Tulipa Species Research Articles

The systematic value of nuclear genome size for “all” species of Tulipa L. (Liliaceae)

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Tulipa L. (Liliaceae). More than 400 accessions representing 123 taxa from mainly wild-collected plants were investigated. Most species of Tulipa have the same basic chromosome number, 2n = 2x = 24. However, the somatic DNA 2C value (2C) is shown to range from 32 to 69 pg for the diploids. The largest genome contains roughly 3.4 × 1010 more base pairs than the smallest and has chromosomes that are more than twice as large. These large differences in the amount of nuclear DNA predict that the hybrids, if any arise, are usually sterile. Depending on the size of the total genome, 1 pg amounts to several thousand genes. Moreover, genome sizes are evaluated here in combination with available morphological, geographical, and molecular data. Therefore, the taxonomy proposed here is not a single-character taxonomy based on genome size alone. The genus Tulipa, as here determined, has 87 species, 29 more than accepted by van Raamsdonk et al. [Acta Hort (ISHS) 430:821–828, 1997], but including 25 species that were not available to them. Of these 87 species, 28 were not seen by Hall (The genus Tulipa, The Royal Horticultural Society, London, 1940) in a living state and placed by him in an addendum. Species of the subgenus Clusianae (Baker) Zonn. differ strongly in nuclear DNA content (DNA 2C value), 32 versus 40–68 pg for all other tulips, and are placed here in a separate subgenus. Also Orithyia, the only group with a style and with only 38–39 pg is placed in a separate subgenus. Therefore, all tulips are attributed to four subgenera, Clusianae (Baker) Zonn., Tulipa, Eriostemones Raamsd., and Orithyia (D. Don) Baker and divided further into 12 sections. Seven of the eight series of section Eichleres (A.D. Hall) Raamsd. are now placed in four sections: (1) section Lanatae (Raamsd.) Zonn., mainly confined to species from the Pamir-Alay and including series Lanatae Raamsd., (2) section Multiflorae (Raamsd.) Zonn. (including series Glabrae Raamsd.), (3) section Vinistriatae (Raamsd.) Zonn. (including series Undulatae Raamsd.), and (4) section Spiranthera Vved. ex Zonn. and Veldk. Triploids, tetraploids, and pentaploids were found in several species. DNA content confirmed the close relationships of the species within the different sections. The rather similar looking and therefore often confused T. armena Boiss. (51.8 pg), T. systola Stapf (56.3 pg), and T. julia K., Koch (61.6 pg) could be clearly distinguished. The same is true for T. biebersteiniana Schult. f. (56.9 pg), T. sylvestris ssp. australis (Link) Pamp. (62.0 pg), and T. primulina Baker (64.6 pg). T. doerfleri Gand. and T. whittalli (Dykes) Hall could be placed as polyploid forms of T. orphanidea Boiss. ex Heldr. On the basis of DNA content, a systematic association between T. julia K. Koch and the triploid T. aleppensis Boiss. and between T. systola Stapf and the triploid T. praecox Tenore was suggested. The new species T. lemmersii Zonn., Peterse, and de Groot is described, and four possible new species are indicated. Genome size as measured by using flow cytometry may conveniently be used to produce systematic data. It is applicable even in the case of dormant bulbs or sterile plants for monitoring the trade in bulbous species.

GISH ANALYSIS OF HYBRIDS PRODUCED BY INTERSPECIFIC HYBRIDIZATION BETWEEN TULIPA GESNERIANA AND T. FOSTERIANA

GISH ANALYSIS OF HYBRIDS PRODUCED BY INTERSPECIFIC HYBRIDIZATION BETWEEN TULIPA GESNERIANA AND T. FOSTERIANA

Molecular cloning of Tulipa fosteriana rDNA and subsequent FISH analysis yields cytogenetic organization of 5S rDNA and 45S rDNA in T. gesneriana and T. fosteriana

In this study, Tulipa fosteriana was found to contain 45S rDNA repeat units of 9.7 and 9.5 kb, in which at least 7 types of 45S rDNAs were identified by restriction site analysis. For 5S rDNA, repeat units ranging from 364 bp to 396 bp were identified. The diploid cultivars (2n = 2x = 24) ‘Christmas Dream’ and ‘Queen of Night,’ representing the horticultural group T. gesneriana, and ‘Red Emperor’, belonging to T. fosteriana, were compared cytogenetically using cloned 5S and 45S rDNAs. Fluorescence in situ hybridization (FISH) analysis identified many rDNA sites located on each chromosome in the diploid genomes. For example, we identified 71 sites of 5S rDNA and 10 sites of 45S rDNA in ‘Red Emperor’. Additionally, FISH analyses enabled construction of karyotypes for these cultivars. Karyotype comparison of T. gesneriana cultivars showed conservation of repetitive rDNA unit positioning. A clear difference in chromosome size and signal pattern was observed between T. gesneriana and T. fosteriana cultivars. Here we demonstrate the unique nature of the highly repeated 5S rDNA units in these Tulipa species and the usefulness of FISH karyotyping with cloned 5S and 45S rDNAs to clearly distinguish between chromosomes from T. gesneriana and T. fosteriana.

DIFFERENCES IN EPICUTICULAR WAX LAYER IN TULIP CAN INFLUENCE RESISTANCE TO BOTRYTIS TULIPAE

Botrytis tulipae is one of the major diseases of tulip. The pathogen is a necrotroph and infection normally results in host cell death, resulting in serious damage to plant tissues and culminating in rotten plants. Several defense strategies are required to counter attack this aggressive invader. Two different methods were used to proof if tulip wax layer had an influence on Botrytis resistance. Wet inoculation method (standard method) with removal of wax layer and dry inoculation method with no removal of wax layer were applied in cultivars Christmas Marvel, Leen van der Mark and Ile de France. The wet inoculation showed that Christmas Marvel was susceptible, Leen van der Mark was partial resistant and Ile de France was resistant. However, when dry method was applied in the same cultivars the level of resistance changed, leading to an increase of the level of resistance in Christmas Marvel, a decrease in Ile de France and equal levels in Leen van der Mark compared to the wet method. These results suggest that wet and dry inoculation altered the level of resistance, meaning that differences in the epicuticular wax layer provides an extra defense strategy against the pathogen in Christmas Marvel, enhanced infection in Ile de France and it had no effect in Leen van der Mark. Moreover, differences in the amount of wax layer in several species of the genus Tulipa are present. In general T. fosteriana is more susceptible to B. tulipae than T. gesneriana. The results showed that T. fosteriana had less wax layer than T. gesneriana. However, there was no complete correlation between amount of wax layer and resistance in all tested cultivars

Anthocyanin content of Tulipa species and cultivars and its impact on tepal colours

Flowers of tulips (17 species and 25 cultivars) were subjected to qualitative and relative quantitative examination for anthocyanins. Altogether five anthocyanins were identified as the 3- O-(6″- O-α-rhamnopyranosyl-β-glucopyranoside) of delphinidin ( 1), cyanidin ( 2) and pelargonidin ( 3), and the 3- O-[6″- O-(2‴- O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside] of cyanidin ( 4) and pelargonidin ( 5). The pigments 1– 5 represented 7%, 43%, 12%, 2% and 31%, respectively, of the total anthocyanin amount in the tepals of the Tulipa species, and 20%, 37%, 30%, 6% and 4%, respectively, in the cultivar tepals. Nearly 50% of the samples contained acetylated anthocyanins. The colours of the freeze-dried tepals described by the CIELab coordinates, hue angle ( h ab), saturation ( C*), and lightness ( L*) together with the anthocyanin content were subjected to multivariate analysis. All tepals classified with hue angles described as “blue nuances” were from cultivars. They contained 1 as the major anthocyanin, and no or just traces of pelargonidin derivatives. The species and cultivars having “magenta nuances” showed similar anthocyanin content with increased relative proportions of 2 at the expense of 1. Orange coloured tepals were to a large extent correlated with high relative proportions of the pelargonidin derivatives, 3 and 5. Acetylation of anthocyanins furnished a weak colour effect opposite to the bluing effect previously reported for anthocyanins with aromatic acyl groups. All six species belonging to the section Eichleres (subgenus Tulipa) were after principal component analysis grouped closely together. They were characterized by high concentrations of the pelargonidin derivatives 3 and 5, and orange petal nuances. However, within section Tulipa (subgenus Tulipa), considerable anthocyanin variation was observed. Species in the subgenus Eriostemones were generally characterized by the two anthocyanins 1 and 2, and no pelargonidin derivatives.

Anther-specific Production of Antimicrobial Tuliposide B in Tulips

Strong antimicrobial activity was observed in water extracts of tulip anthers. Purification by column chromatography and structural analysis by various methods, such as liquid chromatography-mass spectrometry (LC-MS), 1H-, and 13C-nuclear magnetic resonance (NMR) revealed that the active compound was 6-tuliposide B (6-O((S)-4', 5'-dihydroxy-2'-methylenebutyryl)-D-glucopyranose). The antimicrobial susceptibility test revealed that it showed a strong growth inhibition against Gram-positive, Gram-negative bacteria and certain fungicide tolerant strains except for a yeast. Among the 22 cultivars (Tulipa gesneriana L.) and 15 Tulipa species, antimicrobial activities of the anthers from cultivars were stronger than those of the wild species. These production abilities of 6-tuliposide B in anthers were not related to pollen fertility. During anther development, the production of 6-tuliposide B was confined for a short period of approximately 7 to 12 days before flowering. Hence, it appeared that the 6-tuliposide B in anthers was produced in a tissue- and stage-specific manner with higher 6-tuliposide B accumulation than that observed in other tissues that produce both 6-tuliposides A and B. These results suggest that a novel defense strategy evolved in tulips to protect pollens from bacterial pollution in the reproductive process by producing an anther-specific 6-tuliposide B.

Isolation and quantification of tuliposides and tulipalins in tulips (Tulipa) by high-performance liquid chromatography.

The content of tuliposides and tulipalins were determined in Tulipa species and cultivars by reversed-phase high-performance liquid chromatography (RP-HPLC), using a water:methanol gradient as mobile phase. The compounds were detected by a diode array detector employed at 208 nm. The investigation revealed, in addition to 1- and 6-tuliposide A, tuliposide D and the lactonized aglycones tulipalin A and (-)-tulipalin B, the new tuliposide F and 6-tuliposide B, the latter being a new acyl derivative of the known 1-tuliposide B. All compounds were isolated by preparative RP-HPLC and identified by NMR and mass spectroscopy. The predominant compounds were 6-tuliposide A and B present in amounts up to 1.5% and 1.3% of fresh weight, respectively. 6-Tuliposide A and tulipalin A seem to be the major allergens in tulips, although tuliposide D and F may also contribute to the allergenic properties. Tulipalin A and (-)-tulipalin B occur in intact tulips and are not only produced in response to fungal attack or after excision of the plants. A few species were found to have very low allergen content and a relatively high level of tuliposide B, indicating it should be possible to breed non-allergenic and disease-resistant tulips.

Variation in the enzyme esterase within and between Tulipa species; usefulness for the analysis of genetic relationships at different taxonomical levels

Abstract Intra- and interspecific variation in bulb scale esterases of Tulipa species were investigated to determine the usefulness of this marker for the assessment of genetic variation within the genus Tulipa . At the population level, esterases appeared to be suitable markers. Different bulbs of a population, which were analyzed for polymorphisms in their esterases using native PAGE next to other accessions from the same species, generally formed a separate group after cluster analysis. In some of these cases, genetically related accessions clustered within the population cluster. Within section Tulipa , which consists of relatively closely related species, esterases also proved to be useful for analysis of genetic variation at the species level, since the accessions from each species formed separate clusters. However, within section Biflores , which consists of more distantly related species, cluster analysis on the basis of esterases did not reveal clustering of all species. Clustering of the accessions within both sections on the basis of bulb scale esterases was compared with relationships found using morphological data. The species of section Tulipa did not form separate clusters after analysis of the morphological data, which is in contrast to the results from esterase analysis. However, all accessions of section Biflores clustered together per species based on the morphological variation.

Isolation and characterization of chitinase isoforms from the bulbs of four species of the genus Tulipa.

Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15-17 Cys residues/mol), II (5-8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

APPLICATION OF THREE POLLINATION TECHNIQUES AND OF HORMONE TREATMENTS FOR OVERCOMING INTERSPECIFIC CROSSING BARRIERS IN TULIPA

In tulip, interspecific crossing is restricted by both pre-fertilization and post-fertilization barriers. In order to introduce traits from wild species into the cultivar assortment these barriers must be bypassed. By application of embryo rescue techniques, unique hybrids have been obtained of several interspecific tulip crosses. Recently, four techniques have been tested on their capacity to overcome interspecific crossing barriers in tulip, namely hormone treatments of ovaries, cut-style method, grafted-ovary method and placental pollination. After treating ovaries with 0.1 percent BAP at 12 days after pollination, seeds were obtained on the plant from the cross T. gesneriana x T. agenensis. This cross has never been given seeds in vivo before. Treating ovaries with 1 percent BAP seemed to have a negative effect on seedset in compatible crosses, 1 percent NAA did not give any effect. After the application of the cut-style method, the percentage pollen tube penetration did not increase in crosses between T. gesneriana and 5 other Tulipa species. Apparently, crossing barriers were not overcome by using the cut-style method, nor after using the grafted-ovary method. After placental pollination, pollen tube penetration percentages were not increased compared to stigmatic pollination, however, most of the penetrated ovules with pollen tube penetration showedd embryo germination.

Interspecific crosses in the genus Tulipa L.: identification of pre-fertilization barriers

Pollen tube growth in the pistil and pollen tube penetration of ovules have both been studied in crosses between cultivars from Tulipa gesneriana L. and 12 tulip species from all eight sections of the genus Tulipa to identify pre-fertilization barriers. Depending on the cross, pollen tubes grew as far as the stigma or the style or continued growing down into the ovary. Pollen tubes penetrated none or only a few percent of the ovules of some crosses, despite the presence of many pollen tubes in the ovary. In other crosses, from which no or only a few hybrids have been obtained after seed maturation on the plant, pollen tube penetration was found in up to 79% of the ovules. Apparently, various kinds of barriers preventing fertilization or normal embryogenesis occur in interspecific tulip crosses.

Cultivar classification inTulipaL. (Liliaceae)

SUMMARY A total of 43 Tulipa gesneriana (section Tulipa) cultivars, 25 cultivars and wild accessions belonging to 11 species of sections Tulipa and Eichleres and three species hybrids were subjected to multivariate analysis based on data of 30 morphological characters recorded in two subsequent years. The main division according to earliness of flowering, which has been in use since Clusius made this division early in the 17th century, was confirmed by the distribution of the accessions in the principal component plots. The repeatability of measurements is high for most characters, as is deduced from the Pearson correlation coefficient per character between years. A cultivar classification system is proposed with two levels. The acceptance of a two-level cultivar classification is based on the structure of variation encountered and is a continuation of four centuries of tulip cultivar classification. The top level should include three supergroups for Early, Mid-season and Late flowering cultivars. Recommendations are made concerning the restoration of certain cultivar-groups and the placement of some cultivars. It can be concluded that multivariate methods provide basic insight in the diversity among cultivars. This information can be used as a basis for the construction of cultivar classifications.

Crossability analysis in subgenus Tulipa of the genus Tulipa L.

A range of almost 1400 cross combinations has been carried out between 31 species of Tulipa subgenus Tulipa. Most of the sections are well separated reproductively from each other. The designated species aggregates in section Clusianae appeared to be intersterile. Also in section Kolpakowskianae the species, as far as used, cannot be combined. Some crosses between species of section Tulipanum succeeded. The species of sections Eichleres and Tulipa (including the cultivated tulip T gesneriana) could be combined in various ways, although the highest level of seed set was achieved within either section. The crossability data support the current taxonomic division. The level of reproductive isolation between sympatric species is discussed, and the importance of crossability studies for plant breeding in Tulipa pointed out briefly.

Genetic analysis of glutamate oxaloacetate transaminase from leaves of Cretan tulip species

SummaryAn efficient procedure for the identification of tulip populations by leaves, based on polymorphism in the isozymes of GOT is described. All the five species analysed by their GOT banding patterns could be uniquely identified by using native PAGE of leaf protein extracts. The isoenzyme analysis of Tulipa saxatilis Sieber ex Sprengel indicates that this species presents a genotypic differentiation in different populations.

Flower pigment composition in Tulipa

Flower pigment compositions of 43 accessions of Tulipa species, 6 species hybrids and 494 tulip cultivars were analysed with respect to the amount of carotenoids, and the anthocyanidins pelargonidin, cyanidin and delphinidin. After principal component analysis, the different pigment compositions appeared to be correlated with flower color. Hybrids were not intermediate between the parents in all cases. The variation found in T. gesneriana extends that of the whole rest of the genus. The biosynthetic pathway as described in Petunia, Anthirrinum and other genera can also be postulated for the genus Tulipa, but additive effects of multiple genes may be assumed, since several levels of concentration were found. Flavonoid synthesis has biological relevance in view of co-evolution with insect pollinators and male sterility.

Biosystematic studies inTulipa sect.Eriostemones (Liliaceae)

Evolutionary relationships and infra- and interspecific variability within the species ofTulipa sect.Eriostemones Boissier were studied. Measurements of 35 morphological characters were used for principal component and canonical variate analyses. Crossing experiments and chromosome counts were carried out. Two axes of morphological variation were encountered within the principal component plot. Variation of subsect.Biflores was traced along one axis, the species of subsect.Australes as well asSaxatiles were found along the other axis. The nomenclature of names of subsect.Biflores was considered on the basis of their geography, specific morphological characteristics and polyploidy. Conspecificity of names of all species was tested based on both morphology and crossability.

Expression of the GUS-gene in the monocot tulip after introduction by particle bombardment and Agrobacterium

Gene transfer to the monocotyledon tulip (Tulipa sp. L.) was obtained both by particle bombardment and Agrobacterium transformation. Using a Particle Delivery System, transient expression of the reporter gene for ßglucuronidase was demonstrated. It was shown that the CAMV 35S as well as the TR2' promoter were active in flower stem expiants. Various wildtype and disarmed Agrobacterium strains, harbouring the 35S GUSintron gene on a binary plasmid, were used for infection of flower stem expiants of 7 cultivars and 7 botanical Tulipa species. In nine genotypes the GUSintron gene was expressed, despite the fact that tulip tissue did not produce detectable amounts of virulence-inducing substances. Agrobacterium rhizogenes appeared to be most effective in gene transfer to tulip tissue.

Interspecific crosses between Tulipa gesneriana cultivars and wild Tulipa species: a survey

Interspecific crosses were made between 76 Tulipa gesneriana cultivars and 28 botanical Tulipa species. Tulipa gesneriana proved fully congruent (all cross-combinations giving hybrid progeny) with members of the subsection Gesnerianae, was moderately affiliated (50% of all cross-combinations giving hybrid progeny) with representatives of the subsection Eichleres, and was found to be incongruent with most other Tulipa species (except T. stapfii of the subsection Oculussolis).

Pigment composition of flowers of Tulipa species as a parameter for biosystematic research

Pigments were analysed in flowers of 17 Tulipa species of sect. Leiostemones and nine of sect. Eriostemones. Flower colour in both sections was determined by carotenoids and the anthocyanidins delphinidin, cyanidin and pelargonidin. Within sect. Leiosternones, most species contained pelargonidin and within sect. Eriostemones, most species contained delphinidin, whilst most Leiostemones species also contained higher carotenoid levels than sect. Eriostemones species. Distinction of Tulipa species on the basis of separate flower pigments did not seem to be useful. When considering pigment combination in a principal component analysis, however, the species of both sections could be separated almost completely.

Occurrence of 4-methyleneglutamine and 2-oxo-4-methyl-3-pentene-1,5-dioic acid in leaves and stem of tulip plants

Abstract The presence of 4-MeGln in the tulip plant was discovered by ZACHARIUS et al. (1954), and it was found that this amide generally occurred in the leaves of almost all the species of genus Tulipa (FOWDEN and STEWARD 1957a). The 4-MeGln compound has been detected in every part of the tulip plant, i.e., bulb scales, roots, basal plate, young shoots, leaves, stern, and flower (FOWDEN and STEWARD 1957a, b; OHYAMA 1986; OHYAMA et al. 1985, 1988a, b; ZACHARIUS et al. 1954, 1957). Especially 4-MeGln was found to be a major soluble N constituent in the leaves and stem of tulip of the flowering stage (OHYAMA et al. 1985; OHYAMA 1986).