中国水仙(Narcissus tazetta var.chinensis)的组织培养

以栽培的中国水仙Narcissus tazetta var.Chinensis鳞茎为实验材料,采取“无液处理”的简捷方法获取外植体,并筛选出中国水仙离体鳞茎培养的最佳培养基。     

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.     

Analysis of B-Class Genes NAP3L3 and NAP3L4 in Narcissus tazetta var. chinensis

Very few flower organ identity genes have been characterized in Chinese narcissus (Narcissus tazetta var. chinensis), which has petaloid sepals. Here, we report the cloning of two full-length B-class genes, namely NAP3L3 and NAP3L4, that are orthologs of the DEFICIENS lineage. Both genes are highly expressed in the second whorl of the perianth and in the stamens. NAP3L4 is also expressed strongly in the ovule. The functions of these two genes were further analyzed using transgenic plants. Ectopic expression of either gene in Arabidopsis gave no obvious floral organ transformation phenotypes. In yeast two-hybrid assays, NAP3L3 and NAP3L4 failed to homodimerize and interacted weakly with each other. The data suggest that these two genes might not be involved in the formation of petaloid sepals. Isolation and functional analysis of other B-class paralogs should be conducted to fully understand petaloid tepal development in Chinese narcissus.     

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.     

中国水仙花基因工程的研究现状

重点综述了中国水仙花香、花色、花发育、抗性和遗传转化等方面基因的研究现状;探讨了基因调控和改善花观赏价值的方法;简要指出了在研究中存在的问题及展望了其应用前景。     

自然光照下普陀水仙的组织培养

1　植物名称　水仙 (Narcissustazettavar.chi nensis)品种普陀水仙。2　材料类别　带鳞茎盘的双鳞片 (twinscale)。3　培养条件　以MS为基本培养基。 ( 1 )诱导小鳞茎培养基 :MS + 6 BA 5mg·L- 1 (单位下同 ) +NAA 0 .2 5～ 0 .5 ;( 2 )生根培养基 :1 /2MS(大量元素减半 ) +NAA 0 .0 5。培养基均加 0 .8%琼脂 (a gar) ,( 1 )加0 .5 %活性炭 (activatedcharcoal)和 4%蔗糖 (sucrose) ,( 2 )加 3%蔗糖 ,pH 5 .6～ 5 .8。培养温度 ( 2 5± 2 )℃ ,置于培养室自然光线下 ,不用灯光照明。4　生长与分化情况4.1　无菌材料的获得　 5、6月…     

Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

漳州水仙ZDS 基因cDNA克隆及其表达分析

采用RACE技术从漳州水仙‘金盏银台’Narcissus tazetta var. chinensis花器官总RNA中克隆ZDS的cDNA序列,用相关软件进行序列分析,并利用实时荧光定量技术检测ZDS基因在各水仙品种不同花器官的表达。序列分析表明,ZDS基因cDNA全长2189 bp,其中包含69 bp 5'非编码区,395 bp 3'非编码区,1725 bp编码区（编码574个氨基酸,分子量约63.6 kDa）,命名为Ntzds （GenBank登录号: EU573238）,其编码的氨基酸序列（ACB87206.1）与喇叭水仙（CAA12062.1）、葡萄（XP_002277348.21）和温州蜜柑（ABC33728.1）ZDS基因编码产物的相似性分别为97%、85%和83%。实时荧光定量PCR分析表明,Ntzds基因在漳州水仙‘金盏银台’、‘Minnow’、‘Fortissimo’中均有表达,且同一品种中副冠的表达量高于花瓣;随着花色的加深,其转录水平也逐渐趋高。     

中国水仙遗传转化研究进展(综述)

中国水仙是我国传统名花,其栽培种一直存在三个问题：品种单一,繁殖速度有限,病毒积累严重。生物技术的发展为解决中国水仙目前存在的问题提供了新的途径。本文从外植体的预处理、愈伤组织和小鳞茎的诱导与分化、生根及移栽等方面概述中国水仙的组织培养进展,较详尽地阐述中国水仙基因工程的研究进展,并对中国水仙未来研究方向进行展望。     

中国水仙生物技术研究进展

中国水仙是我国传统名花,其栽培中一直存在三个问题:品种单一、繁殖速度有限、病毒积累严重。生物技术的发展为解决中国水仙目前存在的问题提供了新的途径,将成为中国水仙遗传改良、种质资源创新的重要手段。该文从四个方面综述了近年来中国水仙生物技术的研究进展:(1)离体培养;(2)基因工程;(3)离体突变体筛选;(4)分子标记。     

Identification of Genes Involved in Flavonoid Biosynthesis in Sophora japonica Through Transcriptome Sequencing

Sophora japonica is a traditional Chinese medicinal ingredient that is widely used in the medicine, food, and industrial dye industries. Since flavonoids are the main components of S. japonica, studying the flavonoid composition and content of this plant is important. This study aimed to identify molecules involved in the flavonoid biosynthetic pathways in S. japonica. Deep sequencing was performed, and 85,877,352 clean reads were filtered from 86,095,152 raw reads. The clean reads were spliced to obtain 111,382 unigenes, which were then annotated with NR, GO, KEGG, eggNOG. Differential expression analysis and NR function prediction revealed 18 differentially expressed unigenes associated with 13 enzymes in flavonoid biosynthetic pathways. Our results reveal new insights on secondary metabolite biosynthesis‐related genes in S. japonica and enhance the potential applications of S. japonica in genetic engineering.     

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.     

多效唑对水仙生长发育的影响

为提高水仙的观赏价值,探讨复合多效唑对水仙生长发育的影响,用不同的多效唑溶液对水仙鳞茎进行处理。结果表明:多效唑能抑制水仙的营养生长,使植株矮化粗壮,叶片碧绿、挺拔,不易倒伏,根白、短而壮;复合多效唑配方使平均叶长减少37.7%,花期延长2d,花朵直径增加18.3%,延缓黄化叶片的出现,减慢叶肉薄壁细胞及其中叶绿体的解体,在后期维管束的结构相对完整,有效提高观赏价值。     

Necessity of high temperature for the dormancy release of Narcissus tazetta var. chinensis

Winter dormancy has been extensively studied in many plants, while much less information is available for summer dormancy. Narcissus tazetta var. chinensis is characterized by a prolonged period of summer dormancy during the annual cycle. In the present study, we characterized the nature of dormancy in the controlled growth conditions and investigated the effects of temperature and ethylene on dormancy release. Cessation of growth and senescence of aerial tissues occurred even under conditions favorable for growth, suggesting an endo-dormancy process. Bulbs failed to sprout when they were exposed to low storage temperatures, while high temperature treatment preceding low storage temperatures, or heating interruption during low storage temperatures, could make bulbs sprouting. These results suggest that high temperatures are necessary for endo-dormancy release. Ethylene application during a later storage stage showed an obvious accelerative effect on bulb sprouting, whereas ethylene application during the middle stage had no effect on sprouting. The biological progression of dormancy was further studied through cytological and physiological analyses. Under natural conditions, the ethylene level was reduced to trace amounts with the transition and progression of dormancy. A transient peak in ethylene release was found when the plugged plasmodesmata (PD) began to re-open and flower initiation began. The most common PD possessed electron-dense deposits in endo-dormancy. These data indicate that endo-dormancy ends when flower initiation begins and ethylene peak occurs. Ethylene application had no effect on dormancy release, while it hastened sprouting only after the release from endo-dormancy by high temperature.     

中国水仙的核型分析和小孢子发生中的细胞学研究

中国水仙(Narcissustazettavar.chinensis)只开花不结实,以鳞茎营养繁殖。对中国水仙的染色体倍性有不同的报道。对中国水仙的核型分析,支持它是三倍体的观点,但其核型也显示出了异源三倍体的倾向。在小孢子母细胞减数分裂过程中,染色体的异常行为多表现为:中期出现单个染色体游离在纺锤体外面、在后期出现的染色体桥和落后染色体、在末期出现的单个染色体游离在细胞核外形成微核的现象。这些异常现象引起小孢子的败育,也支持中国水仙为三倍体植物的观点。     

离体筛选中国水仙抗叶大褐斑病突变体初步研究

选用中国水仙鳞茎块离体培养,以叶大褐斑病原菌的培养滤液作为选择,结合γ-射线物理诱变,从1420块外植体中得到9个抗病再生种球。抗病性测定表明,经病原菌培养滤液筛选处理得到的再生种球,抗病性比对照明显提高。     

崇明水仙根尖体细胞染色体的观察和核型分析

以崇明水仙(Narcissus tazetta L.var.chinensis Roem.)根尖体细胞为实验材料,对适宜于崇明水仙细胞学研究的前处理液和前处理时间进行了筛选,在此基础上,应用根尖压片法对重瓣花型和单瓣花型崇明水仙体细胞染色体数、核型及倍性进行了比较分析.结果显示:适宜的前处理液是对二氯苯饱和溶液,适宜的前处理时间为12 h.重瓣花型和单瓣花型崇明水仙的染色体核型差异较小,相同点为:不对称二型核型,染色体基数x=10,三倍体,体细胞染色体数2n=3x=30,第7号染色体的短臂具随体,核型均属于"3B"型,臂比大于2的染色体比率为90%.不同点为:重瓣花型的第7号和第8号染色体分别为sm和st型,单瓣花型的第7号和第8号染色体分别为st和sm型;前者的核型不对称系数(76.48%)略小于后者(76.71%);前者的相对长度系数为12L+6M2+12S,后者的相对长度系数为12L+3M1+3M2+12S;前者的最长染色体与最短染色体长度的比值(3.10)略小于后者(3.19).重瓣花型的核型公式为2n=3x=30=15st+15sm(3SAT),单瓣花型的核型公式为2n=3x=30=15st(3SAT)+15sm,崇明水仙根尖体细胞染色体的平均核型公式为2n=3x=30=15st(3SAT)+15sm.根据研究结果初步推测崇明水仙为节段异源三倍体.     

霍格兰溶液培养对水仙生长发育的影响

用霍格兰(Hoagland)溶液培养水仙(Narcissus tazettavar.chinensisBeem),结果显示霍格兰溶液培养的水仙植株根系健壮、嫩白,叶片鲜绿厚实。花梗数和每梗花朵数平均比对照增多5.85支/株和2.85朵/穗,花朵直径比对照平均增大2.9 mm。花期延长5 d左右。     

生长素和赤霉素对离体水仙花茎切段伸长的影响

以离体水仙(Narcissustazettavar.chinensis)花茎切段为材料,通过外源吲哚-3-乙酸(Indole-3-aceticacid,IAA)和赤霉素A3(GA3)处理,结合内源激素分析,研究了这两种激素对水仙花茎切段伸长的影响,以及它们之间的相互作用。结果表明:外源50μmol/LIAA和30μmol/LGA3均能促进花茎切段的伸长,其中IAA的促进作用大于GA3。200μmol/L生长素运输抑制剂2,3,5-三碘苯甲酸(2,3,5-Triiodobenzoicacid,TIBA)和65μmol/L赤霉素合成抑制剂烯效唑(Uniconazole,S-3307)均显著抑制花茎切段的伸长。外源50μmol/LIAA处理明显增加内源GA1 3的含量,是对照的3.40倍;外源30μmol/LGA3处理对内源IAA含量影响不明显,说明IAA对维持花茎切段内源活性GA水平起重要作用,IAA和活性GA共同发挥调控花茎切段伸长的作用。     

Variation in the karyotype of three cultivars of Narcissus tazetta L. (Amaryllidaceae)

Three cultivated varieties of Narcissus tazetta, Grand Soleil d'Or, Chinese Sacred Lily and Cypri, are triploid (2n = 3x+30) with the basic number 10. Grand Soleil d'Or has three homomorphic sets, each comprising 2 long submetacentrics, 4 long acrocentrics and 4 short acrocentrics. Karyotypes of the other two varieties are heteromorphic. Both possess one telocentric satellited chromosome. In addition, Cypri shows translocation between two chromosomes belonging to the seventh and eighth triplets. The number of secondary constrictions varies between 3 (Chinese Sacred Lily) and 4 (Grand Soleil d'Or and Cypri) which is also the number of nucleoli observed in the respective varieties.