Dandelion Species Research Articles

Identification of miRNAs and their targets in two Taraxacum species with contrasting rubber-producing ability.

MicroRNAs (miRNAs) are widely involved in various aspects of plant growth and development. However, how miRNAs and their targets regulate natural rubber metabolism remains unclear in the rubber-producing dandelions, which are being developed as alternative commercial sources of natural rubber. Here, we combined small RNA sequencing, degradome sequencing, target gene prediction, and mRNA sequencing to identify miRNAs and their targets in two dandelion species, the high rubber-yielding Taraxacum kok-saghyz (Tk) and the low rubber-yielding T. spadiceum (Ts). A total of 142 miRNAs, including 108 known and 34 novel ones, were discovered, with 53 identified as differentially expressed (DE) between the latex of Tk and Ts. Degradome sequencing identified 145 targets corresponding to 74 miRNAs. TAPIR and psRNATarget, respectively, predicted 165 and 164 non-redundant targets for the 53 aforementioned DE miRNAs. Gene ontology (GO) enrichment analysis indicated the DE miRNAs and their targets might affect natural rubber production via regulating macromolecular biosynthesis and metabolism in latex. Four critical types of regulatory modules, including miR172-AP2/ERF, miR164-NAC, miR160-ARF, and miRN19-protein kinase, were identified and their interaction networks were constructed, indicating a potential involvement in natural rubber production. The findings and the large miRNA dataset presented here are beneficial to further deciphering the roles of miRNAs in the biosynthesis of natural rubber and medicinal metabolites in dandelion.

Beyond natural rubber: Taraxacum kok-saghyz and Taraxacum brevicorniculatum as sources of bioactive compounds

Taraxacum kok-saghyz (TKS) and T. brevicorniculatum (TB) are broadly investigated as natural rubber-producing plants and possible alternative supply sources for this relevant raw material. To fully exploit Taraxacum as a profitable crop, all the potential co-products should be investigated, through in-depth analysis of metabolites present in different organs of the plant. In the present study, natural rubber (NR), inulin, and resin content was measured by accelerated solvent extraction from the roots of TB and TKS, highlighting a 5-fold more content of NR in TKS compared to TB. Moreover, the chemical composition of both acetone and methanolic extracts from the roots and leaves of TKS and TB has been characterized by ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-HRMS) technique and the content of target compounds between TKS and TB was also compared. The analysis resulted in the detection of 55 metabolites, whose identification was discussed based on chemical classes and the extraction method. Thus, sesquiterpenoids, fatty acids and their derivatives, phenolic compounds, mainly caftaric and chicoric acid, were identified. Hence, both the leaves and roots of TB, and especially of TKS, are rich in a wide variety of high-value-added compounds exploitable along with NR and inulin to increase the commercial value of these two dandelion species.

Pinkish-achened form of Taraxacum hybernum Steven – a source of inulin and high molar mass natural rubber

ABSTRACT Taraxacum hybernum Steven (Asteraceae family) native to Southeast Europe is a promising rubber-bearing dandelion species. In the 1930s, several phenotypically distinct forms were described in this dandelion, such as brown-achened and pinkish-achened. More than 70 years after termination of research of temperate zone rubber-bearing plants we were able to find both forms of this species of dandelion in natural habitat. The species is still poorly studied, and there is no information about the differences in the quality and quantity of rubber between brown-achened and pinkish-achened forms. Therefore, the purpose of our study was to analyze the root morphology and yield, the rubber and inulin content, and to determine the molar mass of rubber in brown-achened and pinkish-achened T. hybernum. These two forms of T. hybernum, as well as T. kok-saghyz Rodin and T. brevicorniculatum Korol., were studied. The results show that the root shape most attractive for domestication is typical of brown-achened T. hybernum. The greatest mass of roots was gained for pinkish-achened T. hybernum. The rubber content was about 6% in T. kok-saghyz, 3% in both forms of T. hybernum, and 1.5% in T. brevicorniculatum per dry root mass. Taraxacum kok-saghyz and pinkish-achened T. hybernum turned out to be the leaders in inulin content. Of all four dandelions, the molar mass of rubber was more than 1 million g/mol only for T. kok-saghyz and pinkish-achened T. hybernum. Pinkish-achened T. hybernum is a good alternative for T. kok-saghyz and can be recommended for domestication and cultivation in the temperate zone.

Chemometrics integrated with in silico pharmacology to reveal antioxidative and anti-inflammatory markers of dandelion for its quality control

BackgroundDandelion is an herb with high nutritional and medicinal values, which has been listed in Chinese Pharmacopeia, European Pharmacopoeia and British Pharmacopoeia, gaining increasing acceptance around the world. However, the current quality control of dandelion is lagging behind. Only in Chinese Pharmacopeia, cichoric acid is used as a marker compound for its quality evaluation, whereas, it can not comprehensively reflect the bioactivity of dandelion.MethodsThis study developed a strategy by integrating chemometrics with in silico pharmacology to reveal the bioactive markers of dandelion for its quality control. Firstly, the major chemicals in dandelion were characterized using HPLC-DAD-MS/MS, and the corresponding antioxidant and anti-inflammatory activities were evaluated in vitro. Subsequently, the active components were screened by relating the chemicals and bioactivity of dandelion via grey relational assay and partial least squares regression analysis. The potential active components were then subjected to a validation for their activities. Moreover, in silico pharmacology was utilized to evaluate the contribution of active components to efficacy.ResultsA total of 22 phenolic compounds were characterized. Among them, cichoric acid, caffeic acid and luteolin were identified as quality markers owing to their good correlations with the bioactivities of dandelion. These three markers were quantified in frequently-used dandelion species, viz. Taraxacum mongolicum Hand.-Mazz. (TAM) and T. officinale F. H. Wigg. (TAO). TAM, with acceptably higher content of cichoric acid and caffeic acid, showed better antioxidant activity than TAO. While TAO included higher content of luteolin, presenting slightly more effective in anti-inflammation.ConclusionAn useful strategy for the quality marker discovery was successfully designed. And the results provided more knowledge for the quality evaluation of dandelion.

Differential effect of inter‐ and intraspecific competition on the performance of invasive and native Taraxacum species

AbstractInter‐ and intraspecific competitive abilities are significant determinants of invasive success and the ecological impact of non‐native plants. We tested two major hypotheses on the competitive ability of invasive species using invasive (Taraxacum officinale) and native (T. platycarpum) dandelions: differential interspecific competitive ability between invasive and native species and the kin recognition of invasive species. We collected seeds from two field sites where the two dandelion species occurred nearby. Plants were grown alone, with kin (plants from the same maternal genotype) or strangers (plants from different populations) of the same species, or with different species in a growth chamber, and the performance at the early developmental stage between species and treatments was compared. The invasive dandelions outcompeted the native dandelions when competing against each other, although no difference between species was detected without competition or with intraspecific competition. Populations of native species responded to interspecific competition differently. The effect of kinship on plant performance differed between the tested populations in both species. A population produced more biomass than the other populations when grown with a stranger, and this trend was manifested more in native species. Our results support the hypothesis that invasive plants have better competitive ability than native plants, which potentially contributes to the establishment and the range expansion of T. officinale in the introduced range. Although kin recognition is expected to evolve in invasive species, the competitive ability of populations rather than kinship seems to affect plant growth of invasive T. officinale under intraspecific competition.

Russian dandelion (<i>Taraxacum kok-saghyz</i> Rodin): rubber extraction methods and prospects for biotechnological methods application

Natural rubber is a strategic natural raw material, which is used in the production of tires and military equipment, in medicine and other industries. An alternative to Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. and the most promising rubber plant is the Russian dandelion (Taraxacum kok-saghyz Rodin). The rubber that accumulates in the roots of this dandelion is not inferior in quality to the natural rubber from H. brasiliensis, and its content reaches 27% of the dry weight of roots. The purpose of this paper is to describe the economically important components of T. kok-saghyz roots, the main methods for extracting natural rubber from the roots, as well as the approaches to micropropagation and genetic transformation of kok-saghyz and related species. In the middle of the 20th century, the industrial method of isolating rubber from Russian dandelion in the USSR was based on preliminary treatment of the roots with a 2% solution of alkali, which could negatively affect rubber quality. Therefore, it is important to develop new, rapid, but at the same time, inexpensive methods of rubber extraction from T. koksaghyz. Some of them are considered in this paper. The breeding of Russian dandelion should be aimed at both increasing the root size and the content of rubber. In this regard, the development of laboratory express methods for rubber extraction is also important. The authors have developed and optimized a method for extracting rubber from dry plant tissue using polar solvents (water and acetone), with the final extraction with a non‑polar solvent (hexane). The developed rubber extraction protocol showed results comparable to the literature data. In order to create more productive plant forms, experiments are also being conducted on T. kok-saghyz micropropagation and genetic transformation. However, the number of such works is still very small, probably due to the low regenerative abilities of this dandelion species.

Antioxidant Sources from Leaves of Russian Dandelion.

Taraxacum kok-saghyz (TKS) is a dandelion species native to Kazakhstan, Uzbekistan and north-west China, considered as a promising alternative source of natural rubber from its roots. The aim of this study was to investigate the possible exploitation of TKS leaves, a rubber byproduct, as a source of phenolic compounds with antioxidant properties for potential applications in forage, nutraceutical and pharmacological fields. Two accessions (TKS016, TKS018) grown under Mediterranean conditions of Sardinia were evaluated at vegetative and flowering stages. The leaves of TKS018 had the highest antioxidant capacity (19.6 mmol trolox equivalent antioxidant capacity 100 g-1 ), total phenolic (106.4 g gallic acid equivalent kg-1 ), tannic phenolics (58.5 g gallic acid equivalent kg-1 ) and total flavonoid contents (22.9 g catechin equivalent kg-1 ). At both phenological stages, TKS016 showed significantly lower values than TKS018 in 1,1-diphenyl-2-picrylhydrazyl (DPPH), total phenolic and tannic phenolics. Six individual molecules were identified, namely chlorogenic, cryptochlorogenic, caffeic, sinapic, chicoric and 3,4-dimethoxycinnamic acids. Chicoric (8.53-10.68 g kg-1 DW) and chlorogenic acids (4.18-7.04 g kg-1 DW) were the most abundant. TKS leaves represent a valuable source of chicoric acid with potential application as antioxidant to be used as herbal medicine and nutrition for production of healthy food/feed.

The enzymes OSC1 and CYP716A263 produce a high variety of triterpenoids in the latex of Taraxacum koksaghyz

Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz, thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol (1) and its ketone lup-19(21)-en-3-one (2). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, β-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.

Taraxacum brevicorniculatum rubber elongation factor TbREF associates with lipid droplets and affects lipid turn-over in yeast

A protein named TbREF that is localized on rubber particles of the rubber producing dandelion species Taraxacum brevicorniculatum was expressed in tobacco leaves and in yeast. TbREF fused to fluorescence proteins colocalized on globular, hydrophobic structures, most likely lipid droplets. Furthermore, triacylglycerol, sterol and total lipid content of TbREF expressing yeast was determined by photometric analyses of nile red stainings and GC–MS analyses. Therefore, yeast exposed an enhanced nile red fluorescence as well as an increased TAG and sterol content compared to wildtype and vector control. Altogether, these findings gave new insights into the putative function of TbREF that might be pushing rubber particle production due to its cytotoxic nature and/or shielding and preventing degradation of lipid droplets. Furthermore, these results highlight possible biotechnological applications regarding the accumulation of hydrophobic compounds in lipid droplet like structures.

Variation within Lactuca spp. for Resistance to Impatiens necrotic spot virus.

Lettuce (Lactuca sativa L.) production in coastal California, one of the major lettuce-producing areas of the United States, is regularly affected by outbreaks of Impatiens necrotic spot virus (INSV), a member of the genus Orthotospovirus. Transmission of INSV among lettuce crops in this growing region has been attributed predominantly to the western flower thrips (Frankliniella occidentalis). INSV is acquired by first- or second-instar thrips nymphs feeding on infected host plants (not necessarily lettuce). The virus replicates within the insect vector, and is transmitted to new plants by adult thrips as they feed on epidermal and mesophyll cells of susceptible host plants. All currently grown cultivars of lettuce are susceptible to the disease. Screening lettuce for resistance to INSV under field conditions is problematic because natural infections appear sporadically and the virus is not evenly distributed across infected fields. We have developed a greenhouse-based assay that uses viruliferous thrips in combination with mechanical inoculation that allows dependable, year-round screening for resistance. In all, 89 cultivars, breeding lines, and plant introductions of cultivated lettuce, together with 53 accessions from 11 other Lactuca spp., 4 accessions from two dandelion (Taraxacum) species, and 4 tomato (Solanum lycopersicum L.) lines were evaluated for resistance to INSV. All tested material was susceptible to INSV to varying degrees, with the exception of two tomato lines that carry the Sw-5 gene that confers resistance to Tomato spotted wilt virus, a virus closely related to INSV. In cultivated lettuce, a partial resistance to INSV was observed in cultivars Amazona, Ancora, Antigua, Commodore, Eruption, Iceberg, La Brillante, Merlot, Telluride, and Tinto. Limited comparison of the greenhouse-based screening results with the data from opportunistic evaluations of resistance on 775 lettuce accessions from six field trials indicates consistency of results from both greenhouse and field environments. The most resistant lettuce accessions are being incorporated into our breeding program for introgression of resistance into lettuce breeding lines.

南関東の 10 年後調査から見た在来 2 倍体種タンポポと外来種タンポポの出現状況変化

南関東の 10 年後調査から見た在来 2 倍体種タンポポと外来種タンポポの出現状況変化

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

BackgroundLatex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex.ResultsThe spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections.ConclusionsWe could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots up to an industrially relevant amount. Our data provide insight into the regulation of the MVA pathway in dandelion latex and can be used as a basis for metabolic engineering to enhance the production of isoprenoid end products in this specialized tissue.

Genome size variation among common dandelion accessions informs their mode of reproduction and suggests the absence of sexual diploids in North America

Apomixis is the ability of plants to produce asexual seeds, which are clones of the mother plant. The phenomenon of apomixis is tightly linked to ploidy, where diploids lack apomixis and reproduce sexually, while higher ploidy levels can exhibit apomixis. Taraxacum F.H.Wigg. species (dandelions) commonly exhibit apomixis; however, only limited studies have evaluated genome size variation of the iconic weedy dandelion, Taraxacum officinale F.H.Wigg. (common dandelion), particularly in North America. To evaluate the ploidy and reproductive biology of common dandelion, we created a global collection, with an emphasis on North America and determined genome size by evaluating seeds with flow cytometry. A total of 635 accessions were screened, and with the exception of two accessions from Germany and Austria, all were found to exclusively contain polyploid seed. The mode of reproduction of a sample of 96 of these accessions was observed by conducting emasculations. Our results indicate that the diploid, sexual cytotype of common dandelion may be absent in North America, while diploids were found in previously described areas of Central Europe. This suggests that the clonal lineages of common dandelion in North America may largely be derivative from apomictic plants introduced from abroad. Furthermore, if all North American common dandelion is apomictic, it may be unreceptive to pollen, which may isolate it from other dandelion species and inform the potential for gene flow.

Development of rubber-enriched dandelion varieties by metabolic engineering of the inulin pathway.

SummaryNatural rubber (NR) is an important raw material for a large number of industrial products. The primary source of NR is the rubber tree Hevea brasiliensis, but increased worldwide demand means that alternative sustainable sources are urgently required. The Russian dandelion (Taraxacum koksaghyz Rodin) is such an alternative because large amounts of NR are produced in its root system. However, rubber biosynthesis must be improved to develop T. koksaghyz into a commercially feasible crop. In addition to NR, T. koksaghyz also produces large amounts of the reserve carbohydrate inulin, which is stored in parenchymal root cell vacuoles near the phloem, adjacent to apoplastically separated laticifers. In contrast to NR, which accumulates throughout the year even during dormancy, inulin is synthesized during the summer and is degraded from the autumn onwards when root tissues undergo a sink‐to‐source transition. We carried out a comprehensive analysis of inulin and NR metabolism in T. koksaghyz and its close relative T. brevicorniculatum and functionally characterized the key enzyme fructan 1‐exohydrolase (1‐FEH), which catalyses the degradation of inulin to fructose and sucrose. The constitutive overexpression of Tk1‐FEH almost doubled the rubber content in the roots of two dandelion species without any trade‐offs in terms of plant fitness. To our knowledge, this is the first study showing that energy supplied by the reserve carbohydrate inulin can be used to promote the synthesis of NR in dandelions, providing a basis for the breeding of rubber‐enriched varieties for industrial rubber production.

Chloroplast genome resources and molecular markers differentiate rubber dandelion species from weedy relatives

BackgroundRubber dandelion (Taraxacum kok-saghyz, TK) is being developed as a domestic source of natural rubber to meet increasing global demand. However, the domestication of TK is complicated by its colocation with two weedy dandelion species, Taraxacum brevicorniculatum (TB) and the common dandelion (Taraxacum officinale, TO). TB is often present as a seed contaminant within TK accessions, while TO is a pandemic weed, which may have the potential to hybridize with TK. To discriminate these species at the molecular level, and facilitate gene flow studies between the potential rubber crop, TK, and its weedy relatives, we generated genomic and marker resources for these three dandelion species.ResultsComplete chloroplast genome sequences of TK (151,338 bp), TO (151,299 bp), and TB (151,282 bp) were obtained using the Illumina GAII and MiSeq platforms. Chloroplast sequences were analyzed and annotated for all the three species. Phylogenetic analysis within Asteraceae showed that TK has a closer genetic distance to TB than to TO and Taraxacum species were most closely related to lettuce (Lactuca sativa). By sequencing multiple genotypes for each species and testing variants using gel-based methods, four chloroplast Single Nucleotide Polymorphism (SNP) variants were found to be fixed between TK and TO in large populations, and between TB and TO. Additionally, Expressed Sequence Tag (EST) resources developed for TO and TK permitted the identification of five nuclear species-specific SNP markers.ConclusionsThe availability of chloroplast genomes of these three dandelion species, as well as chloroplast and nuclear molecular markers, will provide a powerful genetic resource for germplasm differentiation and purification, and the study of potential gene flow among Taraxacum species.

Dandelion Taraxacum linearisquameum does not reflect soil metal content in urban localities

Accumulation of selected heavy metals (Cd, Pb, Ni, Cr, Fe, and Zn) and phenolic metabolites (total soluble phenols, cichoric and caftaric acid) in dandelion organs (leaves, roots, inflorescences/anthodia) collected from six localities within the industrial town Košice (eastern Slovakia) were studied. Localities from the vicinity of a steel factory (Cd, Fe) and heavy traffic (Pb, Ni, Cr, Zn) contained the highest amount of individual metals in the soil but a significant correlation between soil and organ metal content was found only for Cr in the leaves (r2 = 0.7679). The amount of Cd and partially Pb differed among localities in all organs and especially in the leaves and anthodia, indicating probably the impact of atmospheric pollution. The bioaccumulation factor was <1 for almost all metals, suggesting that given dandelion species is not metal accumulator. Translocation factor did not reach values close to or over 1 only for Cd, indicating a root-to-shoot movement of Pb, Ni and Zn though the impact of air pollution on leaves cannot be excluded. A strong correlation between leaf Cd and leaf total phenols, cichoric and caftaric acids was observed (r2 = 0.7926, 0.8682 and 0.8830, respectively), indicating that phenolic metabolites act in the protection of dandelion against Cd excess. Overall, our data indicate low pollution of urban soil by Cd (5.53–113.8 ng g−1) and partially by Cr and the suitability of above-ground organs of dandelion species for the monitoring of air pollution mainly by Cd.

Habitat Requirements of Marsh Dandelions (Taraxacum) in Polish and Estonian Coastal Grasslands

ABSTRACT Factors determining the distribution of highly endangered marsh dandelion species in Polish and Estonian coastal grasslands have been investigated. The aim of this study was twofold: (i) to determine which environmental variables are responsible for the vegetation patterns in coastal grasslands with marsh dandelion and (ii) to analyse the ecological spectra of the identified marsh dandelion species. Altogether 51 plots were sampled (in 2013 and 2014). They were used following statistical analysis: DCA, CCA, Spearman's rank correlation test, Kruskal-Wallis test and hierarchical divisive cluster analysis (TWINSPAN). In total, three marsh dandelion species were found in Polish and Estonian coastal grasslands. The only species found in Poland was Taraxacum balticum. In Estonia all three species occur: T. balticum, T. decolorans and T. suecicum. Taraxacum balticum has been found in the widest ranges of all soil properties included, usually on organic, saline, non-carbonate and acid to slightly acid su...

Rapid and hormone-free Agrobacterium rhizogenes-mediated transformation in rubber producing dandelions Taraxacum kok-saghyz and T. brevicorniculatum

Taraxacum kok-saghyz (TK) and Taraxacum brevicorniculatum (TB) are rubber-producing dandelion species under development as potential crops and model systems of rubber biosynthesis. The former is of industrial interest, as it produces a high percentage of high quality rubber in its roots; the latter is an apomictic cousin of TK and is of interest as a model system for rubber biosynthesis and a source of vigor in breeding efforts. Accordingly, there is interest in developing genetic transformation protocols for applied research, such as metabolic engineering. A rapid and hormone-free transformation system was developed for these two species. Dandelions can naturally regenerate from root fragments, and can be vegetatively propagated using root cuttings. Here, we show that root fragments can regenerate entire plants on half strength Murashige and Skoog medium without iterative hormone treatments or manual manipulations. Regeneration efficiency was increased from 36.6% to 65.3% for TK and from 95.2% to 152.3% for TB by inoculation with Agrobacterium rhizogenes wild type strain K599. After root fragments were inoculated with A. rhizogenes harboring kanamycin resistance genes encoding neomycin phosphotransferase II (nptII), as well as green fluorescent protein or cyan fluorescent protein, non-composite transgenic plants were obtained within 8 weeks. A root diameter of at least 1mm was required for efficient regeneration and transformation. Expression of fluorescent proteins in all cells was validated using confocal microscopy. On average, transformation efficiency (number of transgenic plants/number of root fragments) was 24.7% and 15.7% for TK and TB, respectively; about seven independent transgenic events were generated per starting plant for TK and four for TB. Overall, this high efficiency transformation method provides a rapid and simple system for these two dandelions to yield viable transgenic seeds in as little as 20 weeks. Protocols developed in this study allow introduction of genes of interest to facilitate the improvement of these rubber producing plants into domestic crops and provide an avenue to explore rubber biosynthesis and gene functions.

AFLP 분석을 통한 포공영 기원식물 민들레의 유전 다양성 분석

Collected germplasms of five representative dandelion species (Taraxacum ohwianum, T. platycarpum, T. platypecidum, T. officinale, and T. coreanum) were 104 lines from different habitates in Korea and China. Their genetic diversity was analyzed by genomic fingerprinting method using amplified fragment length polymorphism (AFLP). AFLP results of 6 primer combinations were revealed 1,176 total DNA fragments and 523 polymorphic bands with a 44.4% ratio of polymorphism. On the basis of similarity coefficient analysis by unweight pair group method with arithmetic averages (UPGMA), 104 dandelion germplasm lines were ranged from 0.64 to 0.99 and clustered distinct five group depending on the species. Furthermore, a principal coordinate analysis (PCA) by the application of multi-variate analysis indicated significantly greater differences among species than geographical origins.

Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum

The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1–5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40–50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.