Response of selenium pools to drought stress by regulating physio­biochemical attributes and anatomical changes in Gentiana macrophylla.

Selenium (Se), as a vital stress ameliorant, possesses a beneficial effect on mediating detrimental effects of environmental threats. However, the mechanisms of Se in mitigating the deleterious effects of drought are still poorly understood. Gentiana macrophylla Pall. is a well-known Chinese medicinal herb, and its root, as the main medicinal site, has significant therapeutic effects. The purpose of this experiment was to investigate the functions of Se on the seedling growth and physiobiochemical characteristics in G. macrophylla subjected to drought stress. The changes in microstructure and chloroplast ultrastructure of G. macrophylla leaves under drought exposure were characterized by scanning electron microscopy (SEM), scanning electron microscopes and energy dispersive X-Ray spectroscope (SEM-EDX), and transmission electron microscopy (TEM), respectively. Results revealed that drought stress induced a notable increase in oxidative toxicity in G. macrophylla, as evidenced by elevated levels of hydrogen peroxide (H2O2), lipid peroxidation (MDA), enhanced antioxidative response, decreased plant photosynthetic function, and inhibited plant growth. Chloroplasts integrity with damaged membranes and excess osmiophilic granule were observed in the drought-stressed plants. Se supplementation notably recovered the stomatal morphology, anatomical structure damage, and chloroplast ultrastructure of G. macrophylla leaves caused by drought exposure. Exogenous Se application markedly enhanced SPAD, photosynthetic stomatal exchange parameters, and photosystem II activity. Se supplementation significantly promoted the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), while reducing levels of MDA, superoxide anion (O2-.) and H2O2, and improving membrane integrity. Furthermore, the ameliorative effects of Se were also suggested by increased contents of osmotic substances (soluble sugar and proline), boosted content of gentiopicroside and loganinic acid in roots, and alleviated the inhibition in plant growth and biomass. Fourier transform infrared (FTIR) analysis of Se-treated G. macrophylla roots under drought stress demonstrated that Se-stimulated metabolites including O-H, C-H, N-H, C-N, and CO functional groups, were involved in resisting drought stress. Correlation analysis indicated an obvious negative correlation between growth parameters and MDA, O2-. and H2O2 content, while a positive correlation with photosynthetic gas exchange parameters. Principal component analysis (PCA) results explained the total variance into two principal components contributing the maximum (93.50 %) among the drought exposure with or without Se due to the various experiment indexes. In conclusion, Se exerts beneficial properties on drought-induced detrimental effects in G. macrophylla by relieving oxidative stress, improving photosynthesis indexes, PSII activity, regulating anatomical changes, altering levels of gentiopicroside and loganinic acid, and promoting growth of drought-stressed G. macrophylla.

De Novo Transcriptome Analysis Reveals Flowering-Related Genes That Potentially Contribute to Flowering-Time Control in the Japanese Cultivated Gentian Gentiana triflora.

Japanese cultivated gentians are perennial plants that flower in early summer to late autumn in Japan, depending on the cultivar. Several flowering-related genes, including GtFT1 and GtTFL1, are known to be involved in regulating flowering time, but many such genes remain unidentified. In this study, we obtained transcriptome profiling data using the Gentiana triflora cultivar 'Maciry', which typically flowers in late July. We conducted deep RNA sequencing analysis using gentian plants grown under natural field conditions for three months before flowering. To investigate diurnal changes, the plants were sampled at 4 h intervals over 24 h. Using these transcriptome data, we determined the expression profiles of leaves based on homology searches against the Flowering-Interactive Database of Arabidopsis. In particular, we focused on transcription factor genes, belonging to the BBX and MADS-box families, and analyzed their developmental and diurnal variation. The expression levels of representative BBX genes were also analyzed under long- and short-day conditions using in-vitro-grown seedlings, and the expression patterns of some BBX genes differed. Clustering analysis revealed that the transcription factor genes were coexpressed with GtFT1. Overall, these expression profiles will facilitate further analysis of the molecular mechanisms underlying the control of flowering time in gentians.

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian.

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

The impact of climatic variations on the reproductive success of Gentiana lutea L. in a Mediterranean mountain area.

Increases in temperature have been predicted and reported for the Mediterranean mountain ranges due to global warming and this phenomenon is expected to have profound consequences on biodiversity and ecosystem functioning. We hereby present the case of Gentiana lutea L. subsp. lutea, a rhizomatous long-lived plant living in Central-Southern Europe, which is at the edge of its ecological and distributional range in Sardinia. Concretely, we analysed the reproductive success experienced during three phenological cycles (2013/2014, 2014/2015 and 2015/2016) in four representative populations, with particular attention to the phenological cycle of 2014/2015, which has been recorded as one of the warmest periods of the last decades. The Smirnov-Grubbs test was used to evaluate differences in temperature and precipitation regimes among historical data and the analysed years, while the Kruskal-Wallis followed by the Wilcoxon test was used to measure differences between anthesis and reproductive performances among cycles and populations. In addition, generalised linear models were carried out to check relationships between climate variables and reproductive performance. Significant differences among climate variables and analysed cycles were highlighted, especially for maximum and mean temperatures. Such variations determined a non-flowering stage in two of the four analysed populations in 2014/2015 and significant differences of further five reproductive traits among cycles. These results confirmed that in current unstable climatic conditions, which are particularly evident in seasonal climates, reproductive success can be a sensitive and easily observable indicator of climatic anomalies. Considering the importance of this issue and the ease and cost-effectiveness of reproductive success monitoring, we argue that research in this sense can be a supporting tool for the enhancement of future crucial targets such as biodiversity conservation and the mitigation of global warming effects.

Site fidelity by bees drives pollination facilitation in sequentially blooming plant species.

Plant species can influence the pollination and reproductive success of coflowering neighbors that share pollinators. Because some individual pollinators habitually forage in particular areas, it is also possible that plant species could influence the pollination of neighbors that bloom later. When flowers of a preferred forage plant decline in an area, site-fidelity may cause individual flower feeders to stay in an area and switch plant species rather than search for preferred plants in a new location. A newly blooming plant species may quickly inherit a set of visitors from a prior plant species, and therefore experience higher pollination success than it would in an area where the first species never bloomed. To test this, we manipulated the placement and timing of two plant species, Delphinium barbeyi and later-blooming Gentiana parryi. We recorded the responses of individually marked bumble bee pollinators. About 63% of marked individuals returned repeatedly to the same areas to forage on Delphinium. When Delphinium was experimentally taken out of bloom, most of those site-faithful individuals (78%) stayed and switched to Gentiana. Consequently, Gentiana flowers received more visits in areas where Delphinium had previously flowered, compared to areas where Delphinium was still flowering or never occurred. Gentiana stigmas received more pollen in areas where Delphinium disappeared than where it never bloomed, indicating that Delphinium increases the pollination of Gentiana when they are separated in time. Overall, we show that individual bumble bees are often site-faithful, causing one plant species to increase the pollination of another even when separated in time, which is a novel mechanism of pollination facilitation.

Identification and functional analysis of SVP ortholog in herbaceous perennial plant Gentiana triflora: Implication for its multifunctional roles.

Information concerning to regulation of vegetative phase and floral initiation in herbaceous perennial plants has been limited to a few plant species. To know and compare flowering regulation in a wider range of plant species, we identified and characterized SHORT VEGETATIVE PHASE (SVP)-like genes (GtSVP-L1 and GtSVP-L2) from herbaceous perennial gentian (Gentiana triflora). Apple latent spherical virus (ALSV)-mediated silencing of the GtSVP-L1 in G. triflora seedlings resulted in early flowering and shortened vegetative phase by about one-third period of time, without vernalization. This indicated that GtSVP-L1 acts as a negative regulator of flowering and vegetative phase. Seasonal change in the expression of GtSVP was monitored in the overwinter buds (OWBs) of G. triflora. It was found that the levels of GtSVP-L1 mRNA in OWBs increased concomitantly with induction and/or maintenance of dormancy, then decreased toward release from dormancy, while that of GtSVP-L2 mRNA remained low and unchanged. These results implied that, in herbaceous perennial plants, SVP ortholog might concern to activity-dormancy control, as well as negative regulation in flowering. Practically, these results can be applicable to non-time-consuming technologies for breeding.

Apple latent spherical virus vector-induced flowering for shortening the juvenile phase in Japanese gentian and lisianthus plants.

MAIN CONCLUSION: Infection by apple latent spherical virus (ALSV) vectors that promote the expression of Arabidopsis thaliana FLOWERING LOCUS T ( AtFT ) or Gentiana triflora GtFT s accelerates flowering in gentian and lisianthus plants. Apple latent spherical virus (ALSV) has isometric virus particles (25 nm in diameter) that contain two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). ALSV vectors are used for foreign gene expression and virus-induced gene silencing in a broad range of plant species. Here, we report the infection by ALSV vectors that express FLOWERING LOCUS T (AtFT) from Arabidopsis thaliana or its homolog GtFT1 from Gentiana triflora in three gentian cultivars ('Iwate Yume Aoi' [early flowering], 'Iwate' [medium flowering], and 'Alta' [late flowering]), and two lisianthus cultivars ('Newlination Pink ver. 2' and 'Torukogikyou daburu mikkusu') promotes flowering within 90 days post-inoculation using particle bombardment. Additionally, seedlings from the progeny of the early-flowering plants were tested by tissue blot hybridization, and the results showed that ALSV was not transmitted to the next generation. The promotion of flowering in the family Gentianaceae by ALSV vectors shortened the juvenile phase from 1-3 years to 3-5 months, and thus, it could be considered as a new plant breeding technique in ornamental gentian and lisianthus plants.

Selective Pressures Explain Differences in Flower Color among Gentiana lutea Populations.

Flower color variation among plant populations might reflect adaptation to local conditions such as the interacting animal community. In the northwest Iberian Peninsula, flower color of Gentiana lutea varies longitudinally among populations, ranging from orange to yellow. We explored whether flower color is locally adapted and the role of pollinators and seed predators as agents of selection by analyzing the influence of flower color on (i) pollinator visitation rate and (ii) escape from seed predation and (iii) by testing whether differences in pollinator communities correlate with flower color variation across populations. Finally, (iv) we investigated whether variation in selective pressures explains flower color variation among 12 G. lutea populations. Flower color influenced pollinator visits and differences in flower color among populations were related to variation in pollinator communities. Selective pressures on flower color vary among populations and explain part of flower color differences among populations of G. lutea. We conclude that flower color in G. lutea is locally adapted and that pollinators play a role in this adaptation.

Characterization of spermidine synthase and spermine synthase--The polyamine-synthetic enzymes that induce early flowering in Gentiana triflora.

Polyamines are essential for several living processes in plants. However, regulatory mechanisms of polyamines in herbaceous perennial are almost unknown. Here, we identified homologs of two Arabidopsis polyamine-synthetic enzymes, spermidine synthase (SPDS) and spermine synthase (SPMS) denoted as GtSPDS and GtSPMS, from the gentian plant, Gentiana triflora. Our results showed that recombinant proteins of GtSPDS and GtSPMS possessed SPDS and SPMS activities, respectively. The expression levels of GtSPDS and GtSPMS increased transiently during vegetative to reproductive growth phase and overexpression of the genes hastened flowering, suggesting that these genes are involved in flowering induction in gentian plants.

[Genetic polymorphism of Gentiana lutea L. (Gentianaceae) populations from Chornohora Ridge of Ukrainian Carpathians].

The features of genetic structure and level of diversity were investigated for G. lutea populations from Chornohora Ridge of Ukrainian Carpathians using RAPD- and ISSR-PCR. We have shown a high level of genetic diversity for investigated populations. The differences between populations account for 59-72% of the total genetic variation, whereas intrapopulation polymorphism makes up 28-41%. The relationships among genetic variability level and ecological-geographical conditions as well as biological features of the species were assumed to be possible. The obtained results indicate the genetic isolation of G. lutea Chornohora populations from Ukrainian Carpathians. Pozhyzhevska agropopulation was characterized by a high level of polymorphism that means the possibility to use artificial plantings of the investigated species for its conservation.

The gentio-oligosaccharide gentiobiose functions in the modulation of bud dormancy in the herbaceous perennial Gentiana.

Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [ß-D-Glcp-(1→6)-D-Glc] and gentianose [ß-D-Glcp-(1→6)-D-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of ß-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.

Effects of GA3 pregerminative treatment on Gentiana lutea L. var. aurantiaca germination and seedlings morphology.

Gentiana lutea L. is widely used in bitter beverages and in medicine; Gentianae Radix is the pharmaceutical name of the root of G. lutea. These uses have generated a high demand. The wild populations of Gentiana lutea var. aurantiaca (M. Laínz) M. Laínz have been decimated; it is necessary to establish guidelines for its cultivation. Gentian as most alpine species has dormant seeds. Dormancy can be removed by cold and by means of a gibberellic acid (GA3) treatment. However, cold treatments produce low germination percentages and GA3 treatments may produce off-type seedlings. So, the objective was to determine, for the first time, the presowing treatments that allow high germination rate and good seedling morphology. The best pregerminative doses of GA3 to break seed dormancy were 100, 500, and 1000 ppm, while the best doses to optimize the seedling habit were 50 and 100 ppm. This study provides, for the first time, a 100 ppm GA3 dose that led to a high germination rate and good seedling morphology, as the starting point for gentian regular cultivation.

Dehydrins are highly expressed in overwintering buds and enhance drought and freezing tolerance in Gentiana triflora.

Gentians, herbaceous perennials, produce overwintering buds (OWBs) to survive the cold season. Although gentians are known to have strong stress tolerances against drought, cold and freezing, the molecular mechanisms of tolerance are unclear. We explored genes more highly expressed in OWBs than in other tissues and identified two gentian orthologs of dehydrins, denoted GtDHN1 and GtDHN2. These GtDHNs possess several ABA or dehydration responsive elements. Furthermore, GtDHN1 and GtDHN2 transcripts in OWBs accumulated during the winter but decreased prior to spring, suggesting that GtDHNs may be induced by dehydration stress during cold periods and may act as a stress protectant mediated by ABA. Likewise, cultured gentian plantlets accumulated GtDHN transcripts in response to ABA as well as cold and drought stresses. Moreover, transgenic gentian plantlets overexpressing GtDHN1 or GtDHN2 showed improved cold and drought stress tolerance. Metabolome analysis revealed that major antioxidants such as glutathione and ascorbate were accumulated in all transgenic plantlets. Overexpression of GtDHNs also affected the activities of the antioxidant enzymes, ascorbate peroxidase and glutathione peroxidase. Based on the results of this study, GtDHNs are induced by ABA and dehydration stress and have an ability to alleviate dehydration stress, probably via activating antioxidant mechanisms. Accumulation of GtDHNs may be part of the strategy for winter survival of gentian OWBs.

[Cryopreservation and plantlet regeneration of dormant buds of Gentiana straminea by vitrification].

OBJECTIVE: To investigate the detailed techniques for cryopreservation of Gentiana straminea dormant buds by vitrification. METHODS: Dormant buds as an experimental material,the influence of the different size of dormant buds,preculture and PVS etc. on cryopreservation of Gentiana straminea were studied. RESULTS: The optimal procedures were as follows: 10-11 mm long dormant buds which were cultured on MS medium supplemented with different sucrose concentration (0. 3,0. 5 or 0.7 mol/L) for 1 day respectively. The buds were immersed in loading solution for 20 min at 20 degrees C, and then treated in PVS2 solution for 40 min at 0 degrees C and finally plunged into liquid nitrogen quickly. After 24 hours, the buds were rapidly thawed in a water bath at 40 degrees C for 2 - 3 min and washed twice with MS medium supplemented with 1/2 MS liquid medium containing 1. 2 mol/L sucrose. Finally the buds were transferred to regeneration medium (MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 3% sucrose + 0.7% agar), the survival rate was up to 83.3%. CONCLUSION: A high-efficiency cryopreservation protocol of Gentiana straminea is set up.

A single-base substitution suppresses flower color mutation caused by a novel miniature inverted-repeat transposable element in gentian.

We investigated the genetic basis for the derivation of pink coloration in petals from blue flowers in cultivated gentians. Using a revertant blue-flower phenotype that arose spontaneously from a pink-flowered cultivar, we sought to elucidate the molecular mechanism of flower color restoration caused by a suppressor mutation. Detailed sequencing analysis identified three novel deficient flavonoid 3',5'-hydroxylase (F3'5'H) alleles in pink-flowered gentians in addition to two mutations identified previously (Nakatsuka et al. in Mol Genet Genomics 275:231-241, 2006). Among the deficient alleles, one allele that contained a novel miniature inverted-repeat transposable element (GtMITE1) insertion in an intron of F3'5'H was shown to cause missplicing, resulting in abnormal F3'5'H transcripts and the pink-flower phenotype. The other two mutations were identified as a single-nucleotide insertion and gypsy-Ty3 retrotransposon (Tgt1) insertion within exon 1 and exon 2 of the F3'5'H gene, respectively. The blue-flowered revertant mutant contained a single-nucleotide spontaneous mutation immediately 3' of the TAA target site duplication and the GtMITE1 insertion, which caused restoration of normal splicing of F3'5'H and the normal blue-flower phenotype. Transient expression assays in gentian flowers in vivo demonstrated that normal F3'5'H splicing pattern was recovered from missplicing induced by the GtMITE1 insertion by the single-nucleotide substitution. These findings extend our knowledge of genomic evolution by transposable elements and spontaneous mutations in Gentiana species of economic and medical importance.

Shifting phenology and abundance under experimental warming alters trophic relationships and plant reproductive capacity.

Phenological mismatches due to climate change may have important ecological consequences. In a three-year study, phenological shifts due to experimental warming markedly altered trophic relationships between plants and insect herbivores, causing a dramatic decline of reproductive capacity for one of the plant species. In a Tibetan meadow, the gentian (Gentiana formosa) typically flowers after the peak larva density of a noctuid moth (Melanchra pisi) that primarily feeds on a dominant forb (anemone, Anemone trullifolia var. linearis). However, artificial warming of approximately 1.5 degrees C advanced gentian flower phenology and anemone vegetative phenology by a week, but delayed moth larvae emergence by two weeks. The warming increased larval density 10-fold, but decreased anemone density by 30%. The phenological and density shifts under warmed conditions resulted in the insect larvae feeding substantially on the gentian flowers and ovules; there was approximately 100-fold more damage in warmed than in unwarmed chambers. This radically increased trophic connection reduced gentian plant reproduction and likely contributed to its reduced abundance in the warmed chambers.

The gentian orthologs of the FT/TFL1 gene family control floral initiation in Gentiana.

Gentians are herbaceous perennials blooming in summer through autumn. Although they are popular ornamental flowers in Japan, the regulation of their timing of flowering has not been studied. We identified and characterized gentian orthologs of the Arabidopsis FT/TFL1 gene family to elucidate the mechanisms of flowering initiation. We isolated three gentian orthologs of FT and TFL1, denoted GtFT1, GtFT2 and GtTFL1. Since up-regulation of GtFT1 and GtFT2 as well as down-regulation of GtTFL1 promoted floral initiation in gentian plantlets, these genes affected floral initiation in a similar way to Arabidopsis FT and TFL1. The expression levels of GtFT1 and GtFT2 in leaves of late-flowering gentian increased prior to floral initiation, whereas GtTFL1 was highly expressed in shoot apical meristem at the vegetative stage and decreased drastically just before flowering initiation. Comparison of gene expression patterns showed that GtFT1 expression increased earlier in early-flowering than in late-flowering gentian, whereas the timing of the increase in GtFT2 expression was similar in early- and late-flowering plants. The GtTFL1 expression in early-flowering gentian was extremely low throughout the vegetative and reproductive stages. These results indicated that either the up-regulation of GtFT1 or the down-regulation of GtTFL1 may determine flowering time. Furthermore, we found that early-flowering but not late-flowering gentians have a 320 bp insertion in the promoter region of GtTFL1. Thus, the negligible expression of GtTFL1 in early-flowering lines may be due to this insertion, resulting in a shortened vegetative stage.

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids.

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora.

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0-32.6% of cultured ovary pieces and 0-18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.

Somatic embryogenesis pathway for plant regeneration in Qinjiao (Gentiana macrophylla Pall.).

Qinjiao (Gentiana macrophylla Pall.) is a perennial herbage native to northwestern China. It has been taken as a kind of Chinese herbs for more than one thousand years. The major secondary metabolite named as gentiopicroside accounts for 8% of the dry weight of roots. It has been used for medical purpose in the treatment with rheumatism, osteoarthritis, inflammatory or ulceration. Conventionally, Qinjiao is propagated by seeds, but the seeds should be planted in one year after harvesting and the germination rate is extremely low. These confine its spread by seeds after year-round storage. Therefore, plant regeneration from somatic cells will be an ideal way for its propagation. Plant regeneration from somatic cell can be divided into two ways: somatic embryogenesis and organogenesis. Similar to zygotic embryo, somatic embryo has a bipolar structure with both shoot and root poles. Somatic embryogenesis also goes through the same key stages as globular-, heart-, torpedo- and cotyledon-stages Somatic embryogenesis was first observed in carrot callus cells more than 45 years ago. Since then, plant regeneration via somatic embryogenesis has been studied in many important species. Though the plant regeneration of Qinjiao has been previously studied, there is no investigation of somatic embryogenesis reported in this plant. Therefore, the aim of our experiment is to confirm the somatic embryogenesis pathway of plant regeneration in Qinjiao.