The induction of tetraploidy in chicory (Cichorium intybus L. var. Magdebourg) by protoplast fusion

Summary Fusions of chicory leaf protoplasts were realized in order to obtain a higher number of tetraploid plants compared to the traditional doubling techniques with colchicine. Thanks to the fusion technique, using a mixture of PEG, DMSO and a solution of CaCl₂ (pH 10.5), 25% tetraploid (2n=36) plants were obtained. Among the 167 regenerated plants, only one aneuploid of a near tetraploid level (2n=33) was identified.     

Compatibility and incompatibility in witloof chicory (Cichorium intybus L.). 4. Formation of self-seeds on a self-incompatible and a moderately self-compatible genotype after double and triple pollinations

Summary After double or triple pollinations including selfing and the use of one-day-old compatible foreign pollen a severely self-incompatible clone could be forced to develop a reasonable amount of self-seeds per flower head. This number of self-seeds increased when the foreign pollination was delayed for e.g. 240 minutes in the double pollination experiment. Even more self seeds were obtained in the triple pollination experiment with a delayed second selfing.The influence of the old foreign pollen may be caused by a mentor pollen or a pioneer pollen effect.     

The incompatibility system in Italian red chicory (Cichorium intybus L.)

As a result of an incompatibility mechanism which prevents both self-fertilization and intermating between plants with identical genotypes, inbreeding does not occur in Cichorium intybus. This self-incompatibility system complicates the production of inbred lines and the generation of F₁ hybrids in Italian red chicory. Histological observations on pollinated stigmas show that the incompatibility reaction in chicory is very rapid and either prevents pollen adhering to the stigmatic papillae or determines the arrest of pollen-tube growth on the stigmatic surface; incompatible pollen cannot reach the transmitting tissue of the style. Both histological and genetic analyses of F₁ progeny from a reciprocal cross between two unrelated plants suggest the occurrence of a sporophytic incompatibility system in Italian red chicory.     

Phenotypic polymorphism of six enzymes in the grasspea (Lathyrus sativus L.)

Summary The phenotype variation in six enzymes, 6-phosphogluconate dehydrogenase (6-PGD), malate dehydrogenase (MDH), peroxidase (PRX), isocitrate dehydrogenase (IDH), galactose dehydrogenase (GD) and glutamate oxaloacetate transaminase (GOT), was investigated using horizontal starch gel electrophoresis in 52 accessions of the grasspea, Lathyrus sativus. Phenotypic polymorphism was observed for all six enzymes. High phenotypic polymorphism (Pj) was observed for PRX and 6-PGD, while there was little polymorphism for GOT, with only two accessions showing variation. There was no correlation between phenotypic polymorphism and region of origin, or groupings of accessions made on the basis of flower colour. Tentative genetic interpretations of banding patterns are given for five of the enzyme systems. The level of apparent heterozygosity was higher than expected in this predominantly autogamous species. The level of variation in the grasspea is discussed in terms of its potential for exploitation through plant breeding.     

The progamic phase in Cichorium intybus L. Pollen tube growth in the style,incompatibility reaction and gametophytic competition

Summary Pollen germination and tube growth were studied following compatible, incompatible and pseudo-compatible pollinations in chicory. Pollen germination begins 3 minutes after compatible pollinations. The earliest pollen tubes reach the ovary 17 minutes later. Many of the later germinating pollen tubes are arrested and burst at the stigma papillae. In the transmitting tissue inhibitional effects due to negative interactions between pollen tubes are frequently observed. Complete self-incompatibility results in total inhibition of germination. In case of pseudo-self-compatibility, some pollen germinate but germination and stigma penetration are delayed and often result in pollen bursting. There is no self-incompatibility reaction in the transmitting tract but if the pollen tubes reaching this tissue are relatively numerous, negative interactions between them occur as after compatible pollinations. An hypothesis is presented which attributes the negative interactions between pollen tubes to the diffusion of a substance from the growing pollen tubes. This substance would also provoke pollen bursting on the stigma.     

Compatibility and incompatibility in witloof-chicory (Cichorium intybus L.). 3. Gametic competition after mixed pollinations and double pollinations

Summary In witloof-chicory research was carried out on the occurrence of gametic competition after mixed or double pollinations with self-and foreign pollen. In part of the experiments flower heads were emasculated before pollination. Gametic competition was deduced from the percentages of hybrid seeds in the various experiments with flower colour functioning as a marker.Among self pollen parent (SPP) clones large differences occurred for frequency of germinated pollen grains after self pollination. These differences were positively correlated with the degree of self-compatibility (SC) of the clones. No significant differences for pollen germination rate were found.Among SPP clones large differences occurred for general hybrid producing ability (GHPA). There was a significant negative correlation between GHPA and SC of the clones. Among foreign pollen parent (FPP) clones, used for pollination of SPP clones, large differences occurred for general hybrid inducing ability (GHIA). Results from removing the stigma and a part of the style at varying intervals after mixed pollination and results from double pollination with a delayed second, FPP, pollination suggest differences in pollen tube growth rate in the apical part of the style and in the basal part and/or in the ovary. Besides general HPA and general HIA also specific HPA (SHPA) and specific HIA (SHIA) occur.     

Isozyme polymorphism in Festuca rubraL.

Summary Six Festuca rubra populations from Europe and Scandinavia were studied for variation at three isozyme loci; phosphoglucoisomerase (PGI-2), glutamate oxaloacetate transaminase (GOT-3) and superoxide dismutase (SOD-1). Seven alleles were found at the Pgi-2 locus, four at the Got-3 locus and five at the Sod-1 locus. Most plants were heterozygous and up to five alleles were found in the same plant at the Pgi-2 locus. Each population could be distinguished by the presence or absence of certain alleles or by differences in the frequencies of the alleles present. Values for the Shannon diversity index were calculated which showed that there was considerable heterogeneity both within and between loci. In general, 53% of this diversity could be attributed to within population variation.     

Genomic DNA fingerprints as a tool for identifying cultivated types of radicchio (Cichorium intybus L.) from Veneto, Italy

Red or variegated chicory (Cichorium intybus L., 2n = 2 ×= 18) native to, and very extensively cultivated in north‐eastern Italy as a leafy vegetable, locally called ‘radicchio’, includes different types which represent valuable high‐quality crops. The five major types of radicchio cultivated in the Veneto region were investigated by polymerase chain reaction (PCR)‐derived markers. The experimental material was represented by two outbred populations (one of ‘Variegato di Castel‐franco’ (CF) and one of ‘Rosso di Verona’ (VR)) and by eight inbred lines (three of early ‘Rosso di Treviso’ (TVP), three of late ‘Rosso di Treviso’ (TVT) and two of ‘Rosso di Chioggia’ (CH)). A total of 96 individual plant DNAs and 16 bulked DNAs of six plants each were assayed. The different types were well distinguished from one another if analysed by means of bulks using amplified fragment length polymorphism markers at the population level, while they were not if analysed at the individual level using random amplified polymorphic DNA, inter‐simple sequence repeat and Arbitrarily Primed (AP)‐PCR markers. The genetic variation was shown to be much higher within types than between types. This result suggests that, in each radicchio type, populations produced by breeders through controlled intercrossing (VR and CF) or repeated selfing (TVP, TVT and CH) conserved their well‐separated gene pools over the years. The setting up of a molecular reference system seems to be feasible and suitable both for the precise identification of the single types of radicchio and for the evaluation of the extent of natural hybridization that can occur between different types.     

Regeneration of dihaploid chicory (Cichorium intybus L. var. foliosum Hegi) via microspore culture

In order to develop fully inbred chicory plants, dihaploid plants were raised from callus derived from microspores of three selected Witloof, Robin and Treviso types. Microspores were isolated from florets containing pollen at the uninuclear state and cultured in a modified MS medium plus 0.5mg/l 2,4-D, 0.5mg/l IAA and 2.0mg/l zeatin. During culture periods of up to 6 months, gametoplasts emerged from pollen grains, divided and started to form colonies and calli. These were subcultured on the same basal medium supplemented with 0.5mg/l BA and 0.5mg/l IAA. Shoot growth was enhanced on a low salt-containing medium supplemented with 0.4mg/l kinetin and 0.2mg/l IAA. Shoots were rooted on a half-strength Lepoivre medium plus 0.2mg/l IBA and finally transferred to soil. Florets were excised from 34 capitula, but only microspores from four of them developed into plants via callus. More than 450 plants were raised in the greenhouse and the field. Leaves from these plants were subjected to DNA fluorescence analysis via flow cytometry: a range of ploidy levels was detected. The cell composition of 44 of these plants was predominantly haploid, with a diploid background. Regenerant plant phenotypes were compared with the parent genotypes. The value of such haploids in commercial chicory breeding is discussed.     

Genetic variation for nitrate content between cultivars of endive (Cichorium endiviae L.)

Summary A high nitrate content is a negative quality trait in vegetables. Endive (Cichorium endiviae L.) contributes considerably to human nitrate intake in Northwestern Europe. A collection of 125 commercially available cultivars of endive were grown on nutrient solution and evaluated for genetic variation between cultivars for the nitrate concentration in expressed sap. In two subsequent experiments 23 cultivars, representing the range of nitrate levels found in the first experiment, were grown both in soil and on nutrient solution. There were significant differences between cultivars in nitrate accumulation. Cultivar Vicor consistently had the lowest nitrate content. Averaged over all experiments the nitrate concentration in expressed sap of this cultivar was 0.7 g·l^-1 (19%) lower than that of two recommended cultivars. The results indicate that there may be some possibilities to reduce the nitrate content of endive by cultivar choice or breeding. However, the chances of obtaining a substantial reduction of the nitrate content of endive seem less than in some other crops, such as lettuce. The nitrate levels of the cultivars showed interactions with experiments and growth medium. There was no correlation between the cultivar means for the concentrations of nitrate and chloride, or between the concentration of nitrate and the osmolarity of the plant sap.     

Electrophoretic Analysis of Eleven Isozyme Systems and their Possible Use as Biochemical Markers in Breeding of Chicory (Cichorium intybus L.)

Polymorphism and ontogeny of 11 chicory (Cichorium intybus L.) enzymatic systems have been analyzed by native polyacrylamide gel electrophoresis, namely: leucine aminopeptidase, phosphoglucomutase, shikimate dehydrogenase, glutamate oxaloacetate transaminase, superoxide dismutase, isocitrate dehydrogenase, esterase, phosphorylase, glucose-6-phosphate dehydrogenase, 6-phos-phogluconate dehydrogenase and glucose phosphate isomerase. The use of these systems as biochemical markers is discussed.     

Isozyme variation in taro (Colocasia esculenta (L.) Schott) from Asia and Oceania

Summary Isozyme variation was studied in 1,417 cultivars and wild forms of taro collected in Asia and Oceania. Seven polymorphic enzyme systems (MDH, IDH, PGI, 6-PGD, ME, SkDH, and ADH) revealed 143 isozyme phenotypes, or zymotypes, each uniquely characterized by the presence or absence of 56 electromorphs. Results showed greater isozyme variation in Asia than in Oceania, with Indonesia being the area of greatest diversity. No correlations were found between zymotypes and morphotypes or ploidy levels (as described by other investigators). Multivariate analyses of the isozyme data indicated that the majority of the Indonesian cultivars were different from the Philippine and Oceanian taro cultivars. Oceanian cultivars constituted a continuum of clusters and are thought to have originated from a narrow genetic base introduced from Indonesia. If taro breeding is to have any future in Oceania, it is important to exchange genotypes to broaden the base of existing breeding programmes.     

Temperature related instability of a naturally induced cytoplasmic male sterility in industrial chicory (Cichorium intybus L.)

Natural alloplasmic cytoplasmic male sterile (CMS) clones of industrial chicory were obtained after crossing wild chicory with selected breeding lines. We investigated the CMS stability of 10 clones in various environmental growing conditions. CMS was stable under cool growing conditions in most of them. Fertility restoration, based on pollen production scores, was observed in all clones after a period of hot temperatures. The early flower bud stage was sensitive, resulting in fertile flowers 12–17 days after exposure to high temperatures. Experiments under controlled growing conditions at 15°C demonstrated that a heat shock of 2 days at 25 or 30°C was sufficient to restore fertility. Sterile flowers were formed when plants were again grown at lower temperatures. Significant differences between individual clones were observed, indicating the potential of genetic selection to obtain stable CMS parent lines.     

Isozyme variation in a number of populations of Theobroma cacao L. obtained through various sampling regimes

Summary Isozyme polymorphism in four enzyme systems was used to estimate the genetic diversity in nine populations of Theobroma cacao L. The distribution of allelic variation observed in this study was inconsistent with the currently held view that the Upper Amazon region of Peru is the centre of cacao genetic diversity. All the populations analysed from this area of Peru were genetically similar, and contained a low level of isozyme polymorphism. It is argued that this is a result of genuine low diversity in the region rather than a sampling artifact. If a centre of diversity of wild cacao truly exists, this work indicates that it lies further north in Ecuador and Colombia.Although they are morphologically highly variable, Trinitario cacao populations derived from cultivation are currently accorded low priority for conservation. This phenotypic variation was observed to be reflected in high levels of isozyme polymorphism. It is suggested that the present strategy for the conservation of cacao germplasm, which accords priority to the Upper Amazon region of Peru while neglecting the Carribbean should be reconsidered.     

Compatibility and incompatibility in witloof-chicory (Cichorium intybus L.). 2. The incompatibility system

Summary In witloof-chicory fertilization is frequently prevented by the action of an incompatibility mechanism. In two experiments the incompatibility system was investigated. In the IVT phytotron at a constant temperature of 17 C reciprocal crosses were made between unrelated plants followed by diallel crosses between the F1 plants and backcrosses with the parents.Analyses of the results of the above crosses strongly suggest that in witloof-chicory a one locus sporophytic incompatibility system occurs with different dominance and codominance relationships between S-alleles in pollen and style.     

Varietal identification in Cichorium intybus L. and determination of genetic purity of F1 hybrid seed samples, based on RAPD markers

Random amplified polymorphic DNA (RAPD) markers were used to distinguish between several Cichorium intybus genotypes, comprising four white witloof inbred lines, three red witloof experimental inbred lines and a number of F1 hybrids derived from two white parents. Amplification conditions and reproducibility of RAPD patterns were examined. Comparison of polymerase chain reaction (PCR) products obtained by using 100 10-mer arbitrary primers allowed identification of all the lines analysed. With several primers, we defined line-specific RAPD markers, while with others polymorphism was more extensive, revealing several RAPD markers for several lines. All the differences were confirmed both on individual heads and young seedlings for each genotype. Because of the Mendelian segregation of these molecular markers, this method was applied to evaluate the genetic purity of F1 hybrid seed samples.     

PCR-RFLP analysis of the whole chloroplast DNA from three cultivated species of cotton (Gossypium L.)

Variations of the chloroplast DNA (cpDNA) from three of the four cultivated species of cotton (Malvaceae); Gossypium barbadense L., Gossypium hirsutum L., Gossypium arboreum L. and its synonym Gossypium nanking Meyen., were analyzed. Using specific set of primers, the whole circular cpDNAs from the four test species were amplified. These were subsequently digested with the use of seven restriction enzymes. The amplified fragments of the whole cpDNAs of the diploid cultivated cotton G. arboreum and its synonym G. nanking did not show any differences. However, the allotetraploid cultivated cottons G. barbadense and G. hirsutum, showed some fragment length differences directly visible after amplification and two types of restriction fragment length polymorphism (RFLP), the first appeared as slightly lengthened bands and the other as gain or loss of a restriction site. The results also showed that the chloroplast genomes of the allotetraploid cultivated cottons are highly similar to the diploid cultivated cottons tested in terms of length and digestion patterns. The detected amplified length differences, RFLPs and the restriction sites can be considered as species specific markers for the allotetraploid cultivated cottons, which could be a useful tool for future studies of the cpDNA of the genus Gossypium L.     

Genetic variation in the olive tree (Olea europaea L.) cultivated in Morocco

Summary Genetic variation was studied using enzyme polymorphism at 8 loci in ancient olive trees. These were being cultivated in 10 sites along a transect from North (Pre-Rif) to South (Anti-Atlas), involving the main ecological areas where the species is cultivated in Morocco. For the 328 trees studied, 16 alleles and 68 multilocus genotypes, of which one was present in more than half the individuals analysed, were detected. Eighty seven per cent of the total genetic diversity was attributable to within site variation and showed a high proportion of local genotypes. The highest values for both genotype number and heterozygosity were observed in the South of Morocco. Such high variation may be due to the partial domestication of olive material which may be derived from crosses between cultivars or between cultivars and feral or wild olive (oleaster) trees growing frequently in the Southern region. Genetic diversity in Moroccan olive, constitutes an important genetic resource which must be conserved for further breeding.     

Maternal induced haploids in rye (Secale cereale L.)

Summary An inbred line of rye named P8 was found to possess haploid plants in their diploid stock. Phenomenon of spontaneous haploidy was examined in this line. Fifty families derived from this inbred line were screened to determine their ploidy level by mitotic chromosome count taking root tips. Seven families were found to contain haploid plants. Among these seven families, plants from two families which were diploid were crossed reciprocally with the standard out breeding cultivar Conrah. As all of the tested plants of P8 were found aa allele for 6-Phosphogluconic dehydrogenase (6-PGD) isozyme, therefore, only those Conrah plants which have bb alleles for the same isozyme were used in crossing programme.F1 seedlings were screened with the same isozyme marker and out of 1741 F1 seedlings tested, 51 were found to be non-hybrid phenotype for 6-PGD enzyme. Among these, only five plants were found haploid and the rest were diploid. Haploid plants were obtained only from those crosses where P8 was female which indicates that haploids in this line was maternally induced.     

QTL analysis of important pith characteristics in a cross between two inbred lines of chicory (Cichorium intybus var. foliosum): a preliminary study

The genetic basis of pith characteristics in chicory (Cichorium intybus L. var. foliosum Hegi) was investigated. Quantitative trait loci (QTL) were mapped in an F2 population (565 F2 plants) derived from a cross between two inbred chicory lines. A molecular marker linkage map of this cross had previously been constructed based on 129 random amplified polymorphic DNA markers. Each F2 plant was selfed and plant characteristics were measured in the F3 populations. Although variation in pith characteristics was largely environmentally influenced, QTL for the characteristics length of pith, browning of the pith, hollow pith and apple pith were detected in many linkage groups. Interactions between QTL were found for the three characteristics: pith length, browning of the pith and hollow pith. The QTL detected confirmed the early forcing suitability of the one parent inbred line and late forcing suitability of the other.