Quantitative Estimation of Phloem Regeneration in Coleus Internodes

Phloem regeneration in Coleus internodes, earlier wounded so that one or more phloem bundles were severed, is estimated quantitatively by microscopic examination of permanent slides prepared in the following way: The wounded internode is removed from the plant after a given regeneration period, is fixed in Craf III for 24 hr and is transferred to 85% lactic acid for 12-24 hr. While still in lactic acid, a “strip”, which is composed of the phloem and all tissues peripheral to it in the internode, is peeled from the internode, leaving only the xylem-pith cylinder. The strip is stained for 6-12 hr in 0.1% aniline blue in 85% lactic acid, then is transferred to 60% alcohol containing 0.5% HCI. While in the latter solution, the epidermis, scar tissue, and most of the cortical tissue is carefully dissected from the strip while it is observed in a dissecting microscope. The strip is restained for an hour or more and is passed through two 5-10 min changes each of acidified 60% alcohol, absolute alcohol, and xylene, and is then mounted on a glass slide in damar-xylene. Counts of regenerated, interfascicular phloem strands, governed by a counting convention, which were shown to bear a fairly constant relationship to the actual number of regenerated sieve tube members, are made while examining under low and high power magnifications. This method is presently being used to study the physiology of phloem differentiation and its regulation in Coleus.     

云南20种蕨类植物叶表皮微形态特征研究

利用光学显微镜对云南20种不同科、属的蕨类植物的叶表皮形态进行了观察和研究。结果表明：它们的叶片表面存在不同的附属物,而有的种类无附属物;20种蕨类植物共具8种气孔器类型(极细胞型、腋下细胞型、辐射状细胞型、双不等细胞型、无规则四细胞型、共环极细胞型、聚腋下细胞型和不规则型), 它们在气孔器组成上具多型现象,气孔均为下生型,分布方式及大小多样;叶表皮细胞的微形态在科属间表现出一定的差异,主要有不规则型、多边型或规则条状,表皮细胞垂周壁呈波纹状、深波状或波浪状。上述研究结果为蕨类植物的系统分类及演化提供依据。     

Extractive fermentation of lactic acid by immobilizedLactobacillus casei using ion—exchange resin

Summary A novel method of lactic acid fermentation byLactobacillus casei immobilized in Ca—alginate gels is described, in which an ion—exchange resin packed column is attached to a fermentor for separation of lactic acid from fermentative broth. The technique successfully alleviated the restriction imposed by lactic acid on bacterial growth and product formation. As compared to the conventional batch fermentation, the new fermentation technique enhanced the lactic acid productivity and sugar conversion rate from 0.328g/L·h and 88. 2% to 0.482g/L·h and 98.6%, respectively.     

Technic for Studying Chromosome Structure

The methods described are modifications of various technics for the study of spiral structure in chromosomes. They enable permanent preparations to be made with better fixation and allow the use of stains which give clear and more critical definition. The first method described involves the use of ammonium, hydroxide (880 vols.) fumes for the treatment of pollen mother cells before fixation. Anthers of Tradescantia are smeared on a slide and wet in a 3% cane sugar solution. The preparation is then immediately placed in a dish of fixative where it remains for two hours. The slide can then be washed, bleached and stained with gentian violet or hematoxylin. It was found that fumes of nitric acid, hydrochloric acid and glacial acetic acid gave similar results. For the second method, boiling water is used for pre-treatment. A smear is made on a slide and immersed in boiling water for five to ten seconds. The smear is then fixed and treated in the usual manner.     

Subcellular Localization of a UDP-Glucose:Aldehyde Cyanohydrin beta-Glucosyl Transferase in Epidermal Plastids of Sorghum Leaf Blades

Epidermal and mesophyll protoplasts, prepared from leaf blades of 6-day-old light-grown Sorghum bicolor seedlings were separated by differential sedimentation and assayed for a number of enzymes. The epidermal protoplasts contained higher levels of NADPH-cytochrome c reductase (EC 1.6.2.4), triose phosphate isomerase (EC 5.3.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), and a UDP-glucose:cyanohydrin β-glucosyl transferase (EC 2.4.1.85), but lower levels of NADP⁺ triosephosphate dehydrogenase (EC 1.2.1.13) than did mesophyll protoplasts. When protoplast preparations were lysed and applied to linear sucrose density gradients, triosephosphate isomerase was found to be present in epidermal plastids. A significant fraction (41%) of the glucosyl transferase activity was also associated with the epidermal plastids.     

An Improved Technic for the Examination of Chromosomes in Root Tip Smears

Young, vigorous root tips are fixed in aceto-alcohol (1 part glacial acetic acid, 2 parts absolute alcohol) and are left in the fixative from 24 to 48 hours. If it is desired to store the material, the root tips can be transferred to 80% alcohol and be kept indefinitely. In preparing the smears, the root tips are placed on a slide and are sliced as thinly as possible with a sharp razor blade. Then the slices are smeared on the slide and immediately flooded with aceto-carmin, followed by a cover glass. Using absorbent paper and exerting considerable pressure, the excess aceto-carmin can be removed and the material flattened at the same time. Finally the slide is warmed gently to a point slightly below boiling. By sealing with gum mastic and paraffin, such preparations can be kept from 5 to 10 days.     

中国凤仙花科植物叶表皮特征及其分类学意义

利用光学显微镜观察凤仙花科106份样本材料、36种代表植物的叶表皮微形态特征。结果表明:该科植物叶表皮微形态特征种内稳定,对于种间及属间关系的界定具有重要的分类学价值。上表皮细胞的形状及垂周壁的式样种间差异明显,各分类群间有明显的界限,是种间界定的重要依据,因此上表皮微形态特征具有重要的的分类学价值,基于上表皮的微形态特征将研究的36种代表植物划分为5个类型。下表皮的微形态特征虽更为多样,种间差异显著,可用于种间界定,但对于属下划分难以提供有价值的性状。该科植物叶表皮微形态特征与宏观形态特征的相关性较弱,与地理分布格局的相关性较强,关系更为密切。同一地理分布区域内的种类宏观形态特征虽然差别明显,但叶表皮微形态特征却表现出较强的一致性,这似乎也反映了叶表皮微形态这一性状受环境饰变的影响比较明显。综上所述,叶表皮微形态特征可为凤仙花科的系统发育,尤其是凤仙花属种间界定提供有价值的分类学佐证     

A Note on the Detection of Phytopathogenic Bacteria within the Leaf by a Differential Staining Procedure

A simple differential staining procedure for demonstrating infection within the leaf tissue by Pseudomonas tomato, Ps. lachrymans and Xanthomonas vesicatoria has been developed. It is based on (1) clearing of plant tissue with a mixture of glycerol, lactic acid, phenol and water; (2) treating the leaf tissue with boiling KOH; and (3) staining with aniline blue-chloralhydrate. When observed under a light microscope, the bacteria appear dark blue, whereas the leaf tissue appears transparent and colourless.     

Chitosan and lactic acid-grafted chitosan nanoparticles as carriers for prolonged drug delivery

Nanoparticles of approximately 10nm in diameter made with chitosan or lactic acid-grafted chitosan were developed for high drug loading and prolonged drug release. A drug encapsulation efficiency of 92% and a release rate of 28% from chitosan nanoparticles over a 4-week period were demonstrated with bovine serum protein. To further increase drug encapsulation, prolong drug release, and increase chitosan solubility in solution of neutral pH, chitosan was modified with lactic acid by grafting D,L-lactic acid onto amino groups in chitosan without using a catalyst. The lactic acid-grafted chitosan nanoparticles demonstrated a drug encapsulation efficiency of 96% and a protein release rate of 15% over 4 weeks. With increased protein concentration, the drug encapsulation efficiency decreased and drug release rate increased. Unlike chitosan, which is generally soluble only in acid solution, the chitosan modified with lactic acid can be prepared from solutions of neutral pH, offering an additional advantage of allowing proteins or drugs to be uniformly incorporated in the matrix structure with minimal or no denaturization.     

Surface Printing of Plant Leaves for Phylogenetic Studies

A solution of plastic consisting of toluene, 720 ml; methanol, 180 ml; ethyl cellulose (Ethocel, standard 7 CPS), 250 gm; and Dow resin 276 V-2, 75 gm is applied to a leaf surface which has been dampened with toluene. The plastic is spread to a thin film with the edge of a card and allowed to dry. After drying, the plastic may be peeled from the leaf surface and either stored dry in a small envelope or mounted permanently on a microscope slide. Permanent mounts are prepared by placing a small section of the peel from the upper and lower surfaces of the leaf on a microscope slide and covering with a No. 1 cover glass. A small spot of balsam on each corner of the cover glass secures the glass in position. This air mount has proved to be superior to water, glycerol or balsam mounts. Fresh leaves are washed with a mild detergent before application of the plastic. Herbarium specimens are soaked in water overnight to restore the leaf to a semiturgid condition. Five species of different plant families have been illustrated to show the diagnostic features of the surface of the cuticle. An isolated layer of epidermal cells obtained by chemical maceration permitted cell and imprint comparison. The remarkable amount of detail shown by the prints is an aid for phylogenetic studies and may make the recognition of fossil cuticles possible.     

Hot, Acidified Rhodamine B and Methylene Blue; A Differential Stain Dichromatic for Hair Follicular Keratins, Achromatic for Epidermal Keratin

Procedure:Cut paraffin sections and float on a 45-50 C water bath; spread silicone-rubber adhesive (Clear Seal-General Electric) thinly and evenly over 2/3 of the slide; pick up the sections from the floatation water with the coated slide; dry for 1.5 hr at 25 C and at 60 C for 0.5 hr; deparaffinize, and hydrate to water. Place 150 mg of rhodamine B and 150 mg of methylene blue each in separate 100 ml beakers and add 80 ml of 10% HCl to each beaker. Bring both solutions to a boil on a hot plate in a fume hood; immerse tissue sections in the boiling rhodamine B exactly 2 min; rinse in a beaker of 10% HCl 5 sec; immerse in the boiling methylene blue exactly 0.5 min; rinse in distilled water; blot dry; and mount in a silicone-rubber medium (Glass and Ceramic Adhesive—Dow Corning Corp.). Hair shaft keratin stains red; inner root sheath keratin and keratogenous zone of the hair shaft, blue green; epidermal keratin remains unstained. Pilomatrixornas show foci of both red and blue green keratin; epidermal and hair sheath (“sebaceous”) cysts remain unstained.     

Erratum

A solution of plastic consisting of toluene, 720 ml; methanol, 180 ml; ethyl cellulose (Ethocel, standard 7 CPS), 250 gm; and Dow resin 276 V-2, 75 gm is applied to a leaf surface which has been dampened with toluene. The plastic is spread to a thin film with the edge of a card and allowed to dry. After drying, the plastic may be peeled from the leaf surface and either stored dry in a small envelope or mounted permanently on a microscope slide. Permanent mounts are prepared by placing a small section of the peel from the upper and lower surfaces of the leaf on a microscope slide and covering with a No. 1 cover glass. A small spot of balsam on each corner of the cover glass secures the glass in position. This air mount has proved to be superior to water, glycerol or balsam mounts. Fresh leaves are washed with a mild detergent before application of the plastic. Herbarium specimens are soaked in water overnight to restore the leaf to a semiturgid condition. Five species of different plant families have been illustrated to show the diagnostic features of the surface of the cuticle. An isolated layer of epidermal cells obtained by chemical maceration permitted cell and imprint comparison. The remarkable amount of detail shown by the prints is an aid for phylogenetic studies and may make the recognition of fossil cuticles possible.     

中国梅花草属植物的叶表皮特征及其系统学意义

利用光学显微镜和扫描电镜对梅花草属Parnassia 30种植物的叶表皮进行了观察。结果表明:气孔器普遍存在于叶的下表皮,少数种的上表皮也有分布,均为无规则型。叶表皮细胞形状为多边形或不规则形;垂周壁式样可区分为近平直、浅波状和波状。在扫描电镜下,叶表皮气孔器外拱盖内缘为近平滑、浅波状或波状;一些种的保卫细胞两端有加厚;角质膜条纹状,有的条纹隆起,有的条纹上附有颗粒或小孔穴。气孔器类型及下表皮细胞形状的一致性表明梅花草属是一个自然分类群;sect. Saxifragastrum叶表皮特征具有多样性显示该组可能是一个复合群;突隔梅花草P. delavayi属于subsect. Xiphosandra,其气孔下陷,与其细胞学特征相似,支持独立为一组;此外,气孔器的分布、保卫细胞两端加厚、气孔器外拱盖内缘形态以及角质膜等特征对该属部分种的区分有一定的参考价值。     

天南星科叶表皮研究

利用光学显微镜对天南星科18属27种及菖蒲科1属1种植物的叶表皮微形态进行观察,同时用扫描电镜对具代表性的14种植物作了研究,结果显示：天南星科气孔类型变异较大,有不规则型,辐射型,平列型,胞环型及平列型和胞环型间的过渡类型,副卫细胞数目0－12个;表皮细胞长宽近相等,平周壁具条纹或否,垂周壁平直,弧形或波浪形,虽然气孔类型对天南星科分类上的意义不大,但与表皮细胞垂周壁形状,副卫细胞角质层纹饰等特征相结合对种间分类有一定意义,天南星科与菖蒲科叶表皮微形态明显不同,从而支持菖蒲属从天南星科中分出另立为科的观点。     

Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves

Background and Aims

Epidermal phenolic compounds (mainly flavonoids) constitute a vital screen that protects the leaf from damage by natural ultraviolet (UV) radiation. The effectiveness of epidermal UV-screening depends on leaf anatomy, the content of UV-screening compounds and their spatial uniformity over the leaf area. To investigate in vivo the spatial pattern of the epidermal UV-screen during leaf development, a fluorescence imaging method was developed to map the epidermal UV-absorbance at a microscopic scale. This study was done on oak (Quercus petraea) leaves that were used as a model of woody dicotyledonous leaves.

Methods

The leaf development of 2-year-old trees, grown outdoors, was monitored, at a macroscopic scale, by in vivo measurements of chlorophyll content per unit area and epidermal UV-absorbance using two optical leaf-clip meters. The distribution of pigments within leaves was assessed in vivo spectroscopically. The microscopic images of UV-induced fluorescence and UV-absorbance acquired in vivo during leaf development were interpreted from spectral characteristics of leaves.

Key Results

At a macroscopic scale, epidermal UV-absorbance was high on the upper leaf side during leaf development, while it increased on the lower leaf side during leaf expansion and reached the adaxial value at maturity. At a microscopic scale, in immature leaves, for both leaf sides, the spatial distribution of epidermal UV-absorbance was heterogeneous, with a pattern depending on the flavonoid content of vacuoles in developing epidermal cells. At maturity, epidermal UV-absorbance was uniform.

Conclusions

The spatial pattern of epidermal UV-screen over the area of oak leaves is related to leaf anatomy during development. In vivo spectroscopy and fluorescence imaging of the leaf surface showed the distribution of pigments within the leaf and hence can provide a tool to monitor optically the leaf development in nature.Key words: Blue-green fluorescence, chlorophyll fluorescence, epidermis, flavonoids, leaf development, microscopic imaging, polyphenols, Quercus petraea     

THE EFFECT OF MODERATE SALT STRESS ON LEAF ANATOMY IN HIBISCUS CANNABINUS (KENAF) AND ITS RELATION TO LEAF AREA

Kenaf responded to salt stress in a manner that was typical of moderately salt tolerant non-halophytes. Increase in leaf area was more sensitive to salinity than either leaf emergence rate or dry matter accumulation. Dry weight was reduced only above a threshold of approximately 37 mM NaCl while leaf area was already significantly reduced at this salt concentration. Measurement of epidermal cell cross sectional area and epidermal cell numbers showed that the salt induced reduction in leaf area was due primarily to smaller epidermal cell size. Epidermal cell numbers were also significantly reduced by salinity. Stomatal density increased with increasing salt stress and there was no effect on leaf thickness.     

鹿药属植物叶表皮特征及其系统学意义

采用光学显微镜和扫描电镜对鹿药属12种植物的叶表皮进行了观察,首次报道了12种鹿药属(Sm ilaci-na)植物叶表皮的微形态特征。结果表明:气孔器普遍存在于叶的下表皮,少数种的上表皮也有分布,均为不规则形。叶表皮细胞形状为多边形或不规则形,垂周壁式样可区分为近平直、浅波状和波状。在扫描电镜下,叶表皮气孔器外拱盖内缘为近平滑、浅波状或波状;角质膜条纹状,有的条纹隆起,有的条纹上附有颗粒和晶簇。气孔器的分布、气孔器外拱盖内缘形态以及角质膜等特征对该属部分种的区分具有一定的参考价值。     

蜡梅科植物的叶表皮特征及其在分类上的意义

用扫描电镜和光学显微镜观察了蜡梅科3属5种植物成熟叶片远轴面的表皮特征,认为这些表皮细胞特征和气孔器特征在分类上有比较重要的意义。蜡梅属、夏蜡梅属和美国蜡梅属植物的叶表皮毛均为单细胞毛、非腺毛,上、下表皮细胞均为多边形,垂周壁呈深波状,气孔器均为平列型．这三个属的亲缘关系密切,应该归属于同一个大类群－蜡梅科。这为蜡梅属、夏蜡梅属、美国蜡梅属的分类提供了有用的性状特征。这三个属气孔器的演化趋势为：气孔器在保卫细胞的两极无"T"型加厚到有"T"型加厚,气孔器由单层外拱盖到双层外拱盖．     

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.     

Variation of hydrophobicity of human urinary epidermal growth factor

Epidermal growth factor is present in human urine in large amounts, but its biological significance is not known. The results of this study indicate that the predominant 6000-dalton form of epidermal growth factor in human urine is divided by hydrophobic interaction chromatography into four fractions; only 3% of the total 6000-dalton epidermal growth factor coeluted with the biosynthetic epidermal growth factor and the rest was separated into three different peaks. These different forms may lack one or two amino or carboxy terminal amino acids from the 53 amino acids present in epidermal growth factor, or they may be products of deamidation or oxidation of amino acid(s). Further knowledge of these micromodifications of epidermal growth factor secreted in urine may reveal the origin and function of epidermal growth factor in humans.