Clover Lines Research Articles

Оценка продуктивности и адаптивности сортообразцов клевера ползучего в условиях лесостепи Среднего Поволжья

Цель исследований — оценка перспективных сортообразцов клевера ползучего по продуктивности и адаптивности в условиях лесостепи Среднего Поволжья. Исследования проводили на опытном поле Обособленного подразделения в г. Пензе ФГБНУ «Федеральный научный центр лубяных культур» в 2016–2019 годах. Объектом исследования были перспективные сортообразцы клевера ползучего в питомнике конкурсного сортоиспытания, заложенном в 2015 году. Агроклиматические условия в годы исследований различались. Оптимальные условия для роста и развития клевера складывались в 2016 (ГТК за период отрастания – созревания семян составил 1,1) и в 2017 годах (ГТК = 1,2); 2018 и 2019 годы были засушливыми, ГТК в эти годы составлял 0,5 и 0,4 соответственно. В среднем за 2016–2019 годы урожайность сухого вещества клевера ползучего колебалась в пределах 2,43–2,76 т/га, лучшим по урожайности оказался сортообразец Пл-90-5 (2,76 т/га). Изменчивость урожайности у всех сортов высокая. Почти все сортообразцы показали высокую экологическую адаптивность и пластичность: значение bi у них варьировалось в пределах 0,95–1,15. Наиболее адаптивными и пластичными сортообразцами были Пл-90-2, Пл-90-3, Пл-90-4 и Пл-90-5 (bi=0,95–1,10, σd2=0,0–0,07). Наиболее высоким показателем уровня стабильности сорта отличился сортообразец Пл-90-5 (116,7%), ещё у двух сортообразцов он составил 110,2–115,5%. Наиболее высокий индекс стабильности для данной культуры (0,06) отмечен у четырёх сортообразцов, в том числе у стандарта. Из изучаемых сортообразцов четыре показали коэффициент адаптивности 1,0–1,07, наибольший показатель — у Пл-90-5. Образцы Пл-90-2 и Пл-90-3 обладали наибольшей стрессоустойчивостью. Наибольшая генетическая гибкость между генотипом и факторами среды была отмечена у сортообразцов Пл-90-4 и Пл-90-5 (2,74–2,77). The aim of this research was to evaluate promising genotypes of white clover with regard to its productivity and resistance in the forest-steppe of the Middle Volga region. The experiment took place on the trial field of the Penza branch of the Federal Research Center of Fibre Crops in 2016–2019. A competitive variety trial started in 2015. Environmental conditions varied among years of study. Optimal conditions were in 2016 and 2017, hydrothermal coefficient amounted to 1.1 and 1.2, respectively. Weather of 2018 and 2019 was dry, hydrothermal coefficient reached 0.5 and 0.4, respectively. Dry matter (DM) productivity varied within 2.43–2.76 t ha-1. Pl-90-5 line performed the best producing 2.76 t DM ha-1. Yield variation was high among all the genotypes. Plants showed high adaptability and plasticity: bi varied within 0.95–1.15. Such lines as Pl-90-2, Pl-90-3, Pl-90-4 and Pl-90-5 had the best adaptability and plasticity (bi=0.95–1.10, σd2=0.0–0.07). Pl-90-5 had the highest stability of 116.7%. The highest stability index of 0.06 was determined for four genotypes, including standard variety. Four genotypes had adaptability coefficient of 1.0–1.07, Pl-90-5 showed the highest adaptability. Pl-90-2 and Pl-90-3 had the highest stress-resistance. The highest plasticity between genotype and environment was observed for Pl-90-4 and Pl-90-5 (2.74–2.77).

FORAGE YIELD AND QUALITY OF GELEMEN CLOVER (Trifolium meneghinianum Clem.) LINES

The aim of this research was to determine the forage yields and qualities of Gelemen clover (Trifolium meneghinianum Clem.) lines. The experiment design was randomized block design with three replications. In the research, significant differences were determined among dry matter yields (675-1157kg/da), crude protein contents (15.69-18.56%), crude protein yields (112.16-206.97 kg/da), contents of acid detergent fiber (30.4834.32%), neutral detergent fiber (38.36-43.33%), total digestible nutrient (57.05-62.00%), phosphorus (0.380.43%), potassium (1.91-2.61%), calcium (1.39-1.61%) and magnesium (0.33-0.39%) as well as relative feed values (134-158%). According to results it was decided that 1, 3, 4 and 6 numbered Gelemen clover lines were selected to test in region yield experiments.

The first genetic maps for subterranean clover (Trifolium subterraneum L.) and comparative genomics with T. pratense L. and Medicago truncatula Gaertn. to identify new molecular markers for breeding

This study reports on the construction of the first genetic maps of subterranean clover (Trifolium subterraneum L.), a diploid, inbreeding annual pasture legume, and alignment of its linkage groups with those of red clover (T. pratense L.) and Medicago truncatula Gaertn. Transferability of red and white clover (T. repens L.) simple sequence repeat (SSR) markers to subterranean clover was observed. A total of 343 SSR loci were mapped into eight subterranean clover linkage groups, with 6–31 loci per linkage group and 27 loci with similar locations between two distinct F2 mapping populations. Phenotypic data obtained for flowering time, content of three isoflavonoids (formononetin, genistein and biochanin A), hardseededness, leaf markings, calyx pigmentation and hairiness of stems were analyzed, together with genotypic data. Genomic intervals influencing each trait were assigned to one to three chromosome regions, accounting for 5.5–59.8% of the phenotypic variance. Syntenic relationships were observed among subterranean clover, red clover and Medicago truncatula genomes. Comparisons of loci shared between the three species indicated that at least two chromosomal regions have undergone duplications in the subterranean clover genome. Candidate genes for isoflavone content were identified using M. truncatula as a reference genome. Synteny-based segmentation observed in Brassicaceae chromosomes helped to account for the apparent segmental-based relationship between the clover genomes, particularly within the subterranean clover lines. The proposed segmental nature of clover genome could account for the extensive variation observed between the parental genotypes, while not preventing production of fertile intercrosses.

Resistance to race 2 and cross-resistance to race 1 ofKabatiella caulivorainTrifolium subterraneumandT. purpureum

Twelve cultivars and 10 F6-derived breeding lines of subterranean clover (Trifolium subterraneum) and two cultivars of purple clover (T. purpureum) were screened in the field for their response to clover scorch disease caused by race 2 of Kabatiella caulivora that is restricted in distribution in Western Australia but is generally more virulent than race 1. Two of the cultivars (viz. Coolamon subterranean clover and Electra purple clover), and the breeding line SL031 showed outstanding resistance, with disease scores significantly lower than the resistant cultivar Denmark that had a disease score of 3.3 (0–10 scale with 0 = no disease and 10 = death of all plants). A further two cultivars (viz. Goulburn, and Karridale) and four of the breeding lines (viz. SL019, SL027, SL029 and SM033) had similar levels of resistance to Denmark. Host responses to race 2 were compared with those against race 1 of K. caulivora. Genotypes, such as SL029 and SL031, with combined resistances to both races of K. caulivora were identified, and their potential for effective management of K. caulivora, in areas where both races occur, highlighted.

Functional plasticity of Trifolium repens L. in response to sulphur and nitrogen availability

Recent control of atmospheric SO2 pollution is leading to important soil sulphur impoverishment. Plasticity could be a mechanism allowing species to adapt to this rapid global change. Trifolium repens L. is a key grassland species whose performances in community are strongly linked to nitrogen availability. Plasticity of three white clover lines contrasting in their ability to use atmospheric N2 or soil N was assessed by analysing a set of functional traits along a gradient of nitrogen and sulphur fertilisation applied on a poor soil. White clover traits showed high morphological and physiological plasticity. Nitrogen appeared to be the most limiting factor for the VLF (Very Low Fixation) line. S was the element that modulated the most traits for the nitrogen fixing lines NNU (Normal Nitrate Uptake) and LNU (Low Nitrate Uptake). As expected, N fertilisation inhibited white clover fixation, but we also observed that N2 fixation was enhanced when S was added. S fertilisation increased nodule length as well as the proportion of nodules containing leghaemoglobin. S fertilisation, with a direct effect and an indirect effect through N2 fixation, increases white clover performances particularly with regards to photosynthesis and potential vegetative reproduction. The important plasticity in response to S availability should allow it to adapt to a large range of abiotic conditions, but its sensitivity to S nutrition would be a disadvantage for competition in a situation of soil sulphur impoverishment. In contrast, S fertilisation could help maintain this species when nitrogen status is against it.

Symbiotic competence of rose clover (Trifolium hirtum All.)

Rose clover (Trifolium hirtum All.) is a forage plant that is well adapted to acidic and mildly alkaline soils of low natural fertility in southern Australia and to climates with a winter-dominant annual rainfall of 300 mm and above. Reports of low concentrations of nitrogen in rose clover foliage have been attributed to poor N2 fixation and may have discouraged its use in Australia. This investigation, conducted in tube culture, examined the ability of four lines of rose clover to nodulate and fix N2 with effective strains of clover rhizobia (Rhizobium leguminosarum bv. trifolii) and with soils (as a source of naturalised rhizobia) collected from field sites in New South Wales and South Australia. Comparisons with other Trifolium spp. were also made. It was confirmed that there was a low concentration of N in the shoots of the rose clover cvv. SARDI Rose and Hykon. This occurred even where rose clover nodulated and fixed N2 effectively with well known inoculant strains of clover rhizobia and with soil samples collected in the field (provided that the populations of resident clover rhizobia in the soil were at least 150/g). Individual plants were uniform in response to inoculation. Rose clover cv. SARDI Rose was closely related to six of the nine other lines of clover with which it was compared. It was concluded that the registered cultivars of rose clover, cvv. SARDI Rose and Hykon, are symbiotically competent plants. It appears that low N in rose clover foliage is an intrinsic characteristic of the species unconnected with its symbiotic characteristics.

Resistance to race 1 of Kabatiella caulivora in subterranean clover (Trifolium subterraneum L.) cultivars and breeding lines

Twenty-eight cultivars and 106 F6-derived breeding lines of subterranean clover (Trifolium subterraneum) were screened in the field for their response to clover scorch disease caused by race 1 of Kabatiella caulivora. Eleven of the cultivars, including Denmark and Goulburn, were classified as resistant. Breeding lines with Denmark parentage had 55% of progeny with resistance, while those of Goulburn had only 19% of resistant progeny, suggesting different modes of inheritance. Selection for resistance to race 2 of K. caulivora in the F4 generation markedly increased the probability of selecting F6-derived lines with resistance to race 1, suggesting linkage between genes for resistance to both races.

Effects of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without a silage inoculant ( Lactobacillus plantarum L54)

Polyphenol oxidase (PPO) has been shown to reduce proteolysis and lipolysis in red clover through deactivation of proteolytic and lipolytic enzymes and/or through formation of protein–phenol–lipid complexes. This experiment investigated the time course of both lipolysis and proteolysis in two red clover lines with different PPO activities with and without addition of a silage inoculant to help understand the action of PPO in the silo, and its potential effects on protein and glycerol-based lipid conservation, and to determine effects of a more rapid pH reduction with inoculation on PPO activity. Four silages were prepared from high or low PPO precision chopped red clover in 60 test-tube-silos, each containing 120 g fresh weight: (a) high PPO red clover without inoculation (H−), (b) low PPO red clover without inoculation (L−), (c) high PPO red clover with inoculation (H+), and (d) low PPO red clover with inoculation (L+). Each treatment had three replicates for each time point of 1, 2, 4, 8 and 90 days. The inoculant used was Lactobacillus plantarum strain L54 applied at a rate of 10 6 CFU/g fresh weight. Silage pH was reduced ( P < 0.001) by inoculation with no effect of PPO. Inoculation had no effect on either lipolysis or free amino acid release, although more ( P < 0.01) soluble protein and less ( P < 0.01) ammonia-N was in inoculated silages. H silages had a lower level of both proteolysis (release of free amino acids, P < 0.05) and lipolysis (loss of membrane lipid, P < 0.01) than L red clover silages. Results indicate that PPO reduced proteolysis and lipolysis in the silo and that inoculation had no adverse effects on PPO activity.

Improving organic phosphate utilization in transgenic white clover by overexpression of Aspergillus niger PhyA gene

Using the cotyledon of white clover as explants, the transgenic white clover lines ectopic expression of the PhyA gene were established based on Agrobacterium tumefaciens-mediated transformation method. It was found that the tested transgenic lines were all Polymerase Chain Reaction (PCR) positive. The transgenic lines 1 to 4 were used for further Southern blot and Northern blot analysis. The lines 1 and 3 with higher level of PhyA expression were used to assay the phytase activities in root and its intercellular space. When the phytate was the sole phosphorus source, the phytase activities in root in lines 1 and 3 were 31.43% and 44.76% higher than those in control (CK), respectively. Meanwhile, the phytase activities in the root intercellular space in lines 1 and 3 were 3.3-fold and 5.12-fold higher than those in CK, respectively. The phosphorus concentration of plants, the accumulative P amount per plant, plant fresh weight, and plant dry weight were all much higher in lines 1 and 3 than in CK. Thus, it is clearly shown that ectopic expression of Aspergillus niger PhyA gene could significantly increase the ability for white clover to utilize organic phosphate under inorganic phosphate (Pi)-deficient condition.

Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen fluid

AbstractThis experiment aims to determine whether polyphenol oxidase (PPO) can reduce the extent of lipolysis and the consequent polyunsaturated fatty acid loss through microbial biohydrogenation in red clover when incubated in the presence of rumen fluid. PPO is involved in the browning reaction of red clover leaves when cut or crushed and exposed to air. It starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins and have been shown to reduce proteolysis and lipolysis in silo. Two lines of red clover (cv. Milvus), a genotypic mutant with reduced PPO activity (L) and the wild type (H) with a high level of PPO activity, were cut 3 cm above soil level, crushed and cut into 1 cm strips before being loaded into incubation bottles. These were then incubated in anaerobic buffer at 39 °C in either the absence (−) or the presence (+) of rumen microorganisms. The incubations were then compared over a 24 h time course in terms of lipolytic activity. Characterization of the tissues showed PPO activities of 25.3 and 5.13 U g−1 fresh weight for H and L, respectively. Lipolysis, measured as the proportional decline in the membrane lipid, was reduced (P &lt; 0.001) with increasing PPO activity in both the presence (+) and absence (−) of rumen microorganisms. However, values were significantly higher (P &lt; 0.001) in the presence of rumen microorganisms, with values after the 24 h incubation of 0.28, 0.42, 0.72 and 0.82 for H−, L−, H+ and L+, respectively. Biohydrogenation of C18:2 and C18:3 polyunsaturated fatty acids were significantly lower in the H+ treatment than the L+ treatment, with mean values after 24 h incubation of 53% and 57% (P &lt; 0.05) for C18:2 and 65% and 74% (P &lt; 0.01) for C18:3, respectively. Changes that occurred in the lipid fractions (membrane lipid, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce polyunsaturated fatty acid loses in the rumen. Copyright © 2007 Society of Chemical Industry

Intraspecific variation in Trifolium pratense: impact on feeding and host location by Sitona lepidus (Coleoptera, Curculionidae)

Red clover (Trifolium pratense L.) is becoming increasingly important in grassland systems because of its high productivity, protein content and nitrogen-fixing ability, but its use has been constrained by losses due to pests and diseases, and this contributed to the decline of red clover usage allowing white clover (T. repens L.) to become the dominant legume of UK grasslands. One of the major pests of red clover is the clover root weevil (Sitona lepidus Gyllenhal, Coleoptera, Curculionidae) which attacks both the shoots and roots, particularly the N-fixing root nodules, of clover plants. This current work investigates the feeding preferences of S. lepidus with respect to 11 varieties (Pawera, Kuhn, Astra, Norseman, Norseman low, Norseman high, Marcom, Merviot, Milvus, Britta, Sabtoron) and 5 lines (AA30, AA31, AA4493, AA4494 and AA4495) of red clover in order to identify relationships between the variation in attractiveness of different red clover varieties for both adult and larval stages of the weevil. Of those tested Norseman high showed potential resistance, being less favoured by both adults and larvae of the weevil. This may be attributed to the potentially high phyto-oestrogen levels in this variety.

Determination of Some Plant and Agricultural Characteristics in Persian Clover (Trifolium resupinatum L.)

Forage legumes have been important livestock feed for centuries as pasture, soilage and conserved forage. Because of the large number of species, their wide adaptation to soil and climatic conditions, and their general ability to reseed readily, Trifolium is one of the two most important legume genera in livestock agriculture. Persian clover (Trifolium resupinatum L.), one of the important species of genus Trifolium, has agronomic potential to be grown in Central Anatolia. This study was carried out to investigate some plant and agronomic characteristics of Persian clover lines. A number of lines of this valuable pasture and hay plant were tested for forage yield potential in Ankara conditions. According to the results, the highest natural plant height was measured in Line 5475 as 51.267 cm, the highest plant height was in Line 5475 as 58.987 cm, biggest leaf number was in Line 5453 as 9.6, the widest leaf was in Line 5475 as 22.3 mm, the longest leaf was in Line 5475 as 33.5 mm, the widest stem diameter was in Line 5475 as 55 mm. The highest fresh and dried hay yield obtained from Line 5475 as 1962 kg/da and 331.37 kg/da while Line 5461 gave biggest dried hay ratio as %19.1. Persian clover could be used as a forage crop in Ankara conditions because of it’s high fresh and dry herbage yield. And it can be recommended that Line 5475 is a suitable genotype in the production of fresh and dried hay in Ankara conditions.

Formononetin in clovers as a feeding deterrent against clover root weevil

The effect of leaf feeding by adult clover root weevils on the isoflavonoid content of red and white clovers was investigated in a glasshouse experiment. Lines of both clovers previously selected for variation in formononetin content were used. A higher percentage of white clover leaves than red clover leaves were damaged by the weevils, and the high formononetin red clover was least readily eaten. The formononetin content of the white clovers did not differ between the selections, was much lower than in the red clovers, and did not change in response to weevil feeding. In both red clover lines, formononetin and biochaninA levels rose in response to weevil feeding, suggesting that both compounds have antifeedant properties against clover root weevil. Because high foliar levels of oestrogenic compounds like formononetin threaten fertility of grazing sheep it is concluded that plant breeding programmes should increase the levels of formononetin and/or biochaninA in clover roots to mitigate the impact of weevil larval feeding on roots and nodules.

Interaction between Sitona lepidus and red clover lines selected for formononetin content

AbstractAdult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.

Invasion and reproduction by rootlesion nematode (Pratylenchus penetrans) differs among selected lines of red clover (Trifolium pratense)

Eighteen cultivars and breeding lines of red clover (Trifolium pratense) were evaluated in a greenhouse study for their relative response to inoculation by the root-lesion nematode (Pratylenchus penetrans). The incidence of plants invaded and the nematode concentration in the roots were generally related but not always. One cultivar (Florex) had a low incidence of plants being invaded, but a high concentration of nematodes in the roots of invaded plants. Three entries (CRS 15, CRS 5, and CRS 11) displayed a low incidence of plants being invaded and a low concentration of nematodes in the root. One cultivar, AC Kingston, was judged to be highly susceptible, while the remaining entries had differing invasion incidences and differing concentrations of root-lesion nematodes in the roots.

Performance of nematode-resistant white clover in field trials

Breeding for improved resistance and tolerance to the clover root-knot nematode, Meloidogyne trifoliophila, and the clover cyst nematode, Heterodera trifolii , has been successful in white c lover, Trifolium repens. White clover lines developed from three independent breeding programmes were established in field trials at Lincoln, Palmerston North, Cambridge and Kerikeri. Plants were established in areas either untreated or treated with nematicide, so as to have contrasting pest loads on the plant material and evaluated for up to 4 years. Plant vigour was scored before each grazing at each site. Clover cyst nematode cysts were counted twice in Palmerston North and all nematodes were counted in stained roots on four occasions in Cambridge. Resistant lines from the clover cyst nematode programme performed better than susceptible lines and as well as most cultivars reflecting the high level of resistance developed in this glasshouse-based programme. This resistance was also reflected in the generally lower number of cysts counted under resistant lines from Palmerston North. The root-knot nematode resistant material performed better than the susceptible and as well as most cultivars. The tolerance selections, developed under field conditions, performed as well as, or better than the cultivars. The selections from the breeding programmes have exhibited strong agronomic potential across locations and years and the best material has been crossed, with progeny being assessed in current field trials. Keywords: Heterodera trifolii, Meloidogyne trifoliophila, nematode, pasture, resistance, tolerance, white clover

Plant‐mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities

AbstractPolyphenol oxidase (PPO) is an enzyme involved in the browning reaction of red clover leaves, when cut or crushed and exposed to air. PPO starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins. The leaf tissue of two lines of red clover (cv. Milvus, a genotypic mutant with reduced PPO activity (LowPPO) and the wild‐type, NormalPPO) were extracted in phosphate–citrate buffer, and a third treatment was prepared by extracting the LowPPO leaves in phosphate–citrate buffer plus 50 mM ascorbate to inhibit PPO activity (AscPPO). These extracts were compared over a 12 h time course in terms of proteolytic and lipolytic activity. Characterization of the tissues showed PPO activities of 9.11, 1.85 and 0 Δ optical density g−1 fresh weight min−1, which were reflected in the extent of phenol (derived from quinones) binding to protein after 12 h incubation 102.3, 83.2 and 45.8 mg bound phenol g−1 protein (p &lt; 0.001) for NormalPPO, LowPPO and AscPPO, respectively. Proteolysis measured as free amino acids released into the incubation was significantly reduced (p &lt; 0.001) with increasing PPO activity, with values after the 12 h incubation of 0.03, 0.08 and 0.14 g g−1 protein for NormalPPO, LowPPO and AscPPO, respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (p &lt; 0.001) with increasing PPO activity, with values after the 12 h incubation of 0.12, 0.20 and 0.22 for NormalPPO, LowPPO and AscPPO, respectively. Changes that occurred in the lipid fractions (polar fraction, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid loses in silo and potentially in the rumen. Copyright © 2004 Society of Chemical Industry

Further investigation of the roles of auxin and cytokinin in the NH4+-induced stimulation of nodulation using white clover transformed with the auxin-sensitive reporter GH3:gusA

Although mineral N (nitrate and ammonium) is believed to have generally negative effects on nodulation in legume-rhizobia symbioses, previous studies have shown that low, static concentrations of ammonium stimulate nodulation in pea, and that this enhancement may be due to an elevation in cytokinin to auxin levels in roots. Here, the effects of ammonium (0.0, 0.1, 0.5 and 2.5mM) on nodulation and auxin levels were investigated in wild-type (WT) white clover (Trifolium repens cv. Haifa) and its transformants (lines 38 and 41) which contain the auxin-sensitive reporter gene (GH3:gusA). The effects of exogenous application (10 -10 , 10 -9 and 10 -8 M) of the cytokinin 6-benzylaminopurine (BAP) were also assessed. Whole-plant nodulation (nodules plant -1 ) and dry weight (DW)-specific nodulation (nodules g -1 root DW) were stimulated (up to 49%) in all white clover lines by 0.1mM NH 4 +. This represents the first confirmation of an NH 4 +-induced stimulation of DW-specific nodulation in a species other than pea. At 2.5mM NH 4 +, the effect was lost on whole-plant nodulation and was inhibitory on DW-specific nodulation. Rhizobial inoculation resulted in a decline in the expression of GH3:gusA in root tips as expected; however, ammonium treatment did not affect GH3 expression in any root zones. Exogenous application of BAP at 10 -9 and 10 -8 M stimulated whole-plant and DW-specific nodulation in wild-type white clover to a similar degree as treatment with 0.1 mM NH 4 +. These results support our previous hypothesis that the stimulation of nodulation by low concentrations of ammonium involves the alteration of the ratio of cytokinin to auxin, specifically by increasing cytokinin.

Effect of generation of inbreeding, cutting treatment and year on agronomic traits in berseem populations

The selfing effect in berseem clover (Trifolium alexandrinum L.) was analysed by comparing generation means for nine agronomic traits (dry matter yield, plant height, leaf-stem ratio, harvest index, seed yield and its components). Parents, S1, S2, S3, and S4 generations of six populations of different geographical origin (3 from Egypt and 3 from southern Italy) were investigated to estimate the relative changes with increasing homozygosity and to prove the effectiveness of selfing in a breeding program. The experiment was carried out at Foggia (Italy) under field conditions for two years (1995 and 1996) adopting two cutting treatments: at fifth internode elongation (A) and flowering (B). The mean values decreased with the advancement in selfing generations in both years and cutting treatments for the most of the traits. Treatment B yielded more biomass and seed yield than treatment A in both years. Cutting treatments did not affect the adaptability of the selfed progenies of populations with different geographical origin, in fact, the Italian populations were more productive for dry matter under both cutting treatments (856.6 g m-2 on average), whereas Egyptian populations proved superior for seed yield particularly under treatment B (62.5 g m-2 on average). Inbreeding depression was greater for forage dry matter and seed yield (−62and −79% on average, respectively), whereas the plant height and 1000-seed weight were the least susceptible traits (−26 and −19% on average, respectively). Estimates of the parameters of the stability analysis showed that not all populations responded to inbreeding level and environment variations to the same degree. Differences were evident under treatment A, only. Development of self-fertile, relatively vigorous inbred lines of berseem clover with different degrees of homozygosity appears possible. Genotypes tolerant to selfing from Egyptian and Italian groups have been obtained by inbreeding and selection for self-fertility; they could be utilised as basis for producing new breeding activities.

Characterization of Rose Clover Germplasm for Flowering Traits

Rose clover (Trifolium hirtum All) cultivars with long‐season (November through May) forage production and cold tolerance are needed to increase pasture availability during the winter in northeast Texas. More information about the genetic and environmental control of rose clover flowering is warranted, before this trait can be manipulated through plant breeding. The objective of this study was to evaluate the variability for flowering traits in the U.S. Plant Introduction Collection of rose clover. Sixty‐six accessions of rose clover, from eight different countries, were evaluated. Observations recorded on individual plants at 4‐d intervals included first elongation of internodes, first bud, first flower, first color, full bloom, and mature seed. In general, rose clover behaved as a long‐day plant and flowered in photoperiods from 12 to 14h. Thirty‐one rose clover lines were identified within the maturity range of 172 to 182 days of the year (DOY) to full bloom, twenty‐seven were identified within the maturity range of 162 to 170 DOY to full bloom, only five rose clover lines reached full bloom in less than 162 DOY, and only three took more than 183 DOY. There was a range of 40 d difference in time to reach maturity for the rose clover germplasm studied. Utilizing principal component analysis and cluster analysis, the germplasm was separated into three different maturing groups: early, midseason, and late maturity groups.