THE CULTURE OF APICAL BUDS OF XANTHIUM AND THEIR USE AS A BIOASSAY FOR FLOWERING ACTIVITY OF ECDYSTERONE

Conditions were developed for the sterile culture of shoot tips of Xanthium pensylvanicum Wallr. for use as a bioassay for flower-controlling chemicals. By using a modified Murashige-Skoog medium (minus the auxin but including kinetin) and light intensity much higher than usual for plant tissue cultures, fast growth and development of the shoot tips was achieved. Under short-day conditions (8 hr day: 16 hr night), the cultures from vegetative shoots flowered and fruited; under noninductive conditions (using a 2 hr light-break in the middle of the dark period), the shoot tips continued vegetative development. Both intact plants and cultured tips could be photoinduced in the first days after germination. Ecdysterone, a potent insect moulting hormone, was tested in the bioassay system. It was without either qualitative or quantitative effect on flowering or vegetative development on either cultured shoot tips or intact plants irrespective of whether they were under inductive or noninductive photoperiodic conditions.     

Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species

Summary A method was developed for plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation was achieved using 3% sodium alginate and 75 mM CaCl₂·2H₂O. Maximum percentage response for conversion of encapsulated shoot tips into plantlets was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant growth regulator. The regrowth ability of encapsulated shoot tips was affected by the concentration of sodium alginate, storage duration, and the presence or absence of MS nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were transferred to pots containing an autoclaved mixture of soilrite and peat moss (1∶1). The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts. Encapsulation of vegetative propagules in calcium alginate beads can be used as an alternative to synthetic seeds derived from somatic embryos.     

Preparative Procedures and Culture Medium Affect the Success of Cryostorage of Holostemma annulare Shoot Tips

Shoot tip cryopreservation of Holostemma annulare, an endangered medicinal plant was carried out using Murashige-Skoog (MS) medium containing mM NH⁺ ₄+NO^– ₃; 20.6+39.4 (MS-1), 2.6+18.8 (MS-2) or 0.0+18.8 (MS-3). The three media combinations were tested during four preparative procedures viz.: development of cultures; preconditioning of shoot tip cuttings; preculture of encapsulated shoot tips; and post-freeze recovery to understand the most critical phase of NH₄NO₃ sensitivity. MS-1 used during the initial three preparative steps supported 10.9–16.6% post-freeze recovery of cryopreserved shoot tips. Development of culture in MS-1 and subsequent passages (2nd, 3rd and 4th preparative steps) in MS-2 or MS-3 improved the recovery rate to 26.4–35.8%. MS-3 used throughout the steps favoured 38.5% recovery. Shoot tips from shoot cultures raised in MS-2 upon preconditioning in MS-2 or MS-3 and subsequent preculture of encapsulated shoot tips and post-freeze recovery culture in MS-3 showed maximum regeneration (55%). MS-2 used throughout the procedure supported 48% regeneration of cryopreserved shoot tips.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN : I. Peripheral Nerve

The dehydration of frog sciatic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. Low magnification electron micrographs of OsO₄-fixed preparations showed marked tissue shrinkage which could be correlated quantitatively with the loss of water during the preliminary drying. KMnO₄-fixation appeared to cause a rehydration of the dried tissue. Higher magnification electron micrographs of the OsO₄-fixed preparations showed a sequence of modifications of the myelin layers which could be correlated with changes in the small-angle x-ray diffraction data which were recorded during drying. An intermediate stage of drying was characterised by a partial collapse of layers and a disappearance of the intraperiod dense line in some regions of the myelin sheath. Continuity between collapsed and non-collapsed layers was maintained throughout the sheath. The fully dried preparation showed two main modifications of the myelin layers. In many regions the layers (principal layers) resembled those of normal preparations, but showed an intensification and frequently a doubling of the intraperiod dense line. In addition, there was a very extensive system of fine (40 A periodicity) dense layers, some of which could be demonstrated to be continuous with the principal layers. In such cases it was observed that two of the fine layers were related to each principal layer. The correlation between diffraction data and electron microscope data is discussed, and some speculations are made concerning the molecular significance of the observations.     

Stem elongation and changes in the levels of gibberellins in shoot tips induced by differential photoperiodic treatments in the long-day plant Silene armeria

The effects of differential photoperiodic treatments applied to shoot tips and mature leaves of the long-day (LD) plant Silene armeria L. on growth and flowering responses, and on the levels of endogenous gibberellins (GAs), were investigated. Gibberellins were analyzed by gaschromatography-mass spectrometry and the use of internal standards. Exposure of mature leaves to LD, regardless of the photoperiodic conditions of the shoot tips, short days (SD), LD, or darkness, promoted elongation of the stems and of the immature leaves. Long-day treatment of the mature leaves modified the levels of endogenous GAs in shoot tips kept under LD, SD, or darkness. In shoot tips kept in LD or darkness the levels of GA₅₃ were reduced, whereas the levels of GA₁₉ and GA₂₀ were increased. The contents of GA₁ were increased in all three types of shoots: SD twofold, LD fivefold, and darkness eightfold. Dark treatment of the shoot tips on plants of which the mature leaves were grown in SD promoted elongation of the immature etiolated leaves and increased the GA₁ content of the shoot tips threefold. However, this treatment did not cause stem elongation. The different photoperiodic treatments applied to the shoot tips did not change the levels of GAs in mature leaves. These results indicate that both LD and dark treatments result in an increase in GA₁ in shoot tips. In addition, it is proposed that LD treatment induces the formation of a signal that is transmitted from mature leaves to shoot tips where it enhances the effect of GA on stem elongation.Abbreviations GA_n gibberellin A_n - LD long day(s) - SD short day(s) We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]-gibberellins and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility, East Lansing, for advice with mass spectrometry. This work was supported, in part, by a fellowship from the Spanish Ministry of Agriculture (Instituto Nacional de Investigaciones Agrarias) to M.T., by the U.S. Department of Energy grant No. DE-FG02-91ER20021, and by the U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

A RECONSTRUCTION OF CELL DEVELOPMENT IN THE SHOOT APEX OF MAIZE

Somatic mutations were induced in maize embryos in order to follow the albino-tissue patterns in mature plants. A reconstruction of cellular development in the shoot apex has been attempted. Two strains of maize were employed, wd/Yg₂ and pastel-8549/y₁ for seed irradiation with gamma rays. After mature plants had developed from this radiated seed, the sectored plants were analyzed in detail for their patterns of albino tissue. The location and frequency of these patterns were correlated with cell number at various sites of the initial shoot apex in order to deduce the number of cells contributing to each frequency class. Various lines of evidence lead to the conclusion that the cellular differentiation in the shoot apex is organized and a relatively stable process. Apparently a few cells in the apical dome provided daughter tissue for the upper half of the maize plant. Various sector patterns are diagrammed and the position of their albino tissue is explained in relation to the location of a specific cell in the apex.     

Penetration of Hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans Without the Formation of Appressoria

In a study using scanning electron microscopy (SEM), the mode of hyperparasitism of Coniothyrium minitans on its host Sclerotinia sclerotiorum was investigated. The SEM micrographs confirm previous reports, from light microscopic studies, that hyphal tips of C. minitans invade the host hypha by direct penetration, without developing appressoria, and that indentation of the host cell wall at the point of penetration is often evident. There is no functional distinction between amain branch and a side branch hypha of the hyperparasite and tips of either type of hyphae are capable of invading host hyphae by direct penetration.     

SOME EPIDERMAL CHARACTERISTICS OF PALEAS OF DICHANTHELIUM,PANICUM, AND ECHINOCHLOA

The apical portion of paleas of the perfect (upper) floret of 11 species of Dichanthelium, 10 species of Panicum, and one species of Echinochloa were examined with a scanning electron microscope. The surfaces of the Dichanthelium palea tips uniformly had simple, rounded papillae, widely spaced in regular rows, whereas the palea tips of most Panicum species had irregularly arranged, compound or aggregated papillae. Unusual epidermal features were observed in P. obtusum, P. hians, and P. hemitomon. The Echinochloa crusgalli palea surface exhibited a pattern of papillae or spicule development similar to that of P. hians. Transverse sections of Dichanthelium palea showed the papillae to be little if at all thickened. An electron beam x-ray microanalysis indicated a high concentration of silicon in the papillae.     

A micropropagation protocol forCinnamomum camphora

Summary A micropropagation protocol was developed forCinnamomum camphora (L.) Sieb., using as initial explants 3–5-mm shoot tips from newly emerged laterals of 2-yr-old trees. Performance of small shoot tips was compared with that of 2.0-cm nodal segments during subculture. Murashige and Skoog medium (MS) supplemented with different concentrations of N₆-benzyladenine (BA) or thidiazuron (TDZ) was used to examine shoot proliferation. In separate experiments, MS was supplemented with 1-naphthaleneacetic acid (NAA) for rooting of shoots, and the commercial preparation EM₂ for prevention of hyperhydricity. BA stimulated shoot formation and callus development, whereas TDZ promoted only callus development. Both cytokinins induced hyperhydricity when small shoot tips were used, with severity being directly related to concentrations. Hyperhydricity was avoided in subcultures by using larger nodal segments. EM₂ did not alter degree of hyperhydricity but suppressed callus development and strongly promoted shoot multiplication. The number of new shoots after a 6-wk subculture was 9 per nodal segment when supplemented solely with 4.4 μM BA and 18 per segment when further supplemented with 1000 mg EM₂ per I. Rooting of shoots occurred best when supplemented solely with 0.54 μM NAA, averaging 7 roots per shoot in 4 wk. Ninety percent of rooted shoots survived transfer to the greenhouse.     

ROLE OF STEM GROWTH IN LINUM USITATISSIMUM LEAF TRACE PATTERNS

Linum usitatissimum stem growth parameters were quantified by computer-assisted analyses of scanning electron micrographs of shoot apical meristems throughout ontogeny. There were progressive decreases in the plastochron and relative plastochron rates of radial and vertical stem growth which resulted in the generation of progressively higher orders of contact parastichy phyllotaxis throughout ontogeny. The change in the relative spacing of primordia initiation on the stem coupled with the iterative differentiation of leaf gap and interfascicular ray parenchyma associated with each leaf primordium resulted in the delimitation of progressively higher orders of leaf trace interconnections throughout ontogeny. A set of developmental rules was generated which should permit simulation of many leaf trace patterns.     

STRUCTURE OF THE SHOOT APEX IN LACTUCA SATIVA

Histological observations of the leaf lettuce ‘Black Seeded Simpson’ showed the dormant embryo to possess two visible leaves and a flkat to slightly depressed plumular apex. Observations conducted over a 12-day period of germination and growth showed the development of L₁ and L₂, emergence of L₃ and L₄, and periodic changes in size of the apex which were associated with leaf emergence. Thus the dormant embryo of Lactuca appears to be considerably more advanced in development than was previously believed. The shoot apex appeared flat to slightly depressed at all developmental stages studied.     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

Initiating cultures ofHalesia andMalus: influence of flushing stage and benzyladenine

Shoot tips ofHalesia carolina L. andMalus domestica Borkh. Golden Delicious collected at various times during the spring growth flush varied considerably in their ability to initiate shoot proliferating cultures. Shoot tips collected during, or at the end of, the period of most rapid shoot elongation exhibited weak shoot proliferation potential, while shoot tips collected before or after this period were capable of strong shoot proliferationin vitro. Benzyladenine concentrations in the culture media above 22.5 µM (Halesia) or 44.5 µM (Malus) were inhibitory during the period between bud break and rapid shoot elongation. Benzyladenine concentrations of 22.5 or 44.5 µM were useful in enhancing shoot proliferation potential in shoot tips collected after the period of rapid shoot elongation, but before the onset of summer dormancy. Benzyladenine concentration did not affect the shoot proliferation potential of shoot tips collected during the rapid shoot elongation phase of the spring growth flush.Halesia andMalus shoot tips behaved similarly in this study. For deciduous woody perennials, the time of explant collection for culture initiation could be refined on the basis of these results.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant <Emphasis Type="Italic">Emmenopterys henryi</Emphasis> Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS₂) for 30 min at 0°C and followed by 100% PVS₂ for 40 min at 0°C. After changing the solution with fresh 100% PVS₂, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l⁻¹, α-naphthaleneacetic acid 0.1 mg l⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.     

ULTRASTRUCTURE OF THE SECRETORY DUCTS OF RHUS GLABRA L.

The infrastructure and development of the secretory ducts were studied in the secondary phloem of Rhus glabra L. The ducts were found to develop schizogenously. The electron microscope observations may explain the view of several previous authors of schizo-lysigenous development of the duct lumen in the Anacardiaceae. The secreted material consists of lipophilic and polysaccharide substances. The electron micrographs suggest that the lipophilic substances arise in plastids, ER cisternae, Golgi vesicles, and mitochondria. The polysaccharide constituents apparently originate from the outer wall layers of the epithelial cells. The wall layers facing the lumen of the duct disintegrate and form, together with the secreted osmiophilic droplets, the gum-resin. Numerous microtubules were found along walls of the epithelial cells.     

Chromoplasts of Tropaeolum majus L.: Structure and development

Summary The fine structure of chromoplasts in epidermal cells of flower petals of Tropaeolum has been investigated by light, polarizing, and electron microscopy at different stages of development. The pale greenish-yellow petals still enclosed in the bud contain barely differentiated chloroplasts with few, irregular grana. The chromoplasts of unfolding petals show differently oriented bundles of tubules with variable diameters (mean: 17 nm). Thylakoid membranes become reduced more and more. The tubular bundles are intermingled with numerous isodiametric bodies of ca. 50 nm diameter; these bodies are better discernible at later stages when the chromoplasts possess a less dense matrix. The chromoplasts of open flowers are in a state of disorganization at a time when the cytoplasm still appears normal. A comparison is made between chromoplast tubules and tubular structures described from other kinds of plastids. The observations are discussed in view of chromoplast typology and with regard to possible processes underlying chromoplast differentiation in flowers.Abbreviations in Figures Chr chromoplast - CT chromoplast tubules - Cy cytoplasm - D dictyosome - IB isodiametric body - M mitochondrion - MT microtubule - oG osmiophilic globule - S S-body - St starch grain - V vacuole All micrographs from glutaraldehyde-OsO₄-fixed material, unless otherwise specified. The bar designates 1 m (multiples or fractions of it indicated).     

Scanning Electron Microscopy of the Nematode-trapping Organs in Arthrobotrys oligospora

The three-dimensional capture organs of the nematodetrapping fungus Arthrobotrys oligospora are presented in scanning electron micrographs. The fungus was grown directly on agar substrates or on millipore filters or glass cover slips placed on agar substrates. To avoid shrinkage of the soft-walled mycelial material, a fixation in O_sO₄-vapour was performed, before freeze-drying and coating with Au-Pd alloy. When capture organs were prepared in this way, it was possible to visualize them in their erect position in the scanning electron microscope. Young capture organs, both spontaneously formed and nematode-induced, remained standing better than old ones. An adhesive substance on the capture organs, with the aid of which nematodes are trapped, seems to be visible in these pictures.     

REGULATION OF CHAMISE SHOOT GROWTH

Nutritional, hormonal, and environmental control of chamise (Adenostoma fasciculatum H. & A.) shoot growth was investigated. In vitro culture of shoot tips demonstrated that 0.18 M sucrose was required for optimum apical growth. Cytokinin (benzyladenine) promoted shoot growth at otherwise growth-limiting sucrose concentrations and induced uptake of sucrose from the basal medium. Abscisic acid inhibited growth of cultured shoot tips induced by high sucrose concentration or cytokinin. In the field, inhibition of shoot growth was a function of water stress. These studies indicate that the effects of water stress on chamise shoot growth may be mediated by changes in carbohydrate, cytokinin, or growth inhibitor levels at the shoot apex.     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.