Tulipa Fosteriana Research Articles

Enhancing biotechnological approaches for the in vitro micropropagation: Protecting endangered wild tulip species in Samarkand, Uzbekistan

Tulipa, a genus of bulbous geophytes, boasts a globally recognized ornamental value. Anthropogenic influences have precipitated a decline in the wild populations of Tulipa, including those in Uzbekistan, necessitating the development of forward-looking conservation strategies. This study focuses on advancing biotechnological strategies for in vitro micropropagation to conserve endangered wild Tulipa species in Samarkand, Uzbekistan. Tulipa ingens and Tulipa fosteriana, listed as vulnerable and near threatened, respectively, on the IUCN Red List, face numerous anthropogenic threats including habitat loss and overexploitation. Our research demonstrates the effectiveness of sterilization protocols, successful micropropagation from seeds, and the importance of adapting to non-sterile conditions. Through meticulous experimentation, a novel sterilization approach involving a 5 percent Domestos solution coupled with brief ethanol exposure yielded the highest viability rates for plant materials. Micropropagation from seeds resulted in successful shoot and microbulb formation, providing a promising conservation strategy for these rare species. The study underscores the urgency of conservation efforts in Uzbekistan, where the extent of occurrence and area of occupancy of these species classify them as endangered. Our findings contribute valuable data for ex-situ conservation measures, offering hope for the preservation of red-listed Tulipa species in the region.

Morphology and distribution patterns of Tulipa fosteriana and Tulipa ingens

Tulips are bulbous geophytes with high ornamental value. Species of Tulipa L. are distributed along western China to Eastern Europe. Central Asia including Uzbekistan is considered as the hotspot center of tulips where over 80 and 34 taxa are described. The current work presents morphology of the species from fresh materials, regional distribution and assessment of the area (AOO) and extent (EOO) of occurrence of the ancestor of cultivated tulips, Tulipa fosteriana and Tulipa ingens from Samarkand region (Uzbekistan). Illustrations of the species from fresh materials are also provided.

Current state of Cenopopulations Iris Magnifica Vved and Tulipa Fosteriana W.Irving in Uzbekistan

The current state of cenotic populations of 2 plant species listed in the Red Book of the Republic of Uzbekistan from the territories of Kashkadarya province was assessed, such as: Iris magnifica Vved. and Tulipa fosteriana W. Irving. The presence or absence of certain ontogenetic groups in coenopopulation is associated with the ecological-phytocenotic growth condition and the degree of cattle grazing. The aim of the research was to describe the coenopopulation of Iris magnifica Vved. and Tulipa fosteriana W. Irving. The scientific justification of the ways of ecological improvement of coenopopulation. In the implementation of scientific work, the methods of traditional geobotanical and cartographic remote study of plant formations were applied. The factors that influence the vegetative processes of plant formations were identified in this study so that the ontogenetic structure of coenopopulation of Iris magnifica Vved. and Tulipa fosteriana W. Irving. were developed.

Comparison of anthocyanin components, expression of anthocyanin biosynthetic structural genes, and TfF3′H1 sequences between Tulipa fosteriana ‘Albert heijn’ and its reddish sport

A reddish flower tulip sport Tulipa fosteriana ‘SN09’ was bred from a pink flower cultivar T. fosteriana ‘Albert heijn’ (AH). To investigate the molecular basis underlying this color difference, SN09 and AH were compared using biochemical and molecular approaches. Ultra performance liquid chromatography (UPLC) profiles revealed that major cyanidin 3-rutinoside and minor pelargonidin 3-rutinoside were accumulated in the petals of AH, whereas major pelargonidin anthocyanins (pelargonidin 3-acetylrutinoside and pelargonidin 3-rutinoside) and minor cyanidin 3-glucoside were detected in SN09. The total anthocyanin content in petals of AH and SN09 were not significantly different at Stage 4, and correlated with the expression patterns of dihydroflavonol 4-reductase (TfDFR1) and anthocyanidin synthase (TfANS1). In addition, more transcripts of chalcone synthase (TfCHS1), chalcone isomerase (TfCHI2), and flavanone 3-hydroxylase (TfF3H1) contributed to higher accumulation of flavone and flavonol in SN09 than in AH. The transcription of flavonoid 3′-hydroxylase (TfF3′H1) was significantly lower in SN09 than in AH throughout flower development. Accordingly, the nucleotide sequences of TfF3′H1 in AH and SN09 were compared. Results showed that the deduced amino acids of TfF3′H1 in AH and SN09 were not different, but an extra fragment (255bp) was found in the promoter of TfF3′H1 in SN09. Consequently, the promoters of TfF3′H1 in AH and SN09 were isolated, fused with the β-glucuronidase (GUS), and then transformed to tobacco and Arabidopsis thaliana. Results demonstrated that the GUS activity in Arabidopsis with TfF3′H1 promoter from SN09 was only 5.4% of that from AH, suggesting that the insert mutation in the TfF3′H1 promoter hampered the accumulation of cyanidin anthocyanins in SN09 through the reduction of TfF3′H1 transcription.

Ploidy manipulation and introgression breeding in Darwin hybrid tulips

Meiotic polyploidisation via crossing with 2n gamete producing genotypes and interploidy crosses are two of the main methods currently used to obtain polyploid tulips. In our study diploid 2n gamete producing F-1 hybrids of Darwin hybrids (Tulipa gesneriana x Tulipa fosteriana) and triploid hybrid resulting from 'Rhodos' x 'Princeps' cross were used as pollen donor and crossed with cultivars of T. gesneriana in the following combination: 2x x 2x, 3x x 2x, 2x x 3x, and 3x x 3x. The progenies resulting from crosses at diploid level were mostly diploid, whereas a few seedlings were triploid. In 3x x 2x crosses aneuploids with chromosome constitution in between triploid and tetraploid (43-45 chromosomes) were predominant, but also one tetraploid (2n = 4x = 48) and four pentaploids (2n = 5x = 60) were obtained. In 2x x 3x crosses most progenies were triploid with the exception of a few aneuploids (3x + 1 and 3x - 1), whereas in 3x x 3x cross diploid and aneuploid genotypes were recorded with chromosome number varied from 27 to 34. These results indicate that triploid parents produced aneuploid as well as euploid (x, 2x, 3x) gametes and that success in ploidy manipulation in tulip depends to a large degree on the ploidy level of the parental genotypes used for hybridization. Genome constitution of selected population of F1 and BC1 hybrids was analyzed through genomic in situ hybridization (GISH). GISH analysis of the BC1 showed a considerable amount of intergenomic recombination which is desirable for introgression breeding.

Genetic diversity and structure in a collection of tulip cultivars assessed by SNP markers

Although tulip is one of the most important bulbous crops worldwide, the genetic background of most cultivars is unclear at present. The purposes of this study are to investigate genetic diversity and to identify the genetic structure and relationships among tulip cultivars. A total of 236 polymorphic single nucleotide polymorphisms (SNPs) were obtained from ‘Kees Nelis’ and ‘Cantata’, from which 121 SNPs with a minor allele frequency above 0.1 were selected for genetic analysis. The total observed heterozygosity (Ho) among the 72 accessions was 0.35, Ho of cultivar groups ranged from 0.22 (Tulipa fosteriana) to 0.43 (Tulipa gesneriana×T. fosteriana hybrids). Rather small genetic distances were found among T. gesneriana cultivar groups which are defined according to flowering time and morphology. In both PCoA (principle coordinate analysis) and STRUCTURE analysis, the 72 accessions were separated into three clusters (FST=0.208, P<0.0001). A significant difference could be detected between early and late flowering cultivars (FST=0.072, P=0.02). T. gesneriana×T. fosteriana hybrid breeding lines were easily identified as admixed individuals in STRUCTURE analysis, by means of which cultivar ‘Purissima’ and ‘Flair’ were also inferred as hybrids.

Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana

Getting basic molecular information of pigment formation in tulip flower is a prerequisite for enhancing the range of flower colors. In this study, six cDNA clones encoding putative anthocyanin biosynthesis enzymes were isolated from petals of tulip (Tulipa fosteriana) ‘Shangnong Zaoxia’ by rapid-amplification of cDNA ends (RACE): chalcone synthase (TfCHS1), chalcone isomerase (TfCHI1), flavanone 3-hydroxylase (TfF3H1), flavonoid 3′-hydroxylase (TfF3′H1), dihydroflavonol 4-reductase (TfDFR1) and anthocyanidin synthase (TfANS1). Southern blot analysis showed they all present as multiple copies in the tulip genome. In parallel, the expression profiles of these genes in different tulip tissues as well as in petals during flower development were measured by real-time quantitative PCR. The expression levels of TfCHS1, TfCHI1 and TfF3H1 were higher in stems and leaves than those in floral tissues (stamens, pistils, petals), the expression of TfF3′H1, TfDFR1 and TfANS1 showed highest levels in petals compared with other tissues. During flower development, the transcript levels of TfCHS1 and TfF3H1 were continuously increased, which were in general constant with the changing pattern of anthocyanin accumulation; the mRNA levels of TfDFR1 and TfANS1 and anthocyanin accumulation were both significantly increased; a low level transcript of TfF3′H1 was detected in petals throughout pigmentation process; TfCHI1 expression level firstly dramatically decreased and then gently increased. These results suggest that the red pigment accumulation in petals of T. fosteriana was positively related to the expression levels of TfCHS1, TfF3H1, TfDFR1 and TfANS1.

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.

The origin of Darwin hybrid tulips analyzed by flow cytometry, karyotype analyses and genomic in situ hybridization

Chromosome morphology was studied in diploid cultivars of Tulipa fosteriana and T. gesneriana (2n = 2x = 24) and triploid Darwin hybrids (2n = 3x = 36) developed from interspecific crosses of T. gesneriana and T. fosteriana. Chromosomes were arranged in the karyotype according to decreasing total length. Based on our karyotypic analysis, we propose that median chromosomes may serve as markers for diploid genotypes. Discriminant analysis with respect to total chromosome length and short arm length showed a significant difference between the size of the larger median chromosomes of T. gesneriana and T. fosteriana Comparison of median chromosome length in Darwin hybrid tulips showed that two larger chromosomes and one smaller chromosome were derived from T. gesneriana and T. fosteriana, respectively. This finding was clearly and unambiguously confirmed by simultaneous hybridization of differentially labeled genomic probes of T. fosteriana and T. gesneriana to metaphase chromosomes of the triploid cultivar ‘Yellow Dover’, thereby enabling us to distinguish between the 24 chromosomes derived from T. gesneriana and 12 chromosomes derived from T. fosteriana. Thus, genomic in situ hybridization and median chromosome analyses can be useful to identify the genome constitution of triploid Darwin hybrid tulips. In addition, their hybridity was readily verified by flow cytometry using vegetative tissue of Darwin hybrid tulips. Our results clarify the process of Tulipa cultivar formation and will be useful for interspecific hybridization breeding.

Oxidation of xenobiotics by plant microsomes, a reconstituted cytochrome P450 system and peroxidase: a comparative study

The microsomal fraction from tulip bulbs ( Tulipa fosteriana, L.) contains cytochrome P450 (CYP 3, EC 1.14.14.1) and peroxidase (EC 1.11.1.7.) enzymes catalyzing the NADPH — and hydrogen peroxide — dependent oxidation of the xenobiotic substrates, N-nitrosodimethylamine (NDMA), N-nitrosomethylaniline (NMA), aminopyrine and 1-phenylazo 2-hydroxynaphthalene (Sudan I), respectively. Oxidation of these model xenobiotics has also been assessed in a reconstituted electron-transport chain with a partially purified CYP fraction, phospholipid and isolated tulip NADPH:CYP reductase (EC 1.6.2.4.). Peroxidase isolated from tulip bulbs (isoenzyme C) oxidizes these xenobiotics, too. Values of kinetic parameters ( K m, V max), requirements for cofactors (NADPH, hydrogen peroxide), the effect of inhibitors and identification of products formed from the xenobiotics by the microsomal fraction, partially purified CYP and peroxidase C were determined. These data were used to estimate the participation of the CYP preparation and peroxidase C in oxidation of two out of the four studied xenobiotics (NMA, Sudan I) in tulip microsomes. Using such detailed study, we found that the CYP-dependent enzyme system is responsible for the oxidation of these xenobiotics in the microsomal fraction of tulip bulbs. The results demonstrate the progress in resolving the role of plant CYP and peroxidase enzymes in oxidation of xenobiotics.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

Cytochrome P-450 from Tulip Bulbs (Tulipa fosteriana L.) Oxidizes an Azo Dye Sudan I (1-Phenylazo-2-hydroxynaphthalene, Solvent Yellow 14) in vitro

The microsomal fraction from tulip bulbs (Tulipa fosteriana L.) contains cytochrome P-450 enzymes catalyzing the NADPH-dependent oxidation of the xenobiotic substrate, an azo dye Sudan I (1-phenylazo-2-hydroxynaphthalene, Solvent Yellow 14). C-Hydroxy derivatives [1-(4-hydroxyphenylazo)-2-hydroxynaphthalene, 1-phenylazo-2,6-dihydroxynaphthalene, 1-(4-hydroxyphenylazo)-2,6-dihydroxynaphthalene] and the benzenediazonium ion are the products of the Sudan I oxidation. The oxidation of Sudan I has also been assessed in a reconstituted electron-transport chain with the isolated cytochrome P-450, isolated plant NADPH-cytochrome P-450 reductase and phospholipid. The results are discussed from the point of view of the role of cytochromes P-450 in the metabolism of xenobiotics in plants.

A reconstituted cytochrome P450 system from tulip bulbs ( Tulipa fosteriana L.) oxidizes xenobiotics

The microsomal fraction from tulip bulbs ( Tulipa fosteriana L.) contains cytochrome P450 enzymes catalyzing the NADPH-dependent N-demethylation of the xenobiotics N-nitrosomethylaniline (NMA), N-nitrosodimethylamine (NDMA) and aminopyrine. A cytochrome P450 has been solubilized and purified from this tissue. Demethylation of the xenobiotics NMA, NDMA and aminopyrine has also been assessed in a reconstituted electron-transport chain with the isolated cytochrome P450, phospholipid and isolated plant NADPH-cytochrome P450 reductase. A molar ratio of 0.65 of cytochrome P450 to the reductase is the most appropriate for the catalysis. Besides N-demethylation of NMA, denitrosation of this substrate is catalyzed by the plant cytochrome P450-dependent reconstituted system. The results demonstrate the progress in reconstitution techniques allowing the recovery of the activity of plant cytochrome P450s oxidizing xenobiotics.

Cytophotometric Studies of Histone and DNA in the Mitotic Cycle of Tulip Endosperm Nuclei

SUMMARYIn the endosperm of Tulipa Fosteriana seeds 20–30 days after pollination the endospermal pellice at any given time contain an ordered sequence of mitotic phases as they proceeds through the all visible phases of mitotis to postmitotic interphase. This living material is a very convenient experimental model for a study of some nuclear processes during mitotic cycle.Using such pellices fixed by 10% formalin and mounted on the slides the DNA and histone content of interphase nuclei and sets of mitotic chromosomes were estimated by cyto photometry of Feulgen reaction or fast green pH 8.2 staining. It was shown that the duplication of amount of both DNA and histone take place simultaneously during interphase and there is no change of histone amont in the chromosomes during visible stages of mitosis.