Induction of root and shoot formation from root meristems ofPetunia hybrida

Summary A procedure has been developed for the induction of root or shoot formation from root meristems of germinated seeds ofPetunia hybrida. Root formation was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine (BA) (0–0.5 mg/l) and naphtaleneacetic acid (NAA) (0.05–2.0 mg/l). Induction of predominantly shoot formation was obtained on MS medium containing the following combinations of hormones (in mg/l): 0.05–0.5 NAA and 0.25–2.0 BA. Complete plant formation was obtained after rooting of the shoots on MS medium supplemented with IAA (0–2.0 mg/l) or NAA (0-0.5 mg/l).     

Nitrogen in shoot litter,root litter and root exudates from nitrogen-fixingAlnus incana

Summary A pot experiment withAlnus incana (L.) Moench growing in sand was set up to compare the amounts of nitrogen released from plants shoot litter with that released below ground as root litter and/or root exudation. No nitrogen fixation by free-living microorganisms was found in the sand and the increased nitrogen content of the plant + soil system was therefore due to nitrogen fixation byFrankia in the alder root-nodules. Most of the nitrogen released from the plants was in the nitrogen-rich leaf and other shoot litter. Only small amounts of nitrogen were found in the drainage water from the pots and were recorded as increased nitrogen content of the sand.     

Thidiazuron-induced de novo shoot organogenesis on seedlings,etiolated hypocotyls and stem segments of Huang-qin

Development of an efficient in vitro propagation system for Huang-qin (Scutellaria baicalensis), a traditional Chinese medicinal plant used in the treatment of a wide range of human ailments, is described. Thidiazuron [TDZ: N-phenyl-N′- (1,2,3-thidiazol-5-ylurea)] effectively induced regeneration on cultured intact seedlings, etiolated hypocotyl explants and sterile stem segments of Huang-qin. Histological examinations of excised hypocotyl or nodal explants revealed that adventitious shoots formed through an intermediate callus. Comparison of TDZ-induced regeneration in the three tissue types indicated that isolation of explants was not essential for optimal regenerative efficiency. Significantly more regenerants formed along hypocotyls of intact seedlings (20 shoots/explant) than were observed on excised hypocotyls (9.7 shoots/explant) indicating that endogenous metabolites produced in adjacent tissues provided resources for the shoot initiation. More than 95% of de novo regenerants formed roots and then intact plantlets under either sterile culture or greenhouse conditions. Regeneration protocols developed in this study may provide the basis for improvement of this crop through the identification of medicinally active constituents and eventual development optimized pharmaceutical products. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

From compartmental analysis of radioisotope elutin measurements, fluxes of Ca²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to complete nutrient solutions containing a range of calcium concentrations ([Ca₀]) from 2 eq l^-1 to 20 meq l^-1, increasing in 10-fold steps for Ca²⁺. Except for the calcium counter-ion (usually NO ₃ ^- , sometimes Cl^- at the highest [Ca₀]), the composition of the nutrient solution was other-wise the same at all calcium concentrations. Compartmental analysis indicated that the cytoplasm had a high content of exchangeable Ca²⁺ but, in the light of evidence from animal studies, ionic activity of calcium in the cytoplasm was assumed to be no greater than 0.002 eq ml^-1. With the Ussing-Teorell flux equation as the criterion, it was concluded that at all values of [Ca₀] tested, Ca²⁺ entered the cytoplasm passively and was actively pumped back into the external solution. Entry of calcium to the vacuole from the cytoplasm was active in all cases. The conclusions regarding the character of ion transport across the plasmalemma were the same as when the whole calcium content of the cytoplasm was taken to contribute to the ionic activity. However, the electrochemical activity gradient was very much steeper than formerly estimated. Calcium was transported to the stele in proportion to the calcium content of the cytoplasm and moved in the xylem almost exclusively in the basipetal direction.     

Abscisic acid distribution in horizontal maize root segments

Using gas chromatography-mass spectrometry (GC/MS) techniques of analyses, it has been found that endogenous abscisic acid (ABA) becomes asymmetrically distributed in the elongation zone of horizontal Zea mays (cv. LG 11) roots which are showing a positive gravitropic response. There is a relative increase in the ABA content of the lower half and a concomitant decrease for the upper half in such roots. Asymmetric distribution of ABA is also detected in the elongation zone of half-decapped roots.Abbreviations IAA indoleacetic acid - ABA abscisic acid - GC/MS gas chromatography-mass spectrometry     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.     

In vitro regeneration of silktree (Albizzia julibrissin) from excised roots

Root segments (1 cm long) were excised from 15–20 day old seedlings of silktree (Albizzia julibrissin) grown on B5 medium. About 50% of the control (no growth regulators added) root explants formed shoot buds within 15 days after placement on the culture medium. After 30 days, there were about 4 shoots per control explant. Addition of low levels of various auxins (0.5 M) did not influence the formation of shoot buds from the explants. Higher concentrations (5M), however, decreased shoot regeneration. Kinetin and 2iP did not influence shoot regeneration at the concentrations tested (1 & 10 M). Addition of benzyladenine, Zeatin, or thidiazuron to the culture medium increased both the percentage of explants that formed shoots and the number of shoots per explant. Thidiazuron was highly effective in stimulating shoot formation at low concentrations (<1 M). At 0.05 M thidiazuron, 95% of the explants produced shoots and about 10 shoots were formed per explant. Compared to TDZ, higher concentrations (10 M) of benzyladenine and Zeatin were required to enhance shoot formation. Upon excision and transfer to B5 medium, regenerated shoots developed into normal rooted plantlets.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - TDZ Thidiazuron - 2ip Isopentenyladenine     

Vacuolar reorganization in the motor cells of Albizzia during leaf movement

During the leaf movements of Albizzia julibrissin Durazzini, volume changes in the motor cells of the pulvinule (tertiary pulvinus) are closely correlated with a reversible reorganization of the vacuolar compartment. Motor cells have central vacuoles when expanded, but become multivacuolate during the time the cell volume decreases. The central vacuole reforms — apparently by fusion of small vacuoles — during motor-cell expansion. The volume changes of the vacuolar compartment account for all of the change in the size of the protoplast, while the cytoplasmic volume remains constant during the leaf movements.     

Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.     

Maize root response to N in the greenhouse and field

Summary Root growth and morphology were compared between seven week old maize plants grown in the greenhouse and in the field. The plants were similar in shoot dry weight and the partioning of N and dry matter to roots were similar except for the field grown plants in 1983. Field grown plants had greater root mass per length and greater calculated diameter than greenhouse plants. Nitrogen fertilization decreased N and dry matter partitioning to the root system in all three environments.     

Regeneration in Phaseolus vulgaris L.: High-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and thidiazuron

Induction of prolific shoot formation in Phaseolus vulgaris L. cv. Kinghorn Wax was achieved by germinating mature seeds and growing seedlings on a medium supplemented with 10 M thidiazuron (TDZ), a substituted phenylurea, or 80 M N⁶-benzylaminopurine (BAP). Culture for 7 d in the presence of 10 M TDZ was sufficient to induce maximal shoot formation, whereas a continuous presence of BAP was required for the induction and development of shoots. The differentiation of adventitious shoots occurred within four weeks of seed culture, from tissues in the regions of axillary buds on the cotyledonary node and also areas surrounding the shoot apex of the intact seedling. The number of shoots regenerated from intact seedlings was significantly higher than that obtained with expiants. Regenerated shoots developed into flowering plants. Similar results were obtained in six other bean cultivars.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - TDZ N-phenyl-N 1-(1,2,3 thiadiazol-yl)urea (thidiazuron) To whom correspondence should be addressedThis research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and the University Research Board Grant Programs of the University of Guelph to P.K.S. We thank Drs. Jean Gerrath and R. Rastogi for helpful discussions. Technical assistance from Sangeeta Saxena is gratefully acknowledged.     

Impact of two fluorescent pseudomonads and an arbuscular mycorrhizal fungus on tomato plant growth,root architecture and P acquisition

The ability of fluorescent pseudomonads and arbuscular mycorrhizal fungi (AMF) to promote plant growth is well documented but knowledge of the impact of pseudomonad-mycorrhiza mixed inocula on root architecture is scanty. In the present work, growth and root architecture of tomato plants (Lycopersicon esculentum Mill. cv. Guadalete), inoculated or not with Pseudomonas fluorescens 92rk and P190r and/or the AMF Glomus mosseae BEG12, were evaluated by measuring shoot and root fresh weight and by analysing morphometric parameters of the root system. The influence of the microorganisms on phosphorus (P) acquisition was assayed as total P accumulated in leaves of plants inoculated or not with the three microorganisms. The two bacterial strains and the AMF, alone or in combination, promoted plant growth. P. fluorescens 92rk and G. mosseae BEG12 when co-inoculated had a synergistic effect on root fresh weight. Moreover, co-inoculation of the three microorganisms synergistically increased plant growth compared with singly inoculated plants. Both the fluorescent pseudomonads and the myco-symbiont, depending on the inoculum combination, strongly affected root architecture. P. fluorescens 92rk increased mycorrhizal colonization, suggesting that this strain is a mycorrhization helper bacterium. Finally, the bacterial strains and the AMF, alone or in combination, improved plant mineral nutrition by increasing leaf P content. These results support the potential use of fluorescent pseudomonads and AMF as mixed inoculants for tomato and suggest that improved tomato growth could be related to the increase in P acquisition.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

Shoot and root growth of hydroponic maize (Zea mays L.) as influenced by K deficiency

Potassium (K) has major biophysical and biochemical functions in plant physiology. However, plant responses to K deficiency at the whole plant level are not always clearly related to these well-known functions of K at the cellular level. The objective of this study was to investigate the morphological response of maize to increasing K deficiency and test to what extent this morphological response can be interpreted in the light of the simple model proposed by Leigh and Wyn Jones, suggesting that biophysical functions are affected first. Maize was grown in a greenhouse under hydroponic conditions. For half of the plants, K was removed from the nutrient solution from the 4th visible leaf stage. The K content in the starved plants dropped from 100 to 30 mM, and was not fully compensated by an increase in other cations. Leaf elongation rates were reduced on K-deprived plants, whereas axile root elongation rates were slightly increased between 45°C days and 75°C days after starvation, and reduced thereafter. During the first part of the starvation period, i.e. under moderate K deficiency (K concentration above 40 mM), all measured variables suggest that the whole plant response may be interpreted as the consequence of the reduced leaf growth, probably due to insufficient turgor pressure or cell-wall extensibility. This general pattern of response is in agreement with the model of Leigh and Wyn Jones. However, during the second part of the starvation period, i.e. under more severe K deficiency (K concentration below 40 mM), malfunction of additional physiological processes (mostly related to biochemical functions like photosynthetic processes) must be considered to explain the plant morphological response.     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L. and Albizia julibrissin Durazz

For experimental induction of crystal cells (=crystal idioblasts) containing calcium-oxalate crystals, the lower epidermis was peeled from seedling leaflets of Gleditsia triacanthos L., exposing the crystal-free mesophyll and minor veins to the experimental solutions on which leaflets were floated for up to 10 d under continous light. On 0.3–2.0 mM Ca-acetate, increasing numbers of crystals, appearing 96 h after peeling, were induced. The pattern of crystal distribution changed with Ca²⁺-concentration ([Ca²⁺]): at low [Ca²⁺], crystals formed only in the non-green bundlesheath cells surrounding the veins, believed to have a relatively low Ca²⁺-extrusion capacity; at higher [Ca²⁺], crystals developed in up to 90% of the mesophyll cells, and at supraoptimal [Ca²⁺], large extracellular crystals formed on the tissue surface. By sequential treatments with solutions of different [Ca²⁺], the following three phases were identified in the induction of crystal cells: (1) during the initial 24-h period (adaptive aging), Ca²⁺ is not required and crystal induction is not possible; (2) during the following 48 h (induction period), exposure to 1–2 mM Ca-acetate induces the differentiation of mesophyll cells into crystal cells; (3) crystal growth begins 72 h after the start of induction. In intact leaflets of Albizia julibrissin Durazz., calcium-oxalate crystals are found exclusively in the bundle-sheath cells of the veins, but crystals were induced in the mesophyll of peeled leaflets floating on 1 mM Ca-acetate. Exposure to inductive [Ca²⁺] will thus trigger the differentiation of mature leaf cells into crystal cells; the spatial distribution of crystals is determined by the external [Ca²⁺] and by the structural and functional properties of the cells in the tissue.     

Calcium oxalate formation is a rapid and reversible process inLemna minor L.

Summary Lemna minor root tips form raphide Ca oxalate crystals in both the root cap and root proper. An in vivo system was developed to examine raphide crystal bundle formation in the root of intact plants. By increasing the exogenous Ca concentration, crystal bundle formation could be induced. Entire new crystal bundles could be formed within 30 minutes of an inductive stimulus. The process was reversible with recently formed crystal bundles being dissolved over a period of about 3 hours. Older, previously existing bundles were more resistant to dissolution. The calmodulin antagonists, chlorpromazine and trifluoperazine (300 M), prevented crystal formation and caused dissolution of some crystal bundles, even in the presence of exogenous Ca. When the antagonists were flushed out and replaced with fresh medium, crystals were formed in cells where dissolution had occurred under the influence of the antagonists. The Ca ionophore A 23187 (20 M) caused slow dissolution of crystal bundles, even in the presence of exogenous Ca. A model describing the control of and physiological significance of Ca oxalate formation in plants is presented and discussed with respect to the results obtained in this study.     

Comparison of maize root mucilages isolated from root exudates and root surface extracts by complementary cytological and biochemical investigations

Summary A strategy to obtain fractions enriched in mucilages secreted by root caps or produced by the rhizodermis of axenicallygrown maize seedlings is proposed. It involves a two-step procedure allowing the successive collection of root exudates and surface extracts from the same set of intact, sterile maize plants. Cytological controls were performed at each phase of collection. Whereas root cap mucilage is easily collected in water after one day's extraction, under conditions favouring secretory activity, rhizodermal mucilage remains tightly adherent to the root surface. It can be better extracted using neutral saline buffer assisted by gentle shaking at low temperature. Acidic saline buffer is unsuitable as it induces cell lysis and release of cell wall components.Biochemical analyses confirm that fractions enriched in root cap mucilage contain very high levels of fucose and galactose, high levels of arabinose, xylose and glucose and trace amounts of mannose. Fractions enriched in rhizodermal mucilage contain large amounts of glucose, moderate amounts of arabinose, xylose, mannose and galactose and trace levels of fucose. Isoelectric focusing and SDS-PAGE indicate that there are numerous similarities in the protein composition of materials enriched in root cap mucilages from root exudates or aqueous root surface extracts. However, specific protein bands that could be characteristic of rhizodermal mucilage are obtained using neutral saline buffer extracts. According to these biochemical data, the two-step procedure used in the present study appears to be useful for further biochemical characterization of both types of mucilages.Abbreviations BSA bovine serum albumin - BSTFA N,O-bis (trimethylsilyl)-trifluoroacetamide - DTT dithiothreitol - i. d. internal diameter - MW molecular weight - PATAg periodic acid-thiosemicarbazide-silver proteinate - PVPP polyvinylpolypyrrolidone - RE root exudates - RSE root surface extracts - TMCS trimethylchlorosilane - TMS trimethylsilyl