Chinese Narcissus Research Articles

Complete genome sequence of narcissus late season yellows virus infecting Chinese narcissus in China

The complete genome sequence of a Chinese narcissus isolate of narcissus late season yellows virus from Zhangzhou, China (NLSYV-ZZ), was determined to be 9,651 nucleotides in length, excluding the 3'-terminal poly (A) tail, by amplification and sequencing of virus RNA. The viral genome contains a single long open reading frame of 9,315 nucleotides encoding a polyprotein of 3,105 amino acids. The polyprotein was predicted to be cleaved into ten mature proteins by three viral proteases. Complete genome sequence comparison and phylogenetic analysis indicated that NLSYV-ZZ was most closely related to narcissus yellow stripe virus (NYSV), which was also isolated from narcissus. These viruses shared 69.9 % identity in their complete nucleotide sequences and 77.0 % identity in their polyprotein amino acid sequences.

Cloning and functional characterization of a GNA‐like lectin from Chinese Narcissus (Narcissus tazetta var. Chinensis Roem)

A full-length cDNA encoding Narcissus tazetta lectin (NTL) was isolated from Chinese narcissus (N. tazetta var. Chinensis Roem). The open reading frame (ORF) was 519 bp long and encoded 172 amino acids with a theoretical isoelectric point of 5.27 and a calculated molecular mass of 18.6 kDa. Conserved domain analysis indicated that it possessed three D-(+)-mannose-binding sites, presumed to be similar to those of Galanthus nivalis agglutinin (GNA)-like lectins. A recombinant (glutathione S-transferase) GST-NTL fusion protein of around 40 kDa was successfully synthesized in vitro. Lysates of cells expressing this recombinant protein exhibited significant hemagglutinating activity [418 hemagglutinating units (HU)], as did the purified protein (265 HU). Sugar specificity assays suggested that mannose is the only sugar that significantly inhibits this hemagglutinating activity, confirming that NTL is a member of the GNA-like lectin family. NTL is highly transcribed in flowers, leaves and roots, but less so in scales. However, similar levels of the NTL protein were observed in all four of these organs by western blotting. A fluorescent NTL-GFP (green fluorescent protein) fusion protein was found to be primarily localized in the vacuole of transformed onion epidermal cells, indicating that NTL may be a vacuolar storage protein. This is the first study in which the function of NTL has been examined and provides a considerable body of data concerning its physiological role in Chinese narcissus. The results obtained may be useful in the molecular engineering of plants with enhanced tolerance of biotic and abiotic stresses. Moreover, they may be relevant to medical applications of lectins.

Ectopic expression of an <em>AGAMOUS</em> homolog <em>NTAG1</em> from Chinese narcissus accelerated earlier flowering and senescence in Arabidopsis

There are two cultivating varieties of Chinese narcissus, named as Yulinglong ( Narcissus tazetta var. chinensis Roem. Florepleno) and Jinzhanyutai ( Narcissus tazetta var. chinensis M. Roem.) well known in China. Yulinglong plants exhibit double flower resulted from petaloid stamens. However, the molecular basis of double flower formation is known little and unclear. Based on the flowering ABCDE model, double flower formation is commonly used to link to C functional genes. In this present paper, the isolation and characterization of NTAG1 gene, an AGAMOUS homolog from Chinese narcissus varieties mentioned above are reported. Sequence and expression pattern of NTAG1 gene exhibited the same in both tested varieties. It expresses only in the reproductive organs. Furthermore, functional analysis by using ectopic tests in Arabidopsis showed that NATG1 might be involved in the carpel identity and floral transition The effects of ectopic expression of NTAG1 mainly include dwarfing , early flowering, losing inflorescence indeterminacy,branch number increasing, and advancing senescence, whereas some of homeotic phenotypes gradually sisappeared in higher generation of transgenic plants. The utilizations of NATG1 gene in the future gene engineering were also discussed in the paper.

Isolation and characterization of the LEAFY homologus gene NTLEAFY in Chinese narcissus (Narcissus tazetta var. chinensis Roem)

Isolation and characterization of the LEAFY homologus gene NTLEAFY in Chinese narcissus (Narcissus tazetta var. chinensis Roem)

Identification of a potyvirus associated with mosaic disease of Narcissus sp. in India

Narcissus tazetta cv. Paperwhite is an important ornamental for the floriculture industry. Narcissus is susceptible to three members of genus Potyvirus, namely Narcissus yellow stripe virus, Narcissus late season yellows virus and Narcissus degeneration virus, plus Narcissus latent virus (genus Macluravirus, family Potyviridae). A disease characterized by severe mosaic and light green to yellow leaf stripes, was observed on Narcissus in an experimental plot at the National Botanical Research Institute, Lucknow. Affected plants also showed extreme reduction in their growth, and loss of yield and bloom quality. Total RNA was extracted from 100 mg of leaf tissue as described by Gibbs & MacKenzie (1997). RT-PCR was performed with potyvirid specific primers Pot1 and Pot2 (Gibbs & MacKenzie, 1997) to amplify part of the NIb gene, the coat protein (CP) gene and the 3′ untranslated region (UTR). The amplified DNA fragment of c. 1·5 kb was cloned into the p-drive cloning vector (QIAGEN) and sequenced (GenBank accession DQ991145). The CP gene nucleotide sequence showed 98% identity with Chinese narcissus potyvirus Chongming isolate (AJ311374·1), 97% with Chinese narcissus potyvirus Zhangzhou isolate (AJ311373·1) and 95% with Lycorsis potyvirus (AF511486·1). The 3′-UTR (148 nt.) shared 100% identity with the Chinese narcissus Chongming and Zhangzhou isolates and 97% identity with Lycoris potyvirus (AF511486·1). Earlier investigations in India (Aminuddin et al., 1989, 1999), based mainly on biological, serological and electron microscopic properties demonstrated the association of an unidentified potyvirus with severe mosaic disease of Narcissus tazetta. However, nucleotide sequence information (CP gene and 3′ UTR) available in this study clearly established the identity of the associated potyvirus, at the species level, as the virus first recorded as Lycoris potyvirus. This is the first specific identification of a potyvirus from narcissus in India. However, Narcissus spp. are prone to mixed viral infections (Chen et al., 2003) so this potyvirus may not be the sole agent causing the mosaic disease.

Effect of radiation on regeneration of Chinese narcissus and analysis of genetic variation with AFLP and RAPD markers

The aim of our study was to establish an efficient in vitro propagation protocol for Chinese narcissus (Narcissus tazetta var. chinensis) to obtain variants of this species using γ-radiation treatment and evaluate the effectiveness of this system for variant induction using amplification fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) analysis. Various doses (5–100 Gy) of gamma rays were applied to investigate the effect of radiation on adventitious bud formation from bulb-scales and the survival rate of plantlets. It was demonstrated that the regeneration of Chinese narcissus was very sensitive to gamma radiation even at low doses. The survival and multiplication rate significantly decreased with an increase of radiation dose. The optimal irradiation dose for survival and mutation induction was approximately 10 Gy. The genetic variations among the regenerants derived from irradiated explants were evaluated by DNA fingerprinting using RAPD and AFLP markers which detected a variation frequency of 8.33% and 15.48% respectively. The high frequency of mutants detected by molecular markers indicated that treatment of in vitro cultures with γ-rays may be an effective way to improve narcissus cultivars.

Characterisation of an isolate of Narcissus degeneration virus from Chinese narcissus (Narcissus tazetta var. chinensis)

A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence (9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses.

Complete nucleotide sequence of the genomic RNA of Narcissus yellow stripe virus from Chinese narcissus in Zhangzhou city, China

Complete nucleotide sequence of the genomic RNA of Narcissus yellow stripe virus from Chinese narcissus in Zhangzhou city, China

Isolation and characterization of the AGAMOUS homologous gene NTAG in Chinese narcissus (Narcissus tazetta var. chinensis Roem)

Amaryllidaceae, a monocot plant family, consists of many important ornamental bulb flower species. Chinese narcissus (Narcissus tazetta var. chinensis Roem), its flowers developed at high temperatures and bloomed at lower temperatures during the Chinese Spring Festival, is a traditional Chinese flower with high economic and ornamental value. To study its flower development, a full length cDNA containing MADS box domain from narcissus was isolated by a reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligo-nucleotide primers derived from a conserved MADS-and K-box domain sequence. The 5′ and the 3′ regions of the gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). The 690 bp open reading frame encodes 230 amino acid residues. A comparison of the deduced amino acid sequence of NTAG with the sequence of other MADS box proteins showed 91.3% amino acid identities with HAG (Hyacinthus orientalis). Sequence analysis and alignment showed significant similarity with other AG homologues. RNA blot analysis indicated that the narcissus NTAG gene was expressed only in reproductive organs, especially in stamens and carpels. These results indicated that the NTAG gene was an AG homologue and that the AG genes appeared to be structurally and functionally conserved between dicots and monocots.

Embryological Studies on Narcissus tazetta var. chinensis

Chinese narcissus (Narcissus tazetta var. chinensis Roem) blooms but has no seeds. Embryological studies on the species were conducted to discover the causes of its sterility. Its anther wall is composed of four layers of cells, and its tapetum is of the secretory type. The cytokinesis of microspore mother cells is of the successive type, and the tetrad is tetrahedral. During meiosis of microspore mother cells, some chromosomes lagged, and several micronuclei were found in tetrads. Only 27.7% of the pollen grains contained full cytoplasm, and 1.3% of them germinated in culture medium. No pollen grain, however, could germinate on the stigma. The ovary is trilocular with axile placenta, and the ovules are bitegmic, tenuinucellate, and anatropous. Its embryo sac is of the polygonum type. Most embryo sacs degenerated, and only about 4.5% of the ovules contained a normal embryo sac with an egg cell, two synergids, three antipodal, and a central cell containing two polar nuclei. One reason for the sterility of Chinese narcissus is the abnormality of microsporogenesis and megasporogenesis, in which only a few functional pollen grains and embryo sacs are produced. The other reason is that the pollen grains cannot germinate on the stigma.

Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem)

Callus culture has, to date, been reported only in a few species of Narcissus. We used anthers of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem) as explants for callus induction and plant regeneration. A high percentage of anthers at the early- to mid-uninucleate microspore stage were responsive on the basal MS medium supplemented with 0.5-1 mg l(-1) 2,4-dichlorophenoxyacetic acid and 0.5-2 mg l(-1) 6-benzyladenine under dark conditions. Calli were initiated from anther connective tissue or anther wall tissue, and no division of microspores occurred during callus formation, as determined by histological observation. Using 20 random amplified polymorphic DNA primers, we verified the genetic integrity of the anther-derived plants of Chinese narcissus with respect to the donor plants. These results suggest that anther culture in vitro can provide an efficient new micropropagation technique for Chinese narcissus as well as a new strategy for in vitro mass propagation of other daffodils.

Molecular Characterization of Carla‐ and Potyviruses from Narcissus in China

AbstractConserved carlavirus and potyvirus primers were used in reverse transcriptase‐polymerase chain reaction (RT‐PCR) to amplify virus fragments from Chinese narcissus (Narcissus tazetta var. chinensis) in China and the fragments were subsequently sequenced and compared in phylogenetic analyses. Samples from Fujiang province and Shanghai contained either one or two potyviruses and a carlavirus. One potyvirus (PY1) showed a distant relationship to Iris severe mosaic virus, Onion yellow dwarf virus and Shallot yellow stripe virus, while the other (only in the Fujiang sample, PY2) was most closely related to Turnip mosaic virus (TuMV). Similar experiments using glasshouse grown Narcissus originating from the UK contained Ornithogalum mosaic virus and another potyvirus (PY3) in the TuMV/PY2 cluster. Comparisons with previously determined sequence fragments indicated that PY2 was probably Narcissus yellow stripe virus and PY3 Narcissus late season yellow virus. Carlavirus fragments from both Chinese sites seemed to be of the same virus, which was most closely related in phylogenetic analyses to Potato virus M, Aconitum latent virus and Hop latent virus. It is most probably a new member of the genus Carlavirus and has been tentatively named Narcissus common latent virus.