Seed structure in Crocus sativus L. ×, C. cartwrightianus Herb., C. thomasii Ten., and C. hadriaticus Herb. at SEM

Crocus sativus L. (Iridaceae) is a sterile triploid geophyte widely cultivated for the production of the spice saffron and only reproduced by means of corms. Extensive research has identified Crocus cartwrightianus Herb. as being a probable progenitor of C. sativus. However, other diploid Crocus species of the same C. sativus group, such as C. thomasii Ten. and C. hadriaticus Herb., have been considered as possible progenitors of saffron. Of the characteristics for distinguishing critical genera, species and intraspecific taxa of angiosperms, the most widely adopted have been seed organisation and patterns of spermoderma microstructure detected at SEM. The aim of this study is to use SEM to analyse the seeds of C. sativus ×, a cross obtained by C. sativus with pollen of C. cartwrightianus Herb. and the seeds of allopollinated C. cartwrightianus, C. thomasii Ten., and C. hadriaticus Herb. Results indicate that the seed surface microstructure of C. sativus × is very similar to that of C. cartwrightianus while being different from those of C. thomasii and C. hadriaticus.     

A study on pollen grains ofCrocus cartwrightianus (Iridaceae)

Crocus cartwrightianus andC. cartwrightianus cv.albus pollen have been studied from structural and ultrastructural points of view and the germination assayed in vivo and in vitro.C. cartwrightianus pollen is regularly shaped and sized, has a low percentage of anomalous grains and has a high germination rate in vitro, whileC. cartwrightianus cv.albus is less regularly shaped with some variation in size and has a high percentage of anomalous grains and a low germination percentage in vitro. Ultrastructural observations have revealed, in the pollen of both the taxa, the presence of a thin elongated vegetative nucleus and a generative cell surrounded by a thin membrane. However,C. cartwrightianus pollen shows a thicker intine, andC. cartwrightianus cv.albus shows numerous pollen germination anomalies which are in common withC. sativus.     

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The amplification of the seven Crocus species DNAs with twenty-one primers provided 217 repeatable and interpretable fragments, which were scored for presence/absence and employed for a cluster analysis. Results indicated that C. sativus is very closely related to C. cartwrightianus and also similar to C. thomasii. This result, concurring with part of the previous evidence, would rule out the hypothesis of close relationships between C. sativus and C. pallasii.     

Pregamic and postgamic self-incompatibility systems inCrocus (Iridaceae)

Ovule fertilization and seed set were investigated inCrocus thomasii andCrocus vernus subsp.vernus, after different matings. The results gave evidence for a strict ovarian barrier inC. thomasii, which prevents self-fertilization either in self- or mixed- and double-pollination. InC. vernus the ovarian barrier to selfing is not so effective, but a further incompatibility reaction is expressed after self-fertilization resulting in a complete failure of embryogenesis. The implications of the present observations in respect to the mechanisms of pregamic- and postgamic rejection, indicate that a gametophytic system of self-incompatibility controls the ovule fertilization, and a not yet defined system of self-incompatibility controls embryogenesis.     

Embryology ofCrocus thomasii (Iridaceae)

The embryology ofCrocus thomasii is described. Male meiosis is of simultaneous type, and gives rise to starchy microspores which develop into lipoid pollen grains; these are two-celled and show a spinulate acolpate, abaculate exine lacking apertures. The tapetum is glandular and its cells become bi- or sometimes multinucleate. The ovule is anatropous and bitegmic; the inner integument forms the micropyle. Megasporogenesis is heteropolar with starch accumulation in the functional chalazal megaspore. Embryo sac development conforms to thePolygonum type. The endosperm development is nuclear. The embryo develops according to the Caryophyllad type. In the ripe seed it is differentiated and enveloped by a starchy cellular endosperm. The embryological characters observed strongly favour a close relation betweenC. thomasii andC. sativus.     

Cytology ofCrocus sativus and its allies (Iridaceae)

Populations ofC. cartwrightianus, C. hadriaticus andC. thomasii from the Balkans have 2n = 16 as has the Middle EasternC. pallasii subsp.haussknechtii. C. dispathaceus andC. pallasii subsp.pallasii have 2n = 14 whileC. pallasii subsp.turcicus has 2n = 12.C. niveus has 2n = 28 andC. sativus is an autotriploid with 2n = 24. Karyotype variation was found between populations ofC. pallasii subsp.pallasii in Central Turkey and also inC. pallasii subsp.turcicus.     

Embryo origin and development in Crocus sativus L. (Iridaceae)

Abstract

Fruit and seed set were obtained in the triploid Crocus sativus (saffron) either by hand crossing with pollen of the diploid C. cartwrightianus or by free pollination. The morphology of the capsules and seeds was similar in both Crocus species. The embryo is comparatively small, whereas the endosperm is formed of numerous layers of cells with hemicellulose-thickened walls and a cytoplasm containing few lipid and protein reserves. Seed germination gave rise to a white prophyll from which a green leaf emerged. At the base of this leaf, a small corm without tunics developed, which, after leaf desiccation, became dormant. These corms sown separately in pots were again capable of growth and emitted a single long green leaf. Vegetative development of new corms was similar in the diploid and hybrid saffron plants. In sites of past saffron cultivation the beds were found to contain degenerated corms and corms at different development stages. The absence of fruit and seed set in emasculated and self- and out-pollinated plants suggests that saffron does not form apomictic embryos.     

Pollen structure and germination of Crocus thomasii Ten. (Iridaceae)

Abstract

Mature pollen of C. thomasii Ten. (Iridaceae) has been studied from a morphological and physiological point of view and compared to that of C. sativus L., C. thomasii pollen is roundish, with a 83 μm diameter and a 14% of anomalous grains. Percentage of the positive alcoholdehydrogenase (ADH +) pollen grains is about 90% whereas the in vitro germinated rate reaches 55%. Exine is homogeneously thickened (2.5 μm) with randomly distributed thinner zones and 5 μm thick intine. Cytoplasm of vegetative and generative cells is very rich of small and large smooth vesicles and vesicles coated by ribosomes. The generative cell shows a thin ondulated pectocellulosic wall. Its nucleus is intensely fluorescent after treatment with the 4,6-diamidino-2-phenylindole (DAPI) fluorochrome. Although many ultrastructural aspects of the C. thomasii pollen are common in C. sativus. L., C. thomasii pollen is smaller, more regularly structured and germinates in vivo and in vitro in higher percentage than that of C. sativus.     

Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP)

Background and Aims: Saffron (Crocus sativus) is a sterile triploid (2n = 3x = 24) cultivated species, of unknown origin from other diploid and polyploid species in the genus Crocus (Iridaceae). Species in the genus have high morphological diversity, with no clear phylogenetic patterns below the level of section Crocus series Crocus. Using DNA markers, this study aimed to examine the diversity and relationships within and between species of Crocus series Crocus.Methods: Eleven inter-retroelement amplified polymorphism (IRAP) primers were used in 63 different combinations with 35 single-plant accessions of C. sativus and related Crocus species in order to determine genetic variability and to conduct phylogenetic analysis.Key Results: A total of 4521 distinct polymorphic bands from 100 bp to approx. 4 kb were amplified; no fragment specific to all accessions of a single species was amplified. The polymorphic information content (PIC) values varied from approx. 0·37 to approx. 0·05 (mean 0·17 ± 0·1) and the major allele frequency had a mean of 0·87. High levels of polymorphism were identified between accessions of the six species of Crocus series Crocus related to C. sativus, with further variation between the species. In contrast, no polymorphisms were seen among 17 C. sativus accessions obtained in the region from Kashmir through Iran to Spain.Conclusions In contrast to the intraspecific variability seen in other Crocus species, C. sativus has minimal genetic variation, and it is concluded that the triploid hybrid species has most probably arisen only once. The data show that saffron is an allotriploid species, with the IRAP analysis indicating that the most likely ancestors are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics, and show the need for conservation and collection of wild Crocus.     

Pathogenicity of fungi associated with wheat and barley seedling emergence and fungicide efficacy of seed treatment

Presented study focused on the influence of Cochliobolus sativus isolates origin on pathogenicity towards wheat and barley seedlings in comparison with pathogenicity of certain Fusarium species and Microdochium nivale. The efficacy of fungicide seed treatment against C. sativus was estimated. The C. sativus isolates were collected from different locations and were isolated from wheat, barley and sunflower seeds. The pathogenicity of C. sativus, Fusarium species and M. nivale towards germinating seedlings were expressed as germination (GA) retardation and coleoptile growth rate retardation (CGR). Of wheat only, the CGR was significantly influenced by the isolate origin. The C. sativus isolates obtained from sunflower seeds were the most aggressive. Of the barley seeds, the barley isolates were the most aggressive. Barley was significantly more susceptible to damage by C. sativus isolates than wheat. The pathogenicity of tested fungal species declined in the order: F. culmorum, F. graminearum, C. sativus, F. avenaceum, M. nivale, F. poae for both barley and wheat. The results highlighted high pathogenicity potential of C. sativus equal to that of F. avenaceum and M. nivale. The symptoms of C. sativus on coleoptile and roots were very similar or the same as the symptoms caused by Fusarium species and M. nivale, except of white, pink or red colours. Of wheat sprouts, the fungicide efficacy (FE) against C. sativus declined in the order: tebuconazole + thiram, carboxin + thiram, quazatine, difenoconazole, iprodione + triticonazole (in term of GA) and carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of CGR). In barley, the FE declined in the order: carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of GA) and carboxin + thiram, tebuconazole + thiram, difenoconazole, iprodione + triticonazole, quazatine (in term of CGR).     

Polyphenol oxidase activity in dormant saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) corm

Crocus sativus L., cultivated since ancient times as the source of saffron, is a triploid plant that can be propagated only via its corms which undergo a period of dormancy. Understanding the processes taking place in the corm is essential to preserve the plant and improve its quality. Color and taste being of prime importance in the quality of the saffron spice, knowledge on polyphenol oxidase (PPO) activity in the plant is of particular interest given the role of the enzyme in fruit and vegetable browning during processing and during the storage of processed food. In this paper, PPO activity was investigated for the first time in extracts obtained from dormant C. sativus L. corms. PPO activity was detectable using l-DOPA, pyrogallol, catechol or p-cresol as substrate, each being oxidized to its corresponding o-quinone; no activity was detectable with l-tyrosine, tyramine or phenol as substrate. Two pH optima, respectively at 4.5 and 6.7, were observed with all substrates and a third one, at 8.5, was found with l-DOPA and p-cresol. Kinetics parameters studied at pH 6.7 indicated the highest catalytic efficiency (in units mg⁻¹ prot mM⁻¹) with pyrogallol: 150, then catechol: 39, l-DOPA: 6.4 and p-cresol: 4.6. The enzymatic activity was inhibited by 50% in the presence of 0.22, 0.35, 0.5 and 0.7 mM kojic acid with, respectively, catechol, pyrogallol, p-cresol and l-DOPA as substrate. When stained for PPO activity, non-denaturing gel electropherograms of extract revealed three distinct bands, indicating the presence of multiple isoenzymes in dormant C. sativus L. corms.     

In vitro development of parthenocarpic fruits of Crocus sativus L.

In vitro development of parthenocarpic fruits of Crocus sativus L. was induced by culturing ovaries on MS agar medium supplemented with growth-regulators (2,4-D, GA₃ and BAP). Amongh these, 2,4-D was the most effective in promoting fructification. The fructigenic activity was independent of both the stage at which the ovaries were excised (before, during or after anthesis) and pollination of the stigmas. Unlike the above compounds, abscisic acid inhibited fructification.     

Crocus sativus L. genomics and different DNA barcode applications

Crocus sativus L. is a sterile species (3n) whose origin has not been yet clarified. A lot of morphological studies supported the theory that it would have been originated from the evolution, or the hybridization, of other Crocus exemplars, especially C. thomasii, C. hadriaticus and C. cartwrightianus. Crocus sativus stigmas are saffron raw source but, because of their high economic value, sometimes this spice is adulterated. By the application of the DNA barcode technique, we analyzed different Crocus species genomes and we partially clarified some aspects of the phylogeny of this genus: in particular, C. sativus possible genetic derivation was elucidated. Our results also showed that different C. sativus species might have evolved by independent events, probably due to several geographical pressures. We demonstrated that barcoding method, usually adopted for interspecific taxonomic identification, could be also applied to intraspecific and population studies. Finally, we proposed this molecular approach as scientific tool able to discriminate and certificate saffron authenticity.     

Seed coat structure ofCrocus vernus agg. (Iridaceae)

Cytotypes of theCrocus vernus aggregate differ slightly in their seed surface patterns.Crocus albiflorus may be grouped together withCrocus vernus subsp.vernus, both are relatively easily distinguishable by their seed surface patterns from theCrocus scepusiensis—Crocus heuffelianus group.     

Cytogenetic studies of cultivatedCrocus sativus (Iridaceae)

Meiosis and mitosis are described in cultivatedCrocus sativus of Iran. This indicates that this species is an autotriploid and sterile. Karyotype analysis, rare inversions, laggard chromosomes and distribution of chromosomes in the first anaphase are described, and the reasons for its sterility are given.     

The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors

To further understand flowering and flower organ formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned four MIKC^c type II MADS-box cDNA sequences of the E-class SEPALLATA3 (SEP3) subfamily designated CsatSEP3a/b/c/c_as as well as the three respective genomic sequences. Sequence analysis showed that cDNA sequences of CsatSEP3 c and c_as are the products of alternative splicing of the CsatSEP3c gene. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all four cDNA sequences encode for SEP3-like proteins with characteristic motifs and amino acids, and highlighted intriguing sequence features. Phylogenetically, the isolated sequences were closest to the SEP3-like genes from monocots such as Asparagus virgatus, Oryza sativa, Zea mays, and the dicot Arabidopsis SEP3 gene. All four isolated C. sativus sequences were strongly expressed in flowers and in all flower organs: whorl1 tepals, whorl2 tepals, stamens and carpels, but not in leaves. Expression of CsatSEP3a/b/c/c_as cDNAs was compared in wild-type and mutant flowers. Expression of the isolatedCsatSEP3-like genes in whorl1 tepals together with E-class CsatAP1/FUL subfamily and B-class CsatAP3 and CsatPI subfamilies of genes, fits the ABCE “quartet model,” an extended form of the original ABC model proposed to explain the homeotic transformation of whorl1 sepals into whorl1 tepals in Liliales and Asparagales plants such as C. sativus. This conclusion was also supported by the interaction of the CsatSEP3b protein with CsatAP1/FUL and CsatAP3 proteins. In contrast, expression of both B-class CsatAP3 and CsatPI genes and the C-class CsatAGAMOUS genes together with E-class CsatSEP3-like genes in carpels, without any phenotypic effects on carpels, raises questions about the role of these gene classes in carpel formation in this non-grass monocot and requires further experimentation. Finally, taking advantage of the size and sequence differences in amplified genomic sequences of the triploid C. sativus and comparing them with the respective sequences from C. tomasii, C. hadriaticus and C. cartwrightianus, three putative wild-type diploid progenitor species, we examined the origin of CsatSEP3a sequence.     

Selection for resistance to Cochliobolus sativus in tall fescue (Festuca arundinacea Schreb.)

Summary Cochliobolus sativus (Ito and Kurib.) Drechsl. ex Dastur is a major foliar pathogen of tall fescue (Festuca arundinacea Schreb.) which can greatly reduce the quantity and quality of forages available for animal consumption. A greenhouse screening program was initiated to determine the inheritance of resistance to C. sativus in tall fescue over several cycles of mass selection. Resistance to C. sativus in four tall fescue cultivars was increased with 2–3 cycles of mass selection. Realized heritabilities were low to moderate (0.04 to 0.58) indicating that environmental influences on the expression of resistance are quite high. Variances were unchanged by selection, indicating that further improvement should be possible. However, progress with mass selection can be expected to be slow. Lesion size was decreased in each cultivar by selecting for lesion coverage. Lesion size, being independent of inoculum load and therefore less subject to environmental variation, should be considered as an additional selection criteria to improve the rate of progress.Journal article No. 6370 of the Mississippi Agricultural and Forestry Experiment Station     

Cloning and characterization of a glucosyltransferase from Crocus sativus stigmas involved in flavonoid glucosylation

Background  

Flavonol glucosides constitute the second group of secondary metabolites that accumulate in Crocus sativus stigmas. To date there are no reports of functionally characterized flavonoid glucosyltransferases in C. sativus, despite the importance of these compounds as antioxidant agents. Moreover, their bitter taste makes them excellent candidates for consideration as potential organoleptic agents of saffron spice, the dry stigmas of C. sativus.     

Chromosomal differentiation withinCrocus vernus agg. (Iridaceae) in the Carpathian Mts.

Western and eastern Carpathian populations ofCrocus heuffelianus s. lat. (incl.C. scepusiensis) have 2n = 18 but differ in karyotype. While western populations are chromosomally monomorphic, eastern populations exhibit geographical karyotypic differentiation.