A. E. Slinkard

Bi-directional transfer of nitrogen between alfalfa and bromegrass: Short and long term evidence

Transfer of N from legumes to associated non-legumes has been demonstrated under a wide range of conditions. Because legumes are able to derive their N requirements from N2 fixation, legumes can serve, through the transfer of N, as a source of N for accompanying non-legumes. Studies, therefore, are often limited to the transfer of N from the legume to the non-legume. However, legumes preferentially rely on available soil N as their source of N. To determine whether N can be transferred from a non-legume to a legume, two greenhouse experiments were conducted. In the short-term N-transfer experiment, a portion of the foliage of meadow bromegrass (Bromus riparius Rhem.) or alfalfa (Medicago sativa L.) was immersed in a highly labelled 15N-solution and following a 64 h incubation, the roots and leaves of the associated alfalfa and bromegrass were analyzed for 15N. In the long-term N transfer experiment, alfalfa and bromegrass were grown in an 15N-labelled nutrient solution and transplanted in pots with unlabelled bromegrass and alfalfa plants. Plants were harvested at 50 and 79 d after transplanting and analyzed for 15N content. Whether alfalfa or bromegrass were the donor plants in the short-term experiment, roots and leaves of all neighbouring alfalfa and bromegrass plants were enriched with 15N. Similarly, when alfalfa or bromegrass was labelled in the long-term experiment, the roots and shoots of neighbouring alfalfa and bromegrass plants became enriched with 15N. These two studies conclusively show that within a short period of time, N is transferred from both the N2-fixing legume to the associated non-legume and also from the non-legume to the N2-fixing legume. The occurrence of a bi-directional N transfer between N2-fixing and non-N2-fixing plants should be taken into consideration when the intensity of N cycling and the directional flow of N in pastures and natural ecosystems are investigated.

Sources of Resistance to Anthracnose (Colletotrichum truncatum) in Wild Lens Species

Lentil anthracnose (Colletotrichum truncatum (Schwein.) Andrus et W.D. Moore is a potential threat in many lentil (Lens culinaris Medik.) production regions of North America. In the lentil germplasm maintained in Germany and North America, 16 lines were reported to have resistance to race Ct1, but none has resistance reported to race Ct0. The objective of this study was to examine accessions of wild Lens species for their resistance to races Ct1 and Ct0 of lentil anthracnose. Five hundred and seventy-four wild accessions of six species and control lines were screened in two replications under both field and greenhouse conditions using a 1–9 scoring scale (1, highly resistant; 2–3, resistant; 4–5, moderately resistant; 6–7, susceptible; and 8–9, highly susceptible). Indianhead and PI 320937 were resistant while Eston and Pardina were susceptible to race Ct1 as expected. However, none of the check lines were resistant to race Ct0. Among the six Lens wild species tested, accessions of Lens ervoides (Brign.) Grande had the highest level of resistance, 3–5 to race Ct1 and Ct0 followed by L. lamottei Czefr. in the field and greenhouse. Lens orientalis (Boiss.), L. odemensis L., L. nigricans (M. Bieb.) Godron and L. tomentosus L. were highly susceptible, 8–9 to race Ct0 in the greenhouse. The highest frequency of resistance, especially in L. ervoides (Brign.) Grande, was found in accessions originating from Syria and Turkey. The usefulness of these L. ervoides (Brign.) Grande accessions as sources of resistance to the more virulent race of anthracnose in a lentil breeding program is discussed.

Determination of genetic variation and taxonomy in lentil (Lens Miller) species by chloroplast DNA polymorphism

SummaryChloroplast DNA restriction fragment lenght polymorphisms (RFLP) were used to examine the taxonomic relationships of cultivated and wild lentil (Lens Miller) species and identify the extent of genetic variation in this genus. Twelve accessions representing all Lens subspecies were digested with four hexanucleotide-recognizing restriction endonucleases. These digests randomly surveyed 540 base pairs, or 0.4% of the approximately 125 kilobase lentil chloroplast genome. A high degree of gragment length conservation was seen among members of crossability group I, i.e., L. c. ssp. culinaris, L. c. ssp. orientalis and L. c. ssp. odemensis. Accessions of the two subspecies comprising crossability group II, i.e., L. n. ssp. nigricans and L. n. ssp. ervoides, showed the greatest amount of variation when compared to the cultivated lentil, L. c. ssp. culinaris. Limited variation was observed within subspecies except for L. n. ssp. nigricans, where accessions of the normal cytotype were highly polymorphic to those of the differentiated cytotype. Chloroplast DNA RFLPs reaffirm hypotheses that propose L. c. ssp. orientalis as the progenitor to the cultivated lentil. The implications of this study on taxonomy and genetic resources is also discussed.

RAPD and SCAR markers for resistance to acochyta blight in lentil

Resistance to ascochyta blight of lentil (Lens culinaris Medikus),caused by the fungus Ascochyta lentis, is determined by a single recessive gene, ral2, in the lentil cultivar Indian head. Sixty F2 individuals from a cross between Eston (susceptible) and Indian head (resistant) lentil were analyzed for the presence of random amplified polymorphic DNA (RAPD) markers linked to the ral2gene, using bulked segregant analysis (BSA). Out of 800 decanucleotide primers screened, two produced polymorphic markers that co-segregated with the resistance locus. These two RAPD markers, UBC2271290and OPD-10870, flanked and were linked in repulsion phase to the gene ral2 at 12 cm and 16 cm, respectively. The RAPD fragments were converted to SCAR markers. The SCAR marker developed from UBC2271290 could not detect any polymorphism between the two parents or in the F2. The SCAR marker developed from OPD-10870 retained its polymorphism. The polymorphic RAPD marker UBC2271290 and the SCAR marker developed from OPD-10870 can be used together in a marker assisted selection program for ascochyta blight resistance in lentil.

A modified laboratory canning protocol for quality evaluation of dry bean (Phaseolus vulgaris L)

The effects of calcium (Ca2+ ) level in the soak water, blanch water and brine, blanching temperature, and total seed solids on dry bean canning quality were investigated to optimise a laboratory canning protocol. A linear increase in the Ca2+ level of soak water, blanch water and brine resulted in a linear decrease in hydration coefficient and percent washed drained weight but a linear increase in texture. Low Ca2+ level (10 mg kg-1 ) reduced the hydration time for dry bean seed from 14 to 1 h. Blanching temperatures of 50, 70 and 88 °C had non-significant effects on canning quality traits. Blanching for 30 min at 70 °C for black bean or at 88 °C for navy bean and pinto bean resulted in percent washed drained weight ≥ 60, as required by the Canada Agricultural Products Standards Act. Seed solids levels of 95-97 g per 300 × 407 (14 fl oz) can were sufficient to attain a percent washed drained weight of 60. It was confirmed that the thermal processing conditions (115.6 °C retort temperature, 45 min) used in this study were sufficient to achieve commercial sterility. The optimised lab protocol for evaluation of the canning quality of dry bean breeding lines is as follows. Seed containing 95 g of solids for pinto bean, 96 g for navy bean and 97 g for black bean is soaked in water for 30 min at 20 °C and blanched for 30 min at 70 °C for black bean and 88 °C for navy bean and pinto bean in water containing 10 mg kg-1 of Ca2+ . The seed is then transferred to a 300 × 407 can, filled with brine containing 10 mg kg-1 of Ca2+ , 1.3% (w/v) of NaCl and 1.6% (w/v) of sugar. The can is then sealed, processed in steam at 115.6 °C for 45 min and cooled at 20 °C for 20 min. Cans are stored for at least 2 weeks prior to quality evaluation of the canned product. Canning of dry bean seed according to this protocol results in precise estimation of canning quality traits, particularly percent washed drained weight.

Carbon isotope discrimination and indirect selection for transpiration efficiency at flowering in lentil (Lens culinaris Medikus), spring bread wheat (Triticum aestivum L.) durum wheat (T. turgidum L.), and canola (Brassica napus L.)

SummaryCarbon isotope discrimination has been proposed to indirectly select for transpiration efficiency in several C3 species. To determine the effectiveness of carbon isotope discrimination to indirectly select for transpiration efficiency at flowering we measured: (i) variability for carbon isotope discrimination, (ii) the magnitude of the genotype-by-water regime interaction for carbon isotope discrimination, and (iii) the magnitude of the correlation between carbon isotope discrimination and both transpiration efficiency and dry matter at flowering. Ten lentil (Lens culinaris Medikus) genotypes, ten wheat genotypes (eight spring wheat (Triticum aestivum L.) and two durum wheat (Triticum turgidum L.)), and ten canola (Brassica napus L.) genotypes were grown in a greenhouse at 80, 50 and 30% field capacity. Above ground dry matter was harvested at 80% flowering and dry matter at flowering, water used, and carbon isotope discrimination determined. Genotype variation for carbon isotope discrimination was observed in lentil, spring wheat and canola at each water regime, and when averaged over the three water regimes. The largest range in carbon isotope discrimination among lentil and spring wheat genotypes was observed using the wet regime; whereas, the dry regime provided the largest range for CID in canola genotypes. In all species the genotype-by-water regime interaction for carbon isotope discrimination was nonsignificant. The correlation between carbon isotope discrimination and dry matter at flowering was inconsistent across water regimes and years. In addition, in all three crops, no correlation was observed between carbon isotope discrimination and transpiration efficiency at any of the water regimes, and when averaged over water regimes and years. These results suggests that under the conditions reported here, carbon isotope discrimination cannot be used effectively to indirectly select for transpiration efficiency in lentil, spring wheat, and canola.

Genetic diversity in grasspea (Lathyrus sativus L.)

Genetic diversity was investigated in 348 accessions and subaccessions of grasspea (Lathyrus sativus L.) from 10 geographical regions. Polymorphism for 20 isozymes of 13 enzyme systems was studied to estimate the genetic diversity. The Near East and North Africa regions included the most variability for these isozyme systems, suggesting that the center of diversity (center of origin) for grasspea is in this general area. The lowest variability was found in accessions and subaccessions from South America, followed by those from Sudan–Ethiopia. Diversity was measured for individual loci over regions and EST-1 and SKDH had the highest genetic diversity. The closest genetic diversity was observed for LAP-2, followed by AAT-1 and PGM. The closest genetic distance existed between populations from the Near East and North Africa. Populations from South Asia and Sudan–Ethiopia, though geographically widely separated, exhibited a closer genetic distance from each other than from other regions.

Variation in the ribosomal RNA units of the generaLens andCicer

SummaryInvestigation of the organization of the tandemly repeated 5S and 18–25S ribosomal RNA (rRNA) genes was carried out on members of the generaLens andCicer using restriction endonuclease digestion and Southern hybridization. The 5S rRNA unit (gene+spacer) inLens (0.38 to 0.50 kb) andCicer 0.50 to 0.44 kb) is the largest among the legumes. InL. nigricans andL. culinaris ssp.odemensis a second repeat size was detected. Restriction maps for the 18–25S units ofLens andCicer species were developed. The 18–25S rRNA unit varied from 8.3 to 9.8 kb inLens and 10.5 to 11.4 kb inCicer. The only detectable variability in theLens andCicer 18–25S rRNA unit was in the length of the intergenic spacer (IGS) region, except for a HindIII site in the IGS region ofL. nigricans ssp.nigricans and in allCicer species.Similarities in the size of both 5S and 18–25S rRNA units were noted betweenL. culinaris ssp.culinaris andL. culinaris ssp.orientalis, which supports previous evidence thatL. culinaris ssp.orientalis is the progenitor of the cultivated lentil. Differences in the arrangement of both 5S and 18–25S rRNA units were noted betweenL. nigricans ssp.nigricans andL. nigricans ssp.ervoides, indicating either divergent evolution of these subspecies or alternatively incorrect taxonomy.On the basis of the size of the 5S rRNA unit,C. bijugum, C. chorassanicum andC. echinospermum formed a group with a slightly smaller unit than the otherCicer species. Similarities in the length of both 5S and 18–25S rRNA units ofC. arietinum andC. reticulatum possibly confirmC. reticulatum as the progenitor ofC. arietinum. Cicer cuneatum has a smaller 18–25S rRNA unit than the otherCicer species as a result of a smaller intergenic spacer.

The extent of embryo and endosperm growth following interspecific hybridization between Cicer arietinum L. and related annual wild species

In vivo interspecific pollinations were performed and immature seed development investigated by histological methods in order to study crossability barrier(s) in Cicer L. species wide hybridization. Seven of the eight wild annual Cicer species, belonging to the secondary and tertiary gene pools, were used in reciprocal crosses with the cultivated chickpea. It was confirmed that the zygote was formed in all interspecific crosses. The embryos showed continued and retarded growth at different rate in various crosses, but eventually aborted at an early pro-embryo stage in all crosses except C. arietinum L. ×C. echinospermum Dav. Reciprocal cross differences were observed in early embryo growth rate and could have implications in obtaining hybrids. This study further emphasizes the necessity for developing appropriate and efficient in vitro procedures for rescuing immature globular hybrid pro-embryos, which will make the wild Cicer gene resources amenable to chickpea improvement.

Pollen grain germination and pollen tube growth following in vivo and in vitro self and interspecific pollinations in annual Cicer species

SummaryGermination of pollen grains and growth of pollen tubes were studied to determine the cause of barreness in crosses among annual Cicer species. In vivo and in vitro time-course studies and fluorescent microscopy revealed no pollination incompatibility among the selfs, crosses and reciprocals of C. arietinum L., C. reticulatumLad. and C. cuneatumRich. In general, Cicer pollen grains germinated and grew on styles of Cicer species. Pollen tube growth was characterized by irregularly spaced and intermittent callose deposits. Failure of seed formation in interspecific pollinations may be attributed to the slowness of pollen tube growth or collapse of fertilized ovules. In addition to these causes, shortness of stamens and sparsity of pollen grains were responsible for flower drop in natural selfs. Although the number of pollen tubes entering the micropyle in interspecific pollinations was low, it may be possible to grow the fertilized ovules on an artificial medium to obtain F1 plants.