Controlling hyperhydration of carnations (Dianthus caryophyllus L.) grown in a mist reactor

Carnations (Dianthus caryophyllus L.) grown in vitro often develop physiological abnormalities such as hyperhydration. The amount of hyperhydration and growth was compared between carnations grown in mist reactors and conventional semisolid micropropagation systems (vented or unvented GA7 culture boxes). Plants grown in the mist reactor with long misting times (10 min h(-1)) produced more dry mass than those grown with <10 min h(-1); however, more misting also produced more hyperhydrated plants (70% hyperhydration). Control of hyperhydration in the mist reactor involved either reducing the overall nutrient mist supply or altering the mist supply throughout the culturing period. Stepped decreases in the mist supply throughout the 3-week period or an overall decrease in the duration of misting reduced hyperhydration to 13% and 5%, respectively. However, for both misting regimes, the biomass of normal (healthy) plants (fresh and dry weights) was limited. Further analysis suggested that, although normal plant biomass increased with longer mist exposure, hyperhydration levels also increased while the water content, based on percent dry weight, approached that of hyperhydrated plants. Sufficient normal plant development (fresh weight, leaf and shoot numbers, height, and rooting) with < 50% hyperhydration was obtained by weekly, stepped increases in the nutrient mist supply.     

香石竹叶片离体再生体系的建立

以香石竹(Dianthus caryophyllus Linn.)无菌苗叶片为外植体,从不同细胞分裂素及其他激素配合使用等方面进行筛选,建立香石竹叶片离体再生体系.结果表明,不同的细胞分裂素影响叶片不定芽分化频率,其中较低浓度的6-BA(0.5 mg·L-1)和TDZ(0.001 mg·L-1)配合使用能有效诱导香石竹叶片不定芽分化;添加一定浓度的PP333(4 mg·L-1)可提高叶片不定芽分化频率和平均芽数.香石竹叶片不定芽分化的适宜培养基为:MS 0.002mg·L-1TDZ 0.5 mg·L-16-BA 0.2 mg·L-1IAA 4 mg·L-1PP333;壮苗培养基为:MS 0.2 mg·L-1 6-BA 0.2 mg·L-1IAA;生根培养基为:1/2 MS.不定芽诱导频率达到42.61%,平均芽数为4.53个.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.Abbreviations BAP 6-benzylaminopurine - Kinetin 6-furfurylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.     

Survival of carnation (Dianthus caryophyllus L.) shoot apices frozen to the temperature of liquid nitrogen

Effects of cooling and rewarming rates on the survival of carnationshoot apices frozen to the temperature of liquid nitrogen wereinvestigated. Ten percent dimethyl sulfoxide (DMSO), alone orin combination with 5% glucose, sucrose or sorbitol was mosteffective as a cryoprotectant for carnation shoot apices. Theshoot apices survived slow freezing at about –70?C inthe presence of 10% DMSO. About 80% of the shoot apices survivedfreezing at the temperature of liquid nitrogen after prefreezingat –50?C or below, regardless of the rewarming rates.Shoot apices in the presence of 10% DMSO were cooled at differentrates then rewarmed rapidly. The survival rate gradually decreasedto zero as the cooling rate increased from about 0.5?C/min to50?C/min. At cooling rates higher than 50?C/min, no survivalwas observed even at 5?10⁴?C/min. However, in apices prefrozenat –15?C or below then cooled ultrarapidly at 10⁴?C/min,all remained alive with subsequent rapid rewarming. These apicesdeveloped normal young plants. This ultrarapid cooling methodcombined with prefreezing seems to be useful for the cryopreservationof shoot apices from various plants. ¹Contribution No. 2207 from the Institute of Low TemperatureScience, Hokkaido University. This work was supported in partby a Grant-in-Aid (No. 434035) for Scientific Research fromthe Ministry of Education, Science and Culture. (Received November 13, 1979; )     

Sources of silicon influence photosystem and redox homeostasis-related proteins during the axillary shoot multiplication of Dianthus caryophyllus

The present endeavor has demonstrated the impacts of different sources of silicon (Si) such as potassium silicate (K₂SiO₃) and calcium silicate (CaSiO₃) during the in vitro axillary shoot multiplication of carnation. For the Si treatments, nodal explants were cultured onto the Murashige and Skoog’s medium fortified with 1.0 mg L^?1 of 6-benzyladenine and 0.5 mg L^?1 indole-3-acetic acid with or without K₂SiO₃ and CaSiO₃ in three different concentrations (0, 1.8, or 3.6 mM). After six weeks, the shoot induction ratio, number of shoots produced per explant, expression of photosystem (PS) I and II core proteins, and activities of antioxidant enzymes were examined. Among the Si sources, K₂SiO₃ application enhanced the axillary shoot multiplication and the uptake of Si on comparison with CaSiO₃. Both forms of Si resulted in the enhancement of stomatal density, and PS-related protein such as PsaA and PsbA illustrating the apparent involvement of Si on the photosynthetic process. Nevertheless, addition of Si improved the antioxidant capacity during the in vitro shoot multiplication. Overall, the outcomes of the present study suggested that Si can be utilized as a supplementary source during the in vitro propagation of carnation.     

Characterization of arginine decarboxylase from Dianthus caryophyllus.

Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.     

香石竹毛状根诱导、离体培养及其植株再生

为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。     

香石竹雌蕊特征新发现

对于香石竹雌蕊心皮数目,不同植物志说法略有不同,目前尚无定论。本文对不同花色的香石竹雌蕊心皮数目及形态特征进行观察,发现香石竹的心皮数目随品种变化而异。在两种基本的花形态中,心皮数目存在不同的变异程度。文章讨论了其可能的形成机理,为进一步深入了解提供参考。     

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

 In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l^–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l^–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l^–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l^–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex. Received: 2 February 2000 / Revision received: 30 March 2000 / Accepted: 22 June 2000     

Regeneration of plants using nutrient mist culture

Summary A nutrient mist was used forin vitro culture of plant tissue in a novel bioreactor, wherein the tissues were grown on a biologically inert screen within a sterile chamber which allows excess media to drain away from the tissue. Plants tested includedDaucus, Lycopersicon, Ficus, Cinchona, andBrassica. The latter 4 genera were fully regenerated within the bioreactor. Tissue inocula included callus, anthers, and shoot meristems. All plants grew at least as well in nutrient mists as in agar and always produced a greater quantity of shoots of a higher quality and often faster than agar cultures. Cost analysis estimates showed up to a 65% savings in production costs (labor and materials) could be realized using nutrient mist culture. Nutrient mist culture offers significant improvements in the micropropagation of plants.     

Recovering vitrified carnation (Dianthus caryophyllus L.) shoots using Bacto-Peptone and its subfractions

Vitrified shoots regenerated from carnation petals (Dianthus caryophyllus L. cv. Scania) were recovered by culturing them in a medium containing 3.0 g/l Bacto-Peptone. Wax structures not found on vitrified shoots developed on the abaxial surface of leaves of recovered shoots and on those of normal leaves. Recovered shoots were rooted and successfully acclimatized while vitrified shoots could not survive the acclimatization process. The Bacto-Peptone solution was fractionated and the efficiency of each fraction for the recovery of vitrification was examined. Only basic, non high molecular fractions whose molecular weight was less than 10,000 were effective.     

Micropropagation of Dianthus caryophyllus L. — Control of Vitrification

Vitrification is a morphological and physiological disorder affecting in vitro regenerated plants. Vitrified shoots of carnation (Dianthus caryophyllus L.) regenerated from cultured cotyledons were abnormally glassy, thick and bushy with wider translucent leaves. Such vitrified shoots were recovered by culturing them on a medium supplemented with GA₃. Differentiation of shoot buds from the cultured cotyledons of D. caryophyllus occurred on MS medium supplemented with BAP (2.0 mg/l). Shoot buds subcultured on the same medium resulted in further prolific development of shoot buds and bushy shoot growths. Key words: carnation, shoot morphogenesis, micropropagation, cotyledons vitrification.     

保鲜剂对香石竹切花的保鲜效应

研究了保鲜剂(4％蔗糖 200 mg／L8-HQ 50 mg／L6-BA及4％蔗糖 0．1％明矾 0．02％尿素 0．02％NaCl)对香石竹切花的保鲜效应。结果表明,保鲜剂4％蔗糖 200 mg／L8-HQ 50 mg／L6-BA能明显地缓解切花水分胁迫,改善体内水分平衡,延缓切花衰老,延长切花的寿命。     

Genetic control of chalcone isomerase activity in flowers of Dianthus caryophyllus

In flowers of Dianthus caryophyllus (carnation), the gene I is concerned with a discrete step in flavonoid biosynthesis, Genotypes with recessive (ii) alleles produce yellow flowers, which contain the chalcone isosalipurposide (naringenin-chalcone-2-glucoside) as the major petal pigment, but in genotypes with wild-type alleles flavonols and anthocyanins can be formed and the flowers are white or red. Enzymatic measurements on petal extracts of four strains with different flower coloration revealed a clear correlation between accumulation of chalcone in recessive genotypes and deficiency of chalcone isomerase (E.C. 5.5.1.6) activity. From the chemogenetic and enzymological evidence it can be concluded that naringenin-chalcone is the first product of the synthesis of the flavonoid skeleton and that only the conversion of naringenin-chalcone to naringenin furnishes the substrate for the further reactions to flavonol and anthocyanin.These investigations were supported by a grant from Deutsche Forschungsgemeinschaft.     

康乃馨茎段愈伤组织诱导及植株再生(简报)

以康乃馨无菌苗茎段为外植体,在MS 6-BA 0.5mg/L NAA0.2mg/L培养基上诱导产生愈伤组织,愈伤组织在MS 6-BA 0.5mg/L NAA 0.2mg/L培养基上诱导芽效果较好,芽苗在1/2MS NAA 0.1mg/L培养基上可诱导生根。     

Flavonoids from carnation (Dianthus caryophyllus) and their antifungal activity

A flavonoid glycoside, kaempferol 3-O-β-d-glucopyranosyl (1 → 2)-O-β-d-glucopyranosyl (1 → 2)-O-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (1), along with two known C- and O-flavonoid glycosides (2 and 3, respectively), were isolated from carnation (Dianthus caryophyllus). The structures of the isolated compounds have been elucidated unambiguously by UV, MS, and a series of 1D and 2D NMR analyses. The isolated compounds and other flavonoid glycoside analogues exhibited antifungal activity against different Fusarium oxysporum f.sp. dianthi pathotypes.     

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.