Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry

Stilbenes are grapevine phytoalexins. These highly fluorescent molecules are generally analyzed by HPLC. This technique allows accurate assay of different stilbenes, but it is destructive, time-consuming, and neglects their spatial distribution. This is why we have tested a new method based on in vivo fluorescence using commercial spectrofluorometers that allowed fast and local assessment of stilbene content in grapevine leaves. Stilbene synthesis in grapevine Vitis vinifera var. Muscat Ottonel leaves was induced by Plasmopara viticola inoculation or UV-C irradiation. Fluorescence was measured both from the abaxial and adaxial sides of leaves, then stilbene content was analyzed by HPLC. It varied from 0 in control leaves to 15 mg g-1 dry weight in UV-treated leaves. Highly significant regressions were found between HPLC stilbene content and the corresponding leaf UV-induced blue fluorescence. Thus, in vivo fluorescence is a good tool for a rapid study of stilbenes synthesis in grapevine leaves that can potentially be extended to other fluorescent molecules.     

Stilbene levels in grape cane of different cultivars in southern Chile: determination by HPLC-DAD-MS/MS method

Health benefits of trans-resveratrol and other stilbenes in grapes, must, and wine have been pointed out by numerous authors. Less attention has been paid to the presence of stilbene derivatives in viticultural residues, such as grape canes. The present work reports the first results of a systematic study of stilbene levels in different grape varieties and cultivation areas in Chile, to evaluate their potential as an alternative source of bioactive stilbenes. In all cane samples, the predominant stilbene is trans-resveratrol, followed by ε-viniferin and piceatannol. In canes of Pinot noir up to 5590 ± 172 mg kg(-1) of trans-resveratrol and up to 6915 ± 175 mg kg(-1) of total stilbenes were detected. The observed concentrations of stilbenes in canes of Pinot noir from southern Chile until now are higher than those reported previously for this red variety. However, the highest concentration of total stilbenes observed in the analyzed samples was in the canes of white variety Gewürztraminer with 7857 ± 498 mg kg(-1). Preliminary results indicate that these levels can evolve if canes are left for some months on the vineyard after pruning, observing an increase during the first 2 months and a decrease after this period.     

彩叶芋薄层培养及胚状体发生的组织学研究

以彩叶芋叶柄薄层组织为外植体,接种在附加2.0 mg,L BA 和0.05～0.1 mg/L NAA 的 MS 培养基上,诱导愈伤组织和胚状体的发生。培养4周后,外植体形成瘤状愈伤组织。培养8周后,从愈伤组织分化出胚状体,胚状体进一步发育成正常植株。经12周培养可再生出大量植株。组织学观察表明,其胚状体的发育和结构与典型单子叶植物合子胚的特点很相似。从一至几个细胞原胚、球形胚,至逐渐发育成棒状时,便开始胚芽、胚根、胚轴和子叶的分化,即形成完整的胚状体。胚状体的发育不同步,因而在同一块愈伤组织上可见到不同发育时期的胚状体。愈伤组织内部产生胚状体多于表面。     

Determination of stilbenes (delta-viniferin, trans-astringin, trans-piceid, cis- and trans-resveratrol, epsilon-viniferin) in Brazilian wines

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. We describe a method for the direct determination of stilbenes in several red wines using high-performance liquid chromatography with UV detection. In a survey of 12 commercial wines from the south of Brazil (Rio Grande del Sul), levels of delta-viniferin are reported for the first time in different varieties of red wines. Brazilian red wine contains trans-astringin, trans-piceid, trans-resveratrol, cis-resveratrol (in high quantity: 5 times more than the trans form), epsilon-viniferin, and a compound isolated for the first time in wine, trans-delta-viniferin. Isolation and identification of delta-viniferin was achieved by NMR after extraction and fractionation of red wine phenolics. delta-Viniferin contributes, as well as cis-resveratrol and trans-piceid, to a significant proportion of stilbenes in wine dietary intake, particularly with Merlot varieties containing an average level of 10 mg/L for delta-viniferin, 15 mg/L for cis-resveratrol, and 13 mg/L for trans-piceid. The total stilbene intake from wine origin was estimated for the Brazilian population as 5.3 mg/day per person (on the basis of a regular wine consumption of 160 mL/day). delta-Viniferin can contribute to around 20% of total stilbenes in wine (average of 6.4 mg/L in red Brazilian wines). It would be important in the future to investigate the origins of the differences in wine stilbene levels in relation to the vine varieties, and the bioavailability of the newly extracted stilbene delta-viniferin in plasma after consumption of different types of wines.     

Hypericin and hyperforin production in St. John's wort in vitro culture: influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens

Influence of saccharose in the presence or absence of polyethylene glycol (PEG), methyl jasmonate, and an inactivated bacterial culture of Agrobacterium tumefaciens in cultivation medium on morphology of Hypericum perforatum L. and production of hypericin and hyperforin was studied under in vitro conditions. Production of hypericin and hyperforin was influenced by the presence of different concentrations of saccharose (10-30 g L(-1)) in cultivation medium. Addition of PEG (1.25-5 g L(-1)) in the presence of saccharose (10-30 g L(-1)) increased production of hypericin and hyperforin in the H. perforatum in vitro culture. Synthesis of hypericin and hyperforin was unchanged or reduced for most of the experimental plants at higher contents of PEG (10 and 15 g L(-1)). Concentrations of hypericin and hyperforin in the H. perforatum were on the order 100 and 103 microg g(-1) of dry plant material, respectively. Production of hypericin and hyperforin was stimulated either in the presence of a chemical elicitor (methyl jasmonate) or an inactivated bacterial culture of A. tumefaciens. Morphological changes induced by the abovementioned substances were observed and described in detail. The obtained results will be applied in experimental botany and in the technology of H. perforatum cultivation for pharmaceutical applications.     

Postharvest stilbene-enrichment of red and white table grape varieties using UV-C irradiation pulses

The red table grape varieties Flame, Red Globe, Crimson, and Napoleon, as well as the white varieties Superior, Dominga, and Moscatel Italica, were irradiated with a previously optimized UV-C postharvest irradiation protocol (510 W, 40 cm, 60 s). The induction kinetics of the stilbenes trans-resveratrol, trans-piceid, trans-piceatannol, trans-astringin, and viniferins was followed by using HPLC-DAD/MS/MS. The most inducible stilbenes were trans-resveratrol, trans-piceatannol, and viniferins. Both quantitative and qualitative differences were observed in both the stilbene induction kinetics and stilbene content in the seven table grapes analyzed here. The total resveratrol content ranged from 0.69 mg/100 g fw (Dominga) to 2.3 mg/100 g fw (Red Globe). The net resveratrol induction ranged from 3.4-fold (Flame) to 2315-fold (Red Globe). The highest viniferins content was observed in the variety Flame (0.73 mg/100 g fw), although the variety Red Globe presented the highest viniferins induction (175-fold). The highest content and induction of piceatannol (0.17 mg/100 g fw and 173-fold, respectively) was observed in the variety Flame. It should be stressed that taking into account the health-beneficial effects claimed for stilbenes, the UV-C irradiated table grapes can be considered as new functional fruits that can supply (a serving of unpeeled 200 g table grapes) the resveratrol content (depending on the variety) equivalent to more than seven glasses of red wine ( approximately 1.5 L) with high resveratrol content.     

菊苣高效不定芽直接发生及其植株再生

本研究以菊苣无菌苗叶片为外植体,建立了高效不定芽直接发生及其植株再生体系。在附加不同浓度N-6-benzyladenine(6-BA)或与低浓度α-naphthaleneacetic acid(NAA)组合的MS培养基上,5~7d外植体表面不经过愈伤组织诱导阶段,直接形成不定芽。组织学观察表明,不定芽起源于叶片维管束薄壁细胞,且其微管组织系统与叶片外植体内微管组织系统紧密相连。6-BA是不定芽直接发生所必需的,外植体的发育时期、取材部位和培养基蔗糖浓度对不定芽直接发生有重要影响。在附加2.0mg/L 6-BA,0.5mg/L NAA,100mg/L Vc,100mg/L VB1,300mg/L脯氨酸和40g/L蔗糖的MS培养基上,培养20d龄基部叶片15d时,不定芽直接发生频率最高为100%,每块外植体上产生的不定芽数量也最多,平均为36~38个。在1/2 MS+IBA 0.5mg/L培养基上,再生苗诱导生根频率为97.58%,再生植株移栽于盆土中,100%存活且生长良好,未见形态异常。     

观赏凤梨胚性愈伤组织的诱导、高频再生及超微结构研究

以观赏凤梨‘名宝剑’的短缩茎为外植体,研究了不同激素及浓度组合对愈伤组织诱导和植株分化的影响,并通过形态学和组织细胞学观察了不同类型愈伤组织的超微结构和胚性愈伤组织的分化过程。结果表明：NAA对愈伤组织诱导的影响达到极显著水平（P=0.0019）,其中0.5mg/L NAA最有利于提高愈伤组织的诱导率和胚性愈伤组织的比...     

Effect of jasmonates on ethylene biosynthesis and aroma volatile emission in Japanese apricot infected by a pathogen (Colletotrichum gloeosporioides)

The effects of the application of the jasmonic acid derivative n-propyl dihydrojasmonate (PDJ) on ethylene biosynthesis, volatile compounds, and endogenous jasmonic acid (JA) and methyl jasmonate (MeJA) were examined in Japanese apricot (Prunus mume Sieb.) infected by a pathogen (Colletotrichum gloeosporioides). The fruit were dipped into 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 °C for 6 days. The inoculation induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene, JA, and MeJA. In contrast, PDJ application reduced the endogenous JA, MeJA, and ethylene production and expression of the ACC oxidase gene (PmACO1) caused by the pathogen infection. The lesion diameter with C. gloeosporioides decreased upon PDJ application. The alcohol, ester, ketone, and lactone concentrations and alcohol acyltransferase (AAT) activity increased in the pathogen-infected fruit, but were decreased by PDJ application. These results suggest that PDJ application might influence ethylene production through PmACO1 and that aroma volatile emissions affected by pathogen infection can be correlated with the ethylene production, which is mediated by the levels of jasmonates.     

箭根薯的试管繁殖

本实验用箭根薯成熟种子为材料，培育出无菌幼苗，再用幼叶、叶柄进行试管繁殖。经实验得出各阶段适宜的培养基分别为：（1）种子萌了：MS＋6－BA 1mg/L(单位下同）＋NAA 0．1；（2）诱导愈伤组织：MS＋6－BA 1＋2，4－D 1．5＋KT0．2；（3）丛芽诱导：MS＋6－BA0．5＋NAA0．5；（4）生根培养：1／2MS＋NAA0．5。     

虎杖茎尖离体再生体系的建立和优化

研究了苗龄、接种方式、不同植物生长调节剂、硝酸银和蔗糖等对虎杖(Polygonum cuspidatum)茎尖愈伤组织诱导、不定芽分化和生根的影响。结果表明:愈伤组织诱导的最佳培养基是MS+蔗糖28g/L+琼脂5.5g/L+2,4-D 1.5mg/L+6-BA 1.0mg/L,诱导率为100%;3-7d苗龄的虎杖茎尖愈伤组织诱导能力无显著差异,诱导率均达到95%以上,此后随着苗龄的增加,愈伤组织诱导能力快速下降,苗龄为12d时愈伤组织诱导率只有55.6%;以正插(形态学下端插入培养基)方式接种的外植体愈伤组织诱导率显著大于反插(形态学上端插入培养基)和平放的;不定芽分化最佳培养基为MS+AgNO34.0mg/L+蔗糖30 g/L+琼脂6.0g/L+NAA 0.5mg/L+TDZ 0.8mg/L,分化率为83.9%,增殖系数为7.63;生根培养基选用1/2 MS+蔗糖26g/L+琼脂6.5g/L+活性碳3%+IBA 0.2mg/L+NAA 0.3mg/L,生根率为100%。用透气膜封口比用聚乙烯菌膜生根率明显提高,生根明显提前。     

Chemical composition and antioxidant property of holy basil (Ocimum sanctum L.) leaves, stems, and inflorescence and their in vitro callus cultures

In this study, the chemical constituents and antioxidant property of holy basil (Ocimum sanctum Linn.) field-grown plant parts (leaves, stems, and inflorescence) were compared with those of respective callus cultures induced from each explant in in vitro. The callus cultures were successfully initiated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) (1 mg/L) combined with different concentrations (0.1-0.5 mg/L) of kinetin as plant growth regulators. The distribution of phenolic compounds in these extracts was analyzed using reverse phase high-performance liquid chromatography with reference standards. Interestingly, rosmarinic acid (RA) was found to be the predominant phenolic acid in all callus extracts in comparison with field-grown plant parts. In this study, the antioxidant activity of the extracts was evaluated with six different in vitro antioxidant-testing systems. Their activities of scavenging superoxide anion radicals, 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), hydroxyl radicals, hydrogen peroxide, chelating ferrous iron, and ferric ion reducing potential were assessed. The antioxidant activity was increased in all testing systems with increasing amounts of extract. However, at the same concentration, the callus extracts exhibited higher antioxidant activity in all of the testing systems than the extract obtained from field-grown plant parts. The data obtained from this study suggested the possibility of the isolation of a high content of RA from in vitro callus cultures rather than field-grown plant organs of holy basil.     

红花小孢子培养与胚状体诱导研究

小孢子培养是创造单倍体和双单倍体的重要途径,对提高红花的育种效率具有重要意义。为了研究红花游离小孢子的高效培养体系,用20份红花材料研究花药及小孢子培养中影响愈伤组织生长和胚发生的关键因素。结果表明,小孢子发育时期、红花品种、基本培养基和激素浓度是影响红花诱导愈伤组织的关键因素。花药培养的愈伤及胚诱导率高于游离小孢子,且受基因型影响。单核靠边期管状花长度为 0.45～0.50 cm时,诱导成功率达到68.32%。供试的20份红花材料中,有11份诱导出了愈伤组织,有1份分化出了胚状愈伤,诱导成功率为15.79%。分化培养最适培养基组合为1/2 MS+B5+ 6-BA 1.0 mg/L + NAA 1.0 mg/L;小孢子悬浮培养最适培养基激素浓度以添加6-BA 4.0 mg/L 和NAA 0.5 mg/L效果最好,诱导愈伤率为22.5%。     

小麦幼穗离体培养高效再生系统的建立

选用3个冬小麦(TriticumaestivumL)材料H6756、H311和SP8581,研究了不同取材时期、不同诱导培养基、分化培养基和生根培养基对小麦幼穗离体培养的影响。结果表明,在护颖原基形成期至雌雄蕊原基形成期之间对小麦幼穗进行离体培养都可获得较高的绿苗分化率,而在其它时期进行离体培养绿苗分化率均较低。确定了小麦幼穗离体培养的最佳诱导培养基为MS 2,4-D2mg/L,最佳分化培养基为MS培养基,并发现对分化过程中产生的变态苗增加一个芽苗的诱导及伸长阶段有助于继续分化成苗。最佳生根培养基为1/2MS 0.2mg/LIAA 80g/L蔗糖,在此基础上建立了小麦幼穗离体培养的高效再生系统。     

辐照西洋参培养物皂苷次生代谢调控的研究

研究了不同前体、前体浓度、前体加入时期、前体作用时间、ATP浓度、烟酸浓度对西洋参芽胞愈伤组织生长及皂苷合成的影响。结果表明 :0 1 %甲羟戊酸对生长有抑制作用 ,对皂苷合成有明显的促进作用 ;1 0 0mg L亮氨酸对生长无明显影响 ,对皂苷合成有促进作用 ;444mg L的醋酸镁对培养物生长和皂苷合成都有促进作用 ;在继代开始时加入前体皂苷含量最高 ;甲羟戊酸作用 6h皂苷产量最高 ;1 60mg LATP对生长和皂苷合成均有促进作用 ;1 0mg L烟酸利于培养物生长和皂苷合成 ;用6 0 Coγ射线辐照培养物 ,剂量为 40 0 0Gy时得到皂苷含量高于原愈伤组织 1倍的细胞系。     

Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid

Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.     

日本臭菘体细胞胚胎发生及植株再生体系的建立

以日本臭菘幼叶为外植体,应用均匀设计法筛选其最适合的胚性愈伤组织及胚性细胞复合体诱导、体细胞胚胎发育及植株再生的培养基配方,建立日本臭菘体细胞胚胎发生体系。结果表明,LS+6-BA 1.0 mg/L+2,4-D 2.0 mg/L+IAA 0.1 mg/L适宜日本臭菘幼叶胚性愈伤组织及胚性细胞复合体诱导,诱导率为98%;LS+6-BA 0.50 mg/L+NAA 0.10 mg/L+IAA 0.10 mg/L最适宜体胚发育及植株再生,体胚萌发率为100%,萌发的体胚在发育培养基上继续培养42 d后全部发育成完整植株。对不同阶段培养材料的形态结构及超微结构的观察证明,成功建立了日本臭菘体细胞发生及植株再生体系。     

糯玉米离体培养获取耐盐变异体

为了获取糯玉米耐盐变异体,以120mg/L的平阳霉素预处理糯玉米自交系（S1、S2）种子20h,萌发后用茎尖诱导胚性愈伤组织,比较了激素组合对胚性愈伤组织诱导、继代和分化的影响。结果表明2, 4-D 2.0mg/L和6-BA 0.25mg/L组合诱导胚性愈伤的效果最好;在添加100mg/L CH、6-BA 0.25mg/L和2, 4-D 2.0mg/L的继代培养基上愈伤组织生长良好。将胚性愈伤组织依次在含NaCl 10g/L、15g/L和20g/L培养基中进行筛选,获得耐盐变异体,转入分化培养基中诱导再生植株,自交1代种子在含NaCl 15g/L、20g/L的培养基上萌发率较对照种子高数倍,在20g/L NaCl的胁迫下,变异体自交1代种子幼苗叶片中脯氨酸含量及K+ / Na+比例明显高于对照。     

铁炮百合的胚性愈伤组织诱导和植株再生

以铁炮百合(又称麝香百合)杂种系品种‘White heaven’的组培苗叶片为外植体,研究了不同种类和浓度的激素对愈伤组织诱导和植株再生的影响,并通过叶片位置、接种方式以及光暗等不同处理进一步优化胚性愈伤组织的诱导。结果表明:以组培苗基部叶片为外植体,采用叶片近轴面向上的接种方式,在MS+1.0mg.L-12,4-D+0.1mg.L-16-BA的培养基上暗培养可诱导出胚性愈伤组织,诱导率可达65.74%;经石蜡切片鉴定可观测到球形胚和心形胚。最佳分化和生根培养基分别为MS+0.1mg.L-1NAA和1/2MS+0.5mg.L-1IBA,分化率达81.48%,生根率达91.67%。结果还显示2,4-D在百合胚性愈伤组织的诱导中起到了关键作用。     

水杨酸诱导下人参培养物差异表达基因片段的克隆

本研究以人参愈伤悬浮细胞为材料,在其生长的第28天添加1×10^-3mg／L水杨酸,测定水杨酸添加后,过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶等3种酶在72h内的变化及皂苷含量,结果表明：水杨酸添加后对过氧化物酶和苯丙氨酸解氨酶的活力影响最大,分别在24h和48h达到最大峰值,在18h开始影响多酚氧化酶的活力,培养物生长的第28天添加水杨酸可以明显提高人参愈伤组织中皂苷的合成。确定添加水杨酸后24h提取总RNA,进行cDNA-RDA分析,筛选差异片段。确定差异基因并在GenBank中注册,注册号为FE900130。为探讨水杨酸作为诱导子对人参次生代谢的影响奠定基础。