Demonstration of phenolic compounds in plant tissues by an osmium-iodide postfixation procedure

A simple procedure to stain phenols in plant tissues is described. Postfixation with an aqueous solution prepared by mixing 2 cc of 2% osmium tetroxide and 8 cc of 3% potassium iodide yields brilliant visualization of phenol-containing vacuoles in different tissues of plants (e.g., coffee, oak, tobacco and spruce) bearing high concentration of phenolic compounds. Areas bearing phenols become dark gray to black. Chemical experiments demonstrate that osmium-potassium iodide (Os-KI) mixture reacts rapidly with several naturally occurring plant phenols, developing black solutions from which black solids precipitate. Phenols containing omicron-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, omicron-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.     

Acettc-Orcein: A New Stain-Fixative for Chromosomes

A new stain-fixative method for chromosomes, namely acetic-orcein, is described, which gives results that are equally good in fresh and permanent preparations. A 45% acetic and 1% orcein content is recommended as a standard solution. For salivary glands of Drosopkila a 2% stain gives the best results, and with the two species D. melanogaster and D. miranda the acetic strength has been raised to 70% with advantage. The addition of chloroform proves necessary for hardening in species of Sciara. Acetic-orcein is equally good for rapid chromosome counts. For root tips the addition of 1 cc. of N HC1 solution to 10 cc. of the standard solution together with gentle heating of the tissues in a drop of the mixture assists in the softening and separation of cells necessary for chromosome study. Orcein can also be used successfully in other combinations such as acetic-propionic or acetic-lactic. The latter is useful for making preparations that do not require ringing. Preparations so made keep from 7 to 14 days.     

Responses of the stem borer larval endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) to plant derived synomones: Laboratory and field cage experiments

Laboratory and field cage experiments investigated the response of females of the stem borer larval endoparasitoid Cotesia flavipes to two synthetic synomone components, the terpenoid (E)-β-farnesene and the green leaf volatile, (Z)-3-hexenyl acetate, both compounds identified previously in headspace volatiles of maize plants damaged by stem borer (Chilo partellus). In dose response tests performed in a Y-tube olfactometer, parasitoids were significantly more attracted to the arms bearing 10 or 15 µg of (Z)-3-hexenyl acetate and (E)-β-farnesene than to the control arm. (E)-β-Farnesene was as attractive as the essential oil from the plant Hemizygia petiolata (Lamiaceae) rich in the same compound (80% relative amount). The plant essential oil elicited responses from females of the parasitoid comparable to those elicited by two positive controls, stem borer larval frass and adult parasitoid diet (20% honey solution), tested in the laboratory assays. In field cage trapping experiments, captures in traps baited with the terpenoid, the plant essential oil, (Z)-3-hexenyl acetate and the control of 20% honey solution, were not significantly different relative to captures in unbaited traps. Addition of the green leaf volatile (Z)-3-hexenyl acetate to the plant essential oil to yield a 1:1 two-component blend captured significantly more female parasitoids than traps baited with either of the two components alone. The results show that blends of green leaf volatiles and sesquiterpenoids may have potential in monitoring C. flavipes populations in the field.     

Staining Plant Tissues with Chlorazol Black E and Pianese III-B

The staining schedule was developed for a study of the mycorrhizae of red pine, Pinus resinosa Ait. From 70% alcohol, sections are stained in a saturated solution of chlorazol black E in 70% alcohol, 10-30 min; free dye removed by washing in 95% alcohol; stained 18-24 hr in Pianese III-b; rinsed in 95% alcohol, acidified by the addition of 2 ml of saturated aqueous picric acid per 100 ml, 3-4 changes or until the last change is pale yellow or light green; and rinsing in 95% alcohol to remove the acid. If the acid fuchsin is too intense, a cautious differentiation with 95% alcohol containing 1-3% of a 0.1 N solution of NaOH is made. If too much chlorazol black is removed, the effect can be compensated by overstaining with this dye at the beginning of the process. Sections are dehydrated, cleared, and covered in the usual manner. This stain has applications to plant tissues generally, and is particularly effective for meristematic tissues. It shows details of cytoplasmic structures and gives sharp delineation of primary cell walls.     

Alizarin Red S and Toluidine Blue for Differentiating Adult or Embryonic Bone and Cartilage

This technic has been successfully employed by the author for staining, in toto, the bones and cartilage of mature specimens of Urodela and the developing bone and cartilage of the embryonic human, cat, pig and rat. The differential staining is accomplished by using a modification of Dawson's method of staining bone with alizarin red S following a toluidine blue solution specific for cartilage. Specimens are fixed in 10% formalin, stained one week in a solution of .25 g. of toluidine blue in 100 cc. of 70% alcohol, macerated 5 to 7 days in a 2% KOH solution, counterstained for 24 hours in a 0.001% solution of alizarin red S in 2% aqueous KOH, dehydrated in cellosolve and cleared in methyl salicylate. In the adult and embryonic forms thus treated the soft tissues are cleared while the osseous tissue is stained red, the cartilage blue.     

A New Method of Silver Impregnation for Nerve Cells and Fibers

Fragments of tissue, immediately after death, are fixed in Debaisieux's modification of the Duboscq-Brazil picro-aceticformol fluid, and treated as follows: Hydrate by soaking 2-6 hr. in distilled water with 30 drops of cone. NH₄OH per 100 cc. Freeze and cut sections about 25μ in thickness. Bleach sections about 15 min. in ammoniacal water (52 drops cone. NH₄OH per 100 cc. water). Transfer to 20% AgNO₃ solution and heat at 45° C. till light brown. Add cone. NH₄OH drop by drop till the Ag precipitates and then redisolves into an opalescent solution. Pour solution and sections into a little distilled water and transfer sections quickly to formaldehyde solution (3 cc. formalin to 100 cc. water). Dip sections in distilled water and transfer to 1% aqueous gold chloride till deep blue. Place for about 10 minutes in 5% aqueous sodium thiosulfate solution for fixing and clearing. Wash thoroly in tap water, dehydrate and mount. Special directions are given for applying this technic to delicate material such as insects, and for use with serial sections.     

坡向与龄级对新疆野核桃自然保护区野核桃病害的影响

为了解渐危植物新疆野核桃的病害情况,在新疆野核桃自然保护区调查不同坡向、不同龄级野核桃4种病害的患病比例,分析病害种类、病害等级与野核桃胸径及坡向的相关关系。结果表明: 保护区野核桃的主要病害为核桃褐斑病(95.8%)、核桃枯枝病(90.5%)、核桃黑斑病(74.4%)和核桃腐朽病(7.7%)。4个坡向的野核桃均易患核桃褐斑病,阴坡和半阴坡的野核桃易患核桃枯枝病,阳坡和阴坡的野核桃易患核桃黑斑病,半阳坡和半阴坡的野核桃相对易患核桃腐朽病。4个坡向野核桃的4种病害均随病害等级(1～4级)的增加而病株比例减小。4个坡向核桃枯枝病、核桃黑斑病、核桃褐斑病均以中龄树比例最大,其次是老龄树,再次是小树,未见幼苗患病;核桃腐朽病仅发生在老龄树。核桃枯枝病、核桃腐朽病、核桃黑斑病、核桃褐斑病与野核桃的胸径呈显著正相关,核桃黑斑病与坡向呈显著负相关,核桃枯枝病、核桃腐朽病、核桃黑斑病的不同病害等级与胸径和坡向存在相关性。     

烟草内生真菌多样性和种群结构

从云南“世界烟草品种园”10种烟草的根、茎、叶中分离得到199株内生真菌,根据rDNA-ITS系统发育分析鉴定为17属25种,其中格孢腔菌目Pleosporales内生真菌的种类和数量最多;茎点霉属Phoma、链格孢属Alternaria和镰孢菌属Fusarium为主要优势属,相对频率分别为25.1%、24.6%和11.6%,优势度Y值分别为0.251、0.172和0.104。烟草不同组织内生真菌的种群结构存在显著差异,分离自根的内生真菌的主要优势属为Fusarium和Phoma,Y值分别为0.235和0.123;分离自茎的内生真菌的主要优势属为Phoma和Alternaria,Y值分别为0.186和0.155;分离自叶片的内生真菌的主要优势属为Alternaria,Y值为0.286。Phoma从烟草根茎叶中均可分离得到,而Alternaria只分布在地上部茎叶中,Fusarium只分布在根茎中,表明这3个优势属真菌对根茎叶组织的专化性不同。     

Possible uses of Dioxan in Botanical Microtechnic

Dioxan has been well established as an advantageous dehydrating agent for plant tissues. It dehydrates equally well after fixatives containing formalin, acetic acid, chromic acid, chromates, mercuric chloride, osmic acid, and alcohol. Better infiltration of paraffin after dehydration may be obtained by passing the material thru (1) a cold bath composed of 30 cc. of dioxan, 5 cc. of xylol and 20 cc. of melted soft paraffin and, (2) a warm bath of 50 cc. of dioxan, 50 cc. of paraffin, and 10 cc. of xylol. Transfer from (2) to soft paraffin. A dioxan fixative consisting of dioxan 50 cc., formalin 6 cc., acetic acid 5 cc., water 50 cc. was devised for delicate subjects. The fixed material is transferred directly into dioxan and mounted in dioxan-diaphane or dioxan-balsam. Very delicate objects require dioxan dilution of the balsam and slow concentration of the mounting medium by evaporation.

Entire plant parts or epidermal peelings are fixed in any desired fixative, washed if necessary, transferred to dioxan and mounted in diluted dioxan-balsam or diaphane. Dioxan may be used to mount hyalin objects whose refractive indexes approach those of balsam in media of higher index than balsam. It may be used in place of alcohol in finishing parafin sections, and since it exhibits different stain solubilities than alcohol it offers an important new tool in obtaining and maintaining stain balances.     

Current Status of the Stem-boring Weevil Listronotus setosipennis (Coleoptera: Curculionidae) Introduced Against the Weed Parthenium hysterophorus (Asteraceae) in Australia

Effects of the stem-boring weevil Listronotus setosipennis (Coleoptera: Curculionidae) on different growth stages of the weed Parthenium hysterophorus (Asteraceae) was evaluated in a glasshouse and field cages, and its field prevalence in Queensland, Australia was assessed. In the glasshouse, L. setosipennis reduced the plant height by 51%, number of leaves by 78%, flower production by 63% and plant biomass by 54% in rosette stage plants. Damage by L. setosipennis in rosette stage plants in the field cage reduced the primary stem height by 26% and flower production by 38%, but the impact on total plant height, basal stem width, root length, number of branches, root biomass and total plant biomass was not significant. In both glasshouse and field cages, the impact of L. setosipennis on preflowering and flowering stages of parthenium was not significant. L. setosipennis was recorded in 48% of the parthenium-infested sites (n=132) sampled and 16% of the sites showed high to very high levels of incidence. L. setosipennis was more prevalent on alluvial and black soils than on clay and sandy soils. L. setosipennis is a promising biocontrol agent for regions with prolonged dry periods and erratic rainfall pattern.     

Degradation of Atrazine by Hornwort in Aquatic Systems

Hornwort (Ceratophyllum deinersuin) incubated in the presence of 1 mg L^{-1 14}C atrazine in an aqueous nutrient solution became radioactive. Microscopic autoradiography was used to investigate the localization of ¹⁴C atrazine in a hornwort/epiphyte system. Radioactivity was present within the plant tissue (and also in the nutrient solution), with larger quantities in mature tissue, including stems compared with young tissue. An irregular distribution of black silver granules (which indicate radioactivity) was observable on the plant surface, suggesting the possible involvement of epiphytes (plant surface microorganisms) in the degradation process. Labeled compounds in the extracted plant included atrazine and a major metabolite that may have been an artrazine-glutathione connjugate. Concentrations of atrazine and the metabolite, and the fraction of the metabolite (based on total radioactivity), all in the extracted plant, were dependent on the initial atrazine concentration in the solution. The degradation process was light dependent and the analyses of the nutrient solution indicated that the first half-life of atrazine in the presence of hornwort was 5 days under day/night conditions, while only about 30% of initial atrazine disappeared after 3 weeks under complete dark. The major metabolite in the solution was identified as deethylatrazine.     

Iodophil Components of Insect Cuticle

Deparaffinized sections, after hydration, are successively immersed in the following aqueous solutions: 1% iodine in 2% potassium iodide (10 min), 1% silver nitrate (10 min), any fine-grain commercial photographic developer (10 min), 0.1% gold chloride (15 min), 5% sodium thiosulphate (2 min), and 1 % potassium metabisulphite acidified with a little hydrochloric acid (2 min). Thorough washing after each immersion, to ensure removal of all water-soluble material, is essential before proceeding to the next stage. Only chemically-clean staining vessels should be used. After dehydration, the sections may be mounted in any desired medium. Iodophil regions of the cuticle are stained deep purple to black; the remainder of the cuticle is unstained. Since the stain is formed by metallic deposits, the colors are permanent.     

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.     

Characterization and regulation of the vitamin D hydroxylases

The metabolism of vitamin D is regulated by three major cytochrome P450-containing h hydroxylases—the hepatic 25-hydroxylase, the renal 1-hydroxylase, and the renal and intestinal 24-hydroxylase. In the liver, the 25-hydroxylation reaction is catalyzed by microsomal and mitochondrial cytochrome P450cc25. The microsomal P450 accepts electrons from the NADPH-cytochrome P450 reductase, and the mitochondrial P450 accepts electrons from NADPH-ferredoxin reductase and ferredoxin. In the kidney, the 1- and 24-hydroxylation reactions are catalyzed by mitochondrial cytochromes P450cc1 and P450cc24, respectively. The 24-hydroxylase is also found in vitamin D target tissues such as the intestine. The rat hepatic mitochondrial P450cc25 and the rat renal mitochondrial P450cc24 have been purified, and their cDNAs have been cloned and sequenced. 1,25-Dihydroxyvitamin D, the active metabolite of vitamin D, markedly stimulates renal P450cc24 mRNA and 24-hydroxylase activity in the intact animal and in renal cell lines. This stimulation occurs via a receptor-mediated mechanism requiring new protein synthesis. Despite the availability of a clone, no studies have yet been reported of the regulation of hepatic P450cc25 at the mRNA level. The study of one of the most important enzymes in vitamin D metabolism, the renal 1-hydroxylase which produces the active metabolite, awaits the definitive cloning of the cDNA for the P450cc1.     

Genome size, base composition and karyotype of Jatropha curcas L., an important biofuel plant

In this report, we present the genome size, the base composition and the karyotype of Jatropha curcas L., which is becoming an important oleaginous crop in tropical areas for biofuel production. The genome size and the base composition were obtained by flow cytometry of G₀/G₁ nuclei stained with propidium iodide (for genome size), DAPI (for AT) and chromomycin A3 (for GC), respectively. The karyotype was obtained by root-tip (i) incubation with amiprophos-methyl (microtubule inhibitor), (ii) digestion in enzymatic solution, (iii) squashing on glass slides, (iv) fixation and (v) coloration in Giemsa solution. We found that the genome of J. curcas is relatively small and in the same size range as that of rice. The flow cytometry indicates an average 2C value of 0.85 pg and an average base composition of 38.7% GC. The karyotype of J. curcas is made up of 22 relatively small metacentric and submetacentric chromosomes whose size range from 1.71 to 1.24 μm. The possibility of a polyploidization event in the evolutionary history of J. curcas is discussed.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

Effects of arbuscular mycorrhizal fungi on calorific value and contents of carbon and ash in Robinia pseudoacacia

该试验以根内球囊霉(Glomus intraradices)和地表球囊霉(G. versiforme)为接种剂, 研究了丛枝菌根真菌对刺槐(Robinia pseudoacacia)生物量、热值、含碳量、灰分、能量积累和碳素积累的影响。结果表明, 接种根内球囊霉和地表球囊霉对提高刺槐生物量、热值、能量积累和碳素积累都起到了重要作用。接种根内球囊霉和地表球囊霉后刺槐的总生物量比对照分别增加了89.61%和91.34%, 能量积累分别比对照增加102.20%和94.19%, 碳素积累分别比对照增加93.30%和77.21%; 同时发现刺槐的能量和碳主要分布在根系和叶, 而茎中能量和碳所占的比例较小。接种根内球囊霉提高了刺槐的干重热值, 其根、茎、叶的干重热值分别比对照增加7.72%、8.94%和8.41%; 接种地表球囊霉也显著(p < 0.05)提高了刺槐的干重热值, 但其效果低于根内球囊霉。接种根内球囊霉显著(p < 0.05)提高了刺槐根的含碳量, 对茎和叶的含碳量影响不明显。接种根内球囊霉和地表球囊霉都显著(p < 0.05)提高了刺槐茎和叶的去灰分热值。     

Tannic Acid and Iron Alum with Safranin and Orange G in Studies of the Shoot Apex

A schedule is given for staining the cell walls of young plant tissues in tannic acid and iron alum after the protoplasts have been stained in safranin and orange G. Sections are placed for one minute in 2% aqueous ZnCl₂, and are then stained in a 1/25,000 aqueous solution of safranin O. From this they are placed for five minutes in a bath consisting of orange G (2 g.), tannic acid (5 g.). water (up to 100 cc.) and HC1 (4 drops). This is followed by five minutes in 5% aqueous tannic acid and two minutes in a 1% solution of iron alum. A brief rinse in tap water is given between each stage; the slides are raised and lowered about a dozen times at each change to ensure that the new solution reaches the material quickly. The method was originated for shoot apices but it also works excellently on more mature tissues and on adult material. It has the advantage of allowing extremely easy detection of protophloem in the strands even at the very onset of vascular differentiation.     

New Uses for Calcium Chloride Solution as a Mounting Medium

Fresh cross sections of stems [Psilotum nudum, Coleus blumei, and Pelargonium peltatum] and roots (Setcreasea purpurea) 120 μm thick were fixed in FPA₅₀ (formalin: propionic acid: 50% ethanol, 5:5:90, v/v) for 24 hr and stored in 70% ethanol. The sections were transferred to water and then to 1% phloroglucin in 20% calcium chloride solution plus either hydrochloric, nitric, or lactic acid in the following ratios of phloroglucin-CaCl₂ solution:acid: 25:4, 20:2, or 15:5. The sections were mounted on slides either in one of the three mixtures or in fresh 20% calcium chloride solution. A rapid reaction of the acid-phloroglucin with lignin produced a deep red color in tracheary elements and an orange-red color in sclerenchyma. Fixed and stored leaf pieces from Nymphaea odorata were autoclaved in lactic acid, washed in two changes of 95% ethanol, transferred to water, and treated with the three acid-phloroglucin-calcium chloride mixtures. The abundant astrosclereids stained an orange-red color similar to that of sclerenchyma in the sections. In addition, a new method is reported for specifically staining lignified tissues. When sections or leaf pieces are stained in aqueous 0.05% toluidine blue O, then placed in 20% calcium chloride solution, all tissues destain except those with lignified or partially lignified cell walls. Thus, toluidine blue O applied as described becomes a reliable specific test for lignin comparable to the acid-phloroglucin test.