktB, phbB, phbC) engineered for plastid targeting were introduced into alfalfa by Agrobacterium‐mediated transformation. DNA and RNA blot analyses of transgenic plants indicated integration and expression of the PHB biosynthetic pathway genes. Polyhydroxybutyrate content in the leaves of transgenic plants ranged from ≈0.025 to 1.8 g kg−1 dry weight (DW). Agglomerations of PHB granules 0.2 to 0.4 μm in diameter, similar to bacterial PHB, were located in the chloroplasts of transgenic plants, demonstrating that phb gene products were targeted into the plastids of transgenic alfalfa. Transgenic plants exhibited growth similar to untransformed plants, suggesting that expression of PHB biosynthetic pathway genes at current levels and accumulation of product in the plastids had no deleterious effect on growth and fertility. F1 hybrid progeny, obtained from crosses of PHB transgenic plants with elite alfalfa germplasm, exhibited leaf PHB levels similar to the transgenic parental line, demonstrating that PHB production in alfalfa is a stable and dominantly inherited trait.

Registration of OK 190 Alfalfa Germplasm

Crop ScienceVolume 42, Issue 3 p. 992-993 Registration of Germplasm Registration of OK 190 Alfalfa Germplasm J.L. Caddel, J.L. Caddel Dep. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078Search for more papers by this authorA.A. Zarrabi, A.A. Zarrabi Dep. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078Search for more papers by this authorR.C. Berberet, Corresponding Author R.C. Berberet jlc@mail.pss.okstate.edu Dep. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078Corresponding author (jlc@mail.pss.okstate.edu)Search for more papers by this author J.L. Caddel, J.L. Caddel Dep. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078Search for more papers by this authorA.A. Zarrabi, A.A. Zarrabi Dep. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078Search for more papers by this authorR.C. Berberet, Corresponding Author R.C. Berberet jlc@mail.pss.okstate.edu Dep. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078Corresponding author (jlc@mail.pss.okstate.edu)Search for more papers by this author First published: 01 May 2002 https://doi.org/10.2135/cropsci2002.9920 Contribution from the Oklahoma Agric. Exp. Stn. Registration by CSSA. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume42, Issue3May–June 2002Pages 992-993 RelatedInformation

Production of a Biodegradable Plastic Polymer, Poly-β-Hydroxybutyrate, in Transgenic Alfalfa

Alfalfa (Medicago sativa L.) is primarily used as a source of forage and animal feed. Improving the economic value of alfalfa could be achieved by developing new uses of this perennial crop. To investigate the potential of alfalfa as a source of industrial materials, we employed a genetic transformation approach to produce a biodegradable plastic, poly-β-hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three genes from Ralstonia eutropha (formerly Alcaligenes eutrophus) encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrate-co-hydroxyvalerate, or PHB/V) (bktB, phbB, phbC) engineered for plastid targeting were introduced into alfalfa by Agrobacterium-mediated transformation. DNA and RNA blot analyses of transgenic plants indicated integration and expression of the PHB biosynthetic pathway genes. Polyhydroxybutyrate content in the leaves of transgenic plants ranged from ≈0.025 to 1.8 g kg−1 dry weight (DW). Agglomerations of PHB granules 0.2 to 0.4 μm in diameter, similar to bacterial PHB, were located in the chloroplasts of transgenic plants, demonstrating that phb gene products were targeted into the plastids of transgenic alfalfa. Transgenic plants exhibited growth similar to untransformed plants, suggesting that expression of PHB biosynthetic pathway genes at current levels and accumulation of product in the plastids had no deleterious effect on growth and fertility. F1 hybrid progeny, obtained from crosses of PHB transgenic plants with elite alfalfa germplasm, exhibited leaf PHB levels similar to the transgenic parental line, demonstrating that PHB production in alfalfa is a stable and dominantly inherited trait.