The effect of induced autotetraploidy on resistance to clover rot (Sclerotinia trifoliorum erikss.) in red clover

In infection experiments with clover rot (Sclerotinia trifoliorum Erikss.) on 9 pairs of diploid and corresponding autotetraploid families arisen from chimaera plants the tetraploid families proved to be more resistant than their corresponding diploid families. Significant interaction between family pairs and ploidy level was demonstrated and this is an indication that there may be differences between different genotypes with respect to the effect of autotetraploidy on resistance to clover rot in red clover.

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Samenvatting Bij kunstmatige infectie met klaverkanker van 9 paren (uit chimaere planten van rode klaver afkomstige) diploide en daarmede corresponderende autotetraploide families bleken de tetraploide families grotere resistentie te vertonen dan de daarmede corresponderende diploide families.Uit een vergelijking van de families bleek dat er verschillen in invloed van de autotetraploidie op de resistentie tegen klaverkanker zijn.

     

Genotypic Differences in Root Hair Deformation and Subsequent Nodulation for Red Clover under Different Additions of Starter N Fertilization

Red clover cultivars, including diploid and tetraploid, are commonly used in legume‐based pasture mixtures. However, information on nodulation under different starter N regimens is limited. We hypothesized that there is genetic variability among different red clover cultivars for nodulation. A root hair deformation assay was conducted using three diploid (AC Christie, Tapani and CRS15) and three tetraploid (Tempus, CRS18 and CRS39) red clover cultivars by inoculating them with Rhizobium leguminosarum biovar trifolii. Nodulation and morphological characteristics of two selected red clover cultivars, AC Christie and Tempus, were determined under five starter N concentrations (0, 0.2, 0.4, 0.8 and 1.6 mg per plant). Inoculation with rhizobia increased root hair deformation with significant interaction across cultivars. Nodulation was delayed under high starter N concentrations, and genotypic differences were evident for days‐to‐nodule initiation. There was a positive quadratic response to starter N for AC Christie and a negative quadratic response for Tempus for nodulation. Tempus had more active nodules (92 %) than AC Christie (73 %). The genetic variability of red clover cultivars should be considered in N fixation studies and their response to availability of initial N.     

Influence of flower and flowering characteristics on seed yield in diploid and tetraploid red clover

Tetraploid red clover (Trifolium pratense L.) often produces insufficient seed to be economically interesting. Numerous studies have identified poor pollination due to long corolla tubes as its cause, but other studies disagree. Therefore, we investigated seed yield, corolla tube length and flowering traits in 244 plants from five diploid and five tetraploid cultivars during 2 years. Tetraploids produced fewer seeds, fewer flower heads and fewer seeds per head, but an equal number of flowers per head when compared to diploids. Although corolla tubes were longer in tetraploid than in diploid red clover (P < 0.001), no correlation between corolla tube length and seed yield was detected. Therefore, the corolla tube length is likely neither the main nor only cause of low seed yield in tetraploid red clover. Instead, three putative causes for low seed yield were identified: tetraploids produced less inflorescences per plant, possessed a lower degree of determinacy and had markedly less seeds per head when compared to diploids. Possible causes for such differences include lower pollen viability and higher rates of embryo abortion.     

Interspecific hybridization in sexual diploid Allium senescens var. minor× apomictic tetraploid A. nutans and sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens

Fourteen interspecific hybrids in sexual diploid Allium senescens var. minor× apomictic tetraploid Allium nutans L. crosses, and eight interspecific hybrids in sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens L. crosses were produced. The number of chromosomes was 2n= 24 in interspecific hybrids of diploid × tetraploid, and 2n= 32 in diploid × hexaploid crosses. Triploid and tetraploid interspecific hybrids showed intermediate parental morphological characteristics. Tetraploid interspecific hybrids of A. senescens var. minor×A. senescens crosses formed two groups based on leaf colour and leaf width. Seeds were formed in 11 out of 14 triploid interspecific hybrids under natural conditions. In cytological observations of parthenogenesis, three out of 12 triploid interspecific hybrids and five out of eight interspecific tetraploid hybrids were observed. Parthenogenesis ranged from 26.0% to 86.0% in five tetraploid interspecific hybrids. Non-parthenogenesis to parthenogenesis segregated in a 3:5 ratio in A. senescens var. minor×A. senescens crosses.     

Performance of Diploids and Induced Autotetraploids of Catharanthus roseus under Different Levels of Nitrogen and Plant Spacings

Two bulk experimental strains of induced autotetraploids,‘Purple tetraploid bulk’ (PTB) and ‘White tetraploid bulk’ of Catharanthus roseus were evaluated along with the diploid ‘Purple’ variety (PDB) and a diploid pure line selection PS-3 in four environments consisting of combinations of two levels each of plant spacing and nitrogen at Bangalore and Hyderabad during 1983—84 and in two additional environments at Bangalore during 1984—85. PTB, in general, performed better than PDB at closer plant spacings, especially in the absence of nitrogen application. The highest increase in economic yield (leaf + root yield) over PDB was recorded by PTB with 45 × 30 cm plant spacing and no nitrogen fertilization. The highest economic yield of PTB (obtained with 30 × 30 cm spacing and 100 kg N per ha) was 26 % higher than the highest economic yield of PDB. There were no significant differences between PTB and PDB for content of total alkaloids in leaves within all the environments during both years. Strain × environment interactions were significant for content of total alkaloids in roots during 1983—84. During 1984—85, PTB and PDB did not significantly differ from each other for content of total alkaloids in roots.     

Numbers of chloroplasts and pollen grain pores in diploid and tetraploid varieties of some Trifolium species

Summary In red clover (2n=14), berseem (2n=16) and in white clover (2n=32) the differences in the number of plastids in the leaf stomata and the number of germinal pores in the pollen grains between diploid and induced tetraploid plants was established.In all species the doubling of the chromosomes was followed by an increase in the number of plastids. This increase was about the same in all three species. The number of plastids in the stomata of the amphidiploid white clover was similar to that of the tetraploid types of red clover and berseem.The pollen grains of the diploid plants had almost exclusively three pores. The pollen grains of the chromosome doubled plants had predominantly more than three germinal pores.It was concluded that both characters can serve as useful indicators for establishing the polyploid level in the three clover species studied.     

Contribution of White Clover Varieties in High-Productivity Systems under Grazing and Cutting

Two medium leaf size varieties of white clover (Trifolium repens L.) were grown in mixture with a range of grass species and varieties in a field experiment conducted over 4 years. The clovers were grown singly or as a blend in swards with a tetraploid perennial ryegrass (Lolium perenne L.), a diploid Italian ryegrass (Lolium multiflorum L.) or tetraploid hybrids between these two species (Lolium x boucheanum Kunth). These grasses were also grown in monoculture. Swards were managed in a regime involving two periods of sheep grazing, two periods of cattle grazing and a silage cut in the same year for 4 years. The objectives of this experiment were 3‐fold: (1) to examine the yield and persistency of white clover in highly productive systems with modern perennial, hybrid and Italian ryegrasses; (2) to compare the performance of white clover varieties grown singly in mixture with grasses with their performance together as a blend, and (3) to examine the persistency of new Italian ryegrass varieties, exemplified by AberComo. The clover content of the sward remained high throughout the experiment, with a mean of 31 % overall. Differences in clover yield between plots were largely attributable to the effects of companion grasses. Total yields were very similar in plots containing different clover varieties and in plots where these varieties had been grown singly or as a blend. Grass yields in mixture were greater than those in monoculture but showed similar relative rankings. The diploid Italian ryegrass variety AberComo showed an unexpectedly high yield in the 4th year of the experiment. The implications of these results for strategies for productive, sustainable agriculture using modern white clover and ryegrass varieties are discussed.     

Effects of Inbreeding and Genetic Variation on Forage Quality Traits and Dry Matter Yield in Dactylis glomerata L. Subspecies

Cultivated 4× clones, 2× germplasm and their respective S; progeny were utilized in this study. Dry matter yield and forage quality data (in vitro dry matter disappearance, cell wall constituents, total nitrogen) were obtained on composited vegetative plant material harvested during four successive 4-week growth periods in the greenhouse. There was no detectable average inbreeding effect for any of the traits. The group mean of the diploids was significantly lower for in vitro dry matter disappearance than the tetraploid group mean. However, two out of six diploid 51 families exceeded the highest tetraploid family for this trait. The diploids had significantly higher cell wall constituents than tetraploids except for acid detergent lignin. There was more variation for any trait at the diploid level than at the tetraploid level among S₁ families as well as within families. Dry matter yield and total nitrogen were significantly correlated in diploids and in tetraploids. However, little variation was detected lot total nitrogen at either ploidy level. The results suggest that selection for improved quality in progeny from interploidy crosses may be more efficient by first selecting at the diploid level followed by scaling to the tetraploid level, rather than selection at the tetraploid level after hybridization.     

Screening for resistance to clover rot (Sclerotinia spp.) among a diverse collection of red clover populations (Trifolium pratense L.)

Red clover (Trifolium pratense) is an important perennial forage crop that is widely cultivated in Europe. Clover rot remains a major disease in red clover, but resistance breeding is hampered by the lack of available sources of resistance. Moreover, little is known about the factors that influence clover rot resistance. In this paper we evaluated the variation in clover rot susceptibility among a diverse collection of 113 red clover accessions, with the aim of identifying more resistant accessions. Clover rot susceptibility was assessed with a high throughput bio-test on young plants. We found significant variation in clover rot susceptibility, within and among accessions. ‘Tedi’, ‘Maro’ and ‘No 292’ were the most resistant accessions. Fifteen diploid accessions were more susceptible than the average accession with the cultivar ‘Nemaro’ being the most susceptible. Clover rot susceptibility was not correlated with isoflavone levels from Mullaney et al. (Agronomy abstract. ASA, Madison, p 195, 2000). Cultivars were more resistant than landraces and wild accessions and tetraploid cultivars were more resistant than diploid cultivars. Besides the in-depth analysis for clover rot susceptibility, possible correlations with plant architecture and other diseases were investigated. Growth habit, branching, plant yield, flowering date and susceptibility to mildew, virus and rust diseases were investigated in a 3-year field trial. Unlike previously suggested, clover rot susceptibility was not correlated with branching or with plant yield over three years. On the other hand, late flowering accessions and accessions with erect growth habit were less susceptible to clover rot. Clover rot susceptibility was not correlated with susceptibility to rust disease (Uromyces trifolii) or viral diseases, but negatively with susceptibility to mildew (Erysiphe polygoni). Because no completely resistant accessions were found, the best way to improve clover rot resistance would be to select recurrently for resistant genotypes among diverse cultivars and landraces with lower susceptibility. Tetraploidisation of diploid populations with a higher resistance level can provide an additional level of protection.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of high temperature on the production of 2n pollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization

To integrate the gene pool of a wild species (primarily diploid) into a cultivated pool (primarily tetraploid), a crossing between a dihaploid cultivated rose and a hybrid of Rosa wichurana allowed to obtain interspecific diploid hybrids that produced 2n pollen grains. A return to a tetraploid level sought by breeders can then be considered using sexual polyploidization, obtained by crossing a tetraploid cultivated rose with these hybrids. Application of a high‐temperature regime led to a small but significant increase in the percentage of 2n pollen grains in these hybrids of up to 4.6%. This result was obtained by applying high temperatures close to 32°C during the day to plants cultivated in a glasshouse during recurrent cycles of bloom. Crosses were made between an unreleased tetraploid hybrid tea rose, as a female, and the diploid hybrid that produces the most 2n pollen grains. Tetraploid (42.1%) and triploid (57.9%) offspring were obtained. The use of these 2n pollen grains of the first division restitution type should facilitate the introgression of complex traits of interest.     

Thebaine content and yield in induced tetraploid and triploid plants of Papaver bracteatum Lindl.

Summary The effect of colchicine induced autotetraploidy on the thebaine content and yield of the capsules of Papaver bracteatum was studied. A significant increase in the thebaine content of the capsules was found in tetraploid plants during two consecutive growing seasons, but the thebaine yield per plant was slightly reduced in the autotetraploids. Autotriploid plants showed a very high concentration of thebaine (8.8%) only in the first growing season.Preferential bivalent pairing and a relatively high pollen fertility were observed in the tetraploids, thus making possible the stabilization of tetraploidy by selfing.Tetraploidy seems a most promising approach for developing clones (by vegetative propagation) or lines rich in thebaine.     

Breeding forage grasses for organic conditions

Herbage yield trials are necessary to test if candidate varieties are well suited for organic conditions. In order to elucidate a particular suitability for organic conditions, we run such trials in parallel under both organic and conventional conditions. Here, we report on second and third year results of the sowings 2004 and 2005. These trials are part of a targeted breeding programme for organic conditions. Irregular colonisation of pure grass plots with adventitious plants, in particular white clover, was recognized as a major constraint to the quality of the results obtained under organic conditions. The coefficients of variance in dry matter yield almost doubled when compared to conventional trials, and significant differences in annual yield among varieties were rare. However, when herbage yield was corrected for the estimated fraction of adventitious plant colonization at each cut, the number of instances with statistically significant differences for annual yield among varieties increased more than fourfold. Dry matter yields under organic and conventional conditions were significantly and positively correlated. However, analysis of variance showed a significant culture type by variety interaction for annual dry matter yield in 12 out of 24 trials. When diploid and tetraploid varieties of the same Lolium species were tested in one common trial, only tetraploid varieties were identified as being particularly well suited for organic conditions, while in these trials, the great majority of apparently poorly suited varieties were diploid. This suggests that breeding tetraploid Lolium varieties is promising when aiming at a favourable response to organic agriculture.     

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.     

Marker‐assisted parentage analysis reveals high individual selfing rates in tetraploid red clover genotypes selected for seed yield

Seed yield is a major breeding target in tetraploid red clover. We investigated if marker‐assisted parentage analysis can identify progeny plants with two high seed‐yielding parents in tetraploid red clover and if this technique is more advantageous than traditional half‐sib selection. Parentage analysis was successfully performed on the progeny from the 10% highest seed‐yielding genotypes from a second‐cycle family selection trial: 16.0% of progeny were identified with a high seed‐yielding father. However, progeny plants with two high seed‐yielding parents did not produce more seeds than traditionally selected progeny (27.3 g vs. 30.7 g/plant, respectively). The 10% highest seed‐yielding genotypes displayed on average 2% self‐fertilization. Four genotypes were self‐fertile with individual selfing rates up to 20%. Our results discourage the use of marker‐assisted parentage analysis to improve seed yield in tetraploid red clover when the material has been preselected for seed yield. Breeders should be aware that intensive selection for seed yield in tetraploid red clover may inadvertently lead to selection for increased self‐fertility, which may increase inbreeding in the long term.     

Meiosis in Two New Intergeneric Hybrids between Hordeum and Secale

The diploid hybrid Hordeum californicum×Secale vavilovii and the tetraploid hybrid H. parodii× S. anatolicum, were obtained through embryo rescue techniques at relative frequencies of 0.70 and 5.3 % of pollinated florets, respectively. Means of 11.741 + 1.1311 per cell at metaphase I of meiosis were obtained in the diploid hybrid and 14.74¹+ 6.3l¹¹+ 0.22¹¹¹+ 0.02^IV in the tetraploid hybrid with a maximum of 6.0¹+ 4.0¹¹ and 9.0¹¹+2.0¹¹¹+ 1.0^IV respectively. In the diploid hybrid there was preponderance of intragenomic pairing (86.7 %). Increased pairing in the tetraploid hybrid was attributed to autosyndesis among Hordeum chromosomes.     

Vitamin C,flower color and ploidy variation of hybrids from a ploidy-unbalanced <Emphasis Type="Italic">Actinidia</Emphasis> interspecific cross and SSR characterization

Seedlings derived from an Actinidia interspecific cross between the hexaploid Actinidia chinensis var. deliciosa ‘Jinkui’ and the diploid male A. eriantha × A. chinensis var. chinensis ‘Chaohong’ hybrid were analyzed using flow cytometry, SSR markers and phenotypic observations. The results show that the leaf vitamin C content of this hybrid population has a mid-parent heterosis. Separation of flower color in the progeny was also observed, progeny with red flowers lighter than ‘Chaohong’, white flowers as in ‘Jinkui’ and intermediate types with a red base to the petals and white margins were all present. Flow cytometry analysis confirmed that most of the progeny were tetraploids, and molecular marker data showed that most of these tetraploid progeny had three alleles from the hexaploid parent and one allele from the diploid parent. UPGMA analysis based on the SSR markers showed that the diploid parent was completely separated from the hexaploid parent and all the progeny.     

Transformation of Diploid and Tetraploid Potato Clones with the rol C Gene of Agrobacterium rhizogenes and Characterization of Transgenic Plants

Diploid and tetraploid potato clones were transformed with the rol C gene from Agrobacterium rhizogenes under the control of the cauliflower-35-S promoter. Parameters proper to the sink and source attributes and hormone treatments have been considered to study the effect of the gene on yield physiology of the potato plant. The expression of the rol C gene in diploid and tetraploid potato lines induced drastic developmental abnormalities like dwarfism and increased tillering. Hormonal treatments revealed a different behavior of rol C transformams and controls. This supports the conclusion that the gene product of the ml C gene interacts with the hormonal status of the plant. Grafting experiments showed the non-ditfusable nature of rol C gene product. Tuberization was studied in-vitro and in-planta. Relevant differences in tuber shape and eye number were noted between rol C transfonnams and controls. Tuber yields in-planta in greenhouse or in growth chamber resulted higher in the controls; a significant increased number of tubers per plant was found, however, in both diploid and tetraploid rol C transformants. The effect of the rol C gene was apparently too drastic for diploid and tetraploid potatoes; weaker and tissue specific promoters may be necessary to induce only those modifications which are wanted for this crop. The experiments nevertheless proved that one single gene was capable in potato to affect substantially the manifestation of vieldmg attributes.     

Germinal pore number in pollen grains as a selection basis in the production of tetraploid clover

Experiments with the variety Fahl of Berseem clover and with different material of red clover have shown that the occurrence of tetraploid and diploid tissues can be detected in a rapid and reliable way by determining the number and arrangement of germinal pores of the pollen. The advantages over the conventional methods are mentioned and described.The determination of the germinal pore pattern, which is combined with pair crossing of the tetraploid heads, makes it possible to obtain in an efficient way completely tetraploid material with a great genetic variability.Samenvatting Proeven met het ras Fahl van Alexandrijnse klaver en met verschillend materiaal van rode klaver hebben uitgewezen dat het voorkomen van tetraploide en diploide weefsels op een snelle en betrouwbare wijze aangetoond kan worden door middel van bepaling van het kiemporiënbeeld van het stuifmeel.De bepaling van het kiemporiënbeeld gevolgd door paarsgewijze kruising van de tetraploide hoofdjes maakt het mogelijk op een efficiënte wijze te komen tot zuiver tetraploid materiaal met een grote genetische variabiliteit.