Direct PCR detection of phytoplasmas in experimentally infected insects

Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)-infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl-trimethyl-ammonium-bromide (CTAB), and the other was boiling in Tris-EDTA buffer. The chromosomal primers amplified phytoplasma-specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20^oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.     

A comparative analysis of genetic diversity in medicinal Chrysanthemum morifolium based on morphology, ISSR and SRAP markers

The diversity and genetic relationship among 29 populations of Chrysanthemum morifolium, one of Chrysanthemum indicum and one of Chrysanthemum nankingense from China were analyzed using morphological traits and molecular markers. Twenty morphological traits were scored as well as 182 ISSR marker-fragments, as amplified by 22 primers [the percentage of polymorphic bands (PPB): 81.87%], and 243 SRAP marker-fragments as generated by 26 primer pairs (PPB: 75.72%). Mantel’s test indicated significant correlation (r = 0.624) of morphological trait and SRAP. By contrast, the morphological trait showed low correlation with ISSR (r = 0.246). Cluster analysis showed groupings of the accessions according to all four methods correlated well with their geographic region of origin, and most populations from the south of China were classified into one cluster and most populations from the north of China were classified into another cluster. Finally, an appropriate strategy for conserving the C. morifolium germplasm was proposed.     

Genotype Screening of Recipient Resources with High Regeneration and Transformation Efficiency in Chrysanthemum

Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum, which relies on an optimal regeneration and transformation system. However, the regeneration system of different chrysanthemum cultivars varies, and the regeneration time of most cultivars is long. To screen cultivars with highly efficient regeneration, leaves and shoot tip thin cell layers (tTCL) from eight chrysanthemum cultivars with different flower colors and flower types were cultured on Murashige and Skoog media (MS) supplemented with 1.0–5.0 mg L⁻¹ 6-benzylaminopurine (6-BA) and 0.1–1.0 mg L⁻¹ α-naphthaleneacetic (NAA). The results showed that the most efficient regeneration media were MS + 6-BA 1.0 mg L⁻¹ + NAA 0.5 mg L⁻¹ for leaf explants and MS + 6-BA 5.0 mg L⁻¹ + NAA 0.1 mg L⁻¹ for tTCL explants. Subsequently, another 13 chrysanthemum cultivars were screened by using the media, and finally, three cultivars with high regeneration efficiency were obtained from 21 cultivars. Among these, C1 had the highest regeneration efficiency: the regeneration rate of leaf explants reached 80.0% after 42 days of culture, and the regeneration rate of tTCL explants reached 100% after 31 days of culture. Furthermore, we also established the transformation system for C1 as follows: preculturing for one day, infecting with Agrobacterium suspension (OD₆₀₀ = 0.6) for 10 min, and cultivating in the regeneration medium with 350 mg L⁻¹ carbenicillin and 10 mg L⁻¹ kanamycin, thus ultimately achieving a transformation rate of 4.0%. In this study, a new chrysanthemum cultivar with an efficient regeneration and transformation system was screened, which is beneficial to enrich the flower color of chrysanthemum transgenic plant recipients and to the functional research of flower color or type-related genes.     

An NADPH and FAD dependent enzyme catalyzes hydroxylation of flavonoids in position 8

Yellow flavonols contribute to flower pigmentation in Asteraceae. In contrast to common flavonols, they show additional hydroxyl groups in position 6 and/or 8 of the aromatic A-ring in addition to the basic 5,7-hydroxylation pattern. An enzyme introducing a hydroxyl group in position 8 of flavonols and flavones was demonstrated for the first time with enzyme preparations from petals of Chrysanthemum segetum. Flavanones, dihydroflavonols and glucosylated flavonols and flavones were not accepted as substrates. The enzyme was localized in the microsomal fraction and uses NADPH and FAD as cofactors. Experiments with carbon monoxide/blue light and with antibodies specific for cytochrome P450 reductase did not indicate the involvement of a classical cytochrome P450 dependent monooxygenase in the reaction. Thus, the flavonoid 8-hydroxylase represents a novel type of hydroxylating enzyme in the flavonoid pathway. Apart from flavonoid 8-hydroxylase activity, the presence of all enzymes involved in the formation of flavonoid 7-O-glucosides in C. segetum was demonstrated. The pathway leading to 8-hydroxyflavonoids in C. segetum has been derived from enzyme activities and substrate specificities observed.     

几种菊花花器培养及愈伤组织分化频率研究

在诱导愈伤组织培养基上,25℃,每天12h,1500L光照条件下培养不同品种菊花花器官（花瓣、花萼、花托、雌雄蕊,开放前花蕾）,产生愈伤组织。再在分化培养基上,25℃±2℃、2000L全光照条件下,诱导芽的分化,再生植株．不同品种、不同花器官的分化频率不同,而且经此产生的再生植株,与原品种相比,在生长期内形态、花期、叶形等方面,产生了一系列变异．     

菊花不同花期及花序不同部位香气成分和挥发研究

以切花菊品种‘神马’为试材,采用顶空-固相微萃取和气相色谱-质谱联用(GC/MS)技术,分别测定菊花不同花期及花序不同部位的香气成分种类和含量,并利用生物显微镜观察花瓣的表皮细胞和横切面组织细胞的结构特征。结果表明:(1)菊花花蕾期共检测到香气成分24种,始花期31种,盛花期43种,终花期22种;随着花朵的开放和凋谢,酮类、萜烯类和醇类化合物的含量呈先上升后下降的趋势,在盛花期含量达到最高,而酯类、醛类和杂环类化合物则呈持续下降的趋势。(2)盛花期,在舌状花中共检测到香气成分种类31种,在管状花中共检测到50种;舌状花对菊花香气的贡献比管状花大;菊花舌状花由内轮向外轮香气成分种类变化不大,但是同类香气成分含量的变化出现由内轮向外轮逐渐减少的趋势。(3)异环柠檬醛、桉叶醇、α-蒎烯、β-金合欢烯和石竹烯等化合物可能为菊花的主要特征香气成分。(4)显微观察结果表明:舌状花的香气可能是通过表皮细胞间隙释放的,上表皮是菊花释放香气的主要部位。     

杭白菊茎尖组织培养及试管苗繁殖技术研究

采用茎尖组织培养技术,建立了杭白菊中大洋菊(Chrysanthemum morifolium Ramat.)的无菌试管苗体系.通过基本培养基和激素配比实验,筛选出杭白菊试管苗快速繁殖的最佳培养基组成.结果表明:最适宜的外植体为直径0.3 mm的茎尖;诱导丛生芽的最适培养基为:MS 6-BA 0.1 mg*L-1 IAA 0.02 mg*L-1;诱导试管苗生根的最适培养基为:1/2MS IAA 0.7 mg*L-1.用电子显微镜进行病毒检测后,筛选出2个脱病毒株系,脱病毒试管苗可作为今后提供优质种苗的种源.     

菊花病虫害综合防治研究

系统调查鉴定了菊花病毒病、霜霉病、叶斑病、叶面害虫、蚜虫天敌种类及其流行规律,研究了组培脱毒苗、地膜覆盖移栽、与高秆作物套作、摘顶稍、利用昆虫天敌及化学防治等综合治理效果.测定了农药残留量.结果表明,菊花组培苗脱毒率在60%左右,脱毒苗增产50%以上,叶枯病、霜霉病病叶率下降28%～30%,虫口密度下降40%,在收菊花前1个月使用拟菊酯杀虫剂,农药残留量低于允许值,不会对菊花和环境造成污染.     

A protoplast to plant system for the chrysanthemum Dendranthema zawadskii x D. grandiflora

Summary Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - MS Murashige and Skoog (1962) - NAA 1-naphthylacetic acid - Pfr Photon fluence rate - V-KM Binding and Nehls (1977)