Auxin, Cytokinin and Ethylene Differentially Regulate Specific Developmental States Associated with Shoot Bud Morphogenesis in Leaf Tissues of Mangosteen (Garcinia mangostana L.) Cultured in Vitro

Hormonal regulation of de novo shoot bud formation in leaf explantsof mangosteen has been studied from a developmental perspective.This analysis indicates that at least three discrete, experimentallydistinguishable developmental states, namely, morphogenic competence,caulogenic determination and organ differentiation, were expressedduring shoot bud morphogenesis. The state of morphogenic competencein leaf tissues was expressed maximally between days 10 and12 of leaf development. Competent cells in explants requireda minimum of 6 days of BA treatment (20 µM) to becomecaulogenically determined. Such determined cells would continueshoot organogenesis on medium devoid of growth regulators. Delayingof BA exposure for as short as 2 days caused a dramatic declinein tissue competence. The state of competence and the processof caulogenic determination were adversely affected by IAA,but were insensitive to ethylene or its precursor, ACC. Shootbud differentiation was greatly enhanced by BA, but selectivelydelayed by ethylene. IAA also showed an inhibitory effect onshoot bud differentiation, but not mediated through ethylene.The distinct roles of auxin, cytokinin and ethylene on the regulationof shoot bud development in mangosteen leaf explants have beendiscussed on the basis of the current understanding of the conceptof tissue competence, determination and differentiation. (Received August 12, 1996; Accepted October 31, 1996)     

Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae)

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.     

The effect of the gaseous state on bud induction and shoot multiplication in vitro in eastern white cedar

The effect of vessel type and the gaseous phase on the morphogenic response of Thuja occidentalis L. explants in vitro was studied. Explants were cultured in container types that varied in their degree of gas exchange. Traps for ethylene and CO₂ were employed. During shoot bud induction from embryonic explants, the number and elongation of shoot buds improved significantly when gastight, serum-capped flasks were used compared to the foam bung-capped flasks or the regularly used Petri dishes. Elimination of the two gases from the headspace of the flasks either singly or together reduced shoot bud induction and especially elongation of shoots. A similar response was seen during axillary bud development from cultured shoots. Ethylene and CO₂ accumulation promoted development and elongation of axillary shoots. An increase in the zeatin concentration in the medium produced a greater number of axillary shoots and higher levels of ethylene in the culture vessels. Removal of CO₂ caused gradual death of the shoots, while removal of ethylene alone reduced axillary shoot lengths significantly. Inclusion of aminoethoxyvinylglycine in the medium combined with ethylene traps produced an effect similar to the use of ethylene traps alone.     

Anatomy and development of the fern sporophyte

Recent research on the developmental anatomy and morphology of the fern sporophyte is reviewed. Detailed histological and experimental studies of the organization of the fern shoot apical meristem have reconfirmed the recently controversial role of the shoot apical cell as the single apical initial of the meristem. The shoot apical meristem is nevertheless an anatomically and functionally complex structure with a strongly zoned cytohistological organization. Fern shoot apex organization can be compared with that of seed plants. The control of leaf initiation and phyllotaxy remains poorly understood. Studies differ as to whether leaf initiation in ferns involves one leaf mother cell or a multicellular region of the shoot apex. The concept of non-appendicular fronds is refuted for living ferns. The later developmental changes in the determinate leaf apical and marginal meristems of the leaf primordium form an area that is still largely unexplored but could be investigated by methods similar to those used to study shoot and root apices. Branching in ferns is morphologiclaly and developmentally diverse. There is apparently more than one developmental mode of dichotomous branching, and several modes of lateral bud formation have been described, including the phyllogenous initiation of branches at the base of leaf primordia. Developmental changes in bud meristems related to apical dominance, inhibition, and bud activation is another major area for continued study. The traditional concept of the role of the root apical cell has been reestablished by studies similar to those made of the shoot apex. Detailed ultrastructural investigations of the root ofAzolla have given a sophisticated new picture of developmental processes in that organ. Fern roots show remarkably precise patterns of histogenesis in relation to apical segmentation. The formation of secondary vascular tissue inBotrychium suggests that the Ophioglossales may be related to the seed plants. The causal relationship of leaf (and branch and root) formation and the initiation of vascular tissue in the shoot needs more study. Although still poorly understood, protoxylem systems in ferns are variable and may have morphological and systematic significance. Recent investigations of hydraulic conductance in fern stems have found possible correlations of conductance levels with growth forms. The anatomical diversity of ferns makes comparative functional anatomy a promising field for future study.     

Effects of nitrogen forms on dry matter partitioning and nitrogen metabolism in two contrasting genotypes of Catasetum fimbriatum (Orchidaceae)

The effects of either organic (urea and glutamine) or inorganic nitrogen forms (nitrate and ammonium) on dry matter accumulation in shoots and roots and on nitrogen assimilatory enzyme activities were studied in two Catasetum fimbriatum genotypes. Both genotypes, which had inverse patterns of dry matter partitioning between shoots and roots, were aseptically incubated in gelled culture media containing 6 mol m⁻³ of nitrogen and incubated in growth chamber for 30 and 60 days. In vivo nitrate reductase, glutamine synthetase, glutamate dehydrogenase activities as well as free ammonium contents were determined in shoots and roots of plants grown in four different nitrogen sources. Nitrogen assimilatory enzyme activities showed the highest values in the genotype that accumulated dry matter predominantly in the shoots. The nitrogen sources supplied affected dry matter accumulation in shoots and roots of both C. fimbriatum genotypes; however, they were not enough to change the characteristic pattern of dry matter partitioning of each genotype. On the other hand, the differences in the root/shoot ratio found among nitrogen treatments were relatively higher in the genotype that directed dry matter mainly to roots than in the genotype that allocates biomass to shoots. Our results suggest that NADH-dependent glutamate dehydrogenase plays an important role in ammonium assimilation in C. fimbriatum plants, particularly in the root system. Nitrogen metabolism and the dry matter partitioning of the two genotypes are discussed.     

Abscisic acid and auxin accumulation in Catasetum fimbriatum roots growing in vitro with high sucrose and mannitol content

Endogenous contents of indolyl-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in excised roots of Catasetum fimbriatum (Orchidaceae) cultured in vitro on solidified Vacin and Went medium with 1, 2, 4, 6, 8 and 10 % sucrose, as well as 2 % sucrose plus mannitol. Maximum root growth was observed in media with 4 % sucrose and 2 % sucrose plus 2.2 % mannitol, suggesting that a moderate water or osmotic stress promotes orchid root growth. Contents of both ABA and IAA increased in parallel to increasing sucrose concentration and a correlation between root elongation and the ABA/IAA ratio was observed. Incubating isolated C. fimbriatum roots with radiolabeled tryptophan, we showed an accumulation of IAA and its conjugates.     

Regulation of root development in <Emphasis Type="BoldItalic">Arabidopsis thaliana</Emphasis> by phytohormone-secreting epiphytic methylobacteria

In numerous experimental studies, seedlings of the model dicot Arabidopsis thaliana have been raised on sterile mineral salt agar. However, under natural conditions, no plant has ever grown in an environment without bacteria. Here, we document that germ-free (gnotobiotic) seedlings, raised on mineral salt agar without sucrose, develop very short root hairs. In the presence of a soil extract that contains naturally occurring microbes, root hair elongation is promoted; this effect can be mimicked by the addition of methylobacteria to germ-free seedlings. Using five different bacterial species (Methylobacterium mesophilicum, Methylobacterium extorquens, Methylobacterium oryzae, Methylobacterium podarium, and Methylobacterium radiotolerans), we show that, over 9 days of seedling development in a light-dark cycle, root development (hair elongation, length of the primary root, branching patterns) is regulated by these epiphytic microbes that occur in the rhizosphere of field-grown plants. In a sterile liquid culture test system, auxin (IAA) inhibited root growth with little effect on hair elongation and significantly stimulated hypocotyl enlargement. Cytokinins (trans-zeatin, kinetin) and ethylene (application of the precursor ACC) likewise exerted an inhibitory effect on root growth but, in contrast to IAA, drastically stimulated root hair elongation. Methylobacteria are phytosymbionts that produce/secrete cytokinins. We conclude that, under real-world conditions (soil), the provision of these phytohormones by methylobacteria (and other epiphytic microbes) regulates root development during seedling establishment.     

Shoot inversion-induced ethylene in pharbitis nil induces the release of apical dominance by restricting shoot elongation

Shoot inversion induces outgrowth of the highest lateral bud (HLB) adjacent to the bend in the stem in Pharbitis nil. In order to determine whether or not ethylene produced by shoot inversion plays a direct role in promoting or inhibiting bud outgrowth, comparisons were made of endogenous levels of ethylene in the HLB and HLB node of plants with and without inverted shoots. That no changes were found suggests that the control of apical dominance does not involve the direct action of ethylene. This conclusion is further supported by evidence that the direct application of ethylene inhibitors or ethrel to inactive or induced lateral buds has no significant effect on bud outgrowth. The hypothesis that ethylene evolved during shoot inversion indirectly promotes the outgrowth of the highest lateral bud (HLB) by restricting terminal bud (TB) growth is found to be supported by the following observations: (1) the restriction of TB growth appears to occur before the beginning of HLB outgrowth; (2) the treatment of the inverted portion of the shoot with AgNO₃, an inhibitor of ethylene action, dramatically eliminates both the restriction of TB growth and the promotion of HLB outgrowth which usually accompany shoot inversion; and (3) the treatment of the upper shoot of an upright plant with ethrel mimics shoot inversion by retarding upper shoot growth and inducing outgrowth of the lateral bud basipetal to the treated region.     

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.     

Growth regulators in changing apical growth at transition to flowering

Reorganization of growth in the shoot apex ofChenopodium rubrum during transition to flowering is described. Growth and morphogenic changes — a rise in cell division rate, changes in leaf and bud formation and changes in directions of cellular growth — are viewed from the aspect of a possible role of growth hormones in controlling these changes. Growth and morphogenic effects of exogenous growth regulators in the shoot apex ofChenopodium are summarized and their floral effects explained in terms of changing apical growth correlations. New evidence concerning the timing of increased cell division rate and showing the limited requirement of axillary cell division and a shift to more vertical direction of growth in the apex in the floral developmental pathway was obtained in experiments with kinetin application and by surgical treatments.     

Gravistimulus Direction, Ethylene Production and Shoot Elongation in the Release of Apical Dominance in Pharbitis nil

Prasad, T. K. and Cline, M. G. 1985. Gravistimulus direction,ethylene production and shoot elongation in the release of apicaldominance in Pharbitis nil.—. exp. Bot. 36: 1969–1975.Release of apical dominance can be induced in Pharbitis nilby the inversion of the upper shoot. This promotion of outgrowthof the highest lateral bud adjacent to the bend of the stemappears to be mediated by ethylene inhibition of growth of theinverted main shoot. In the present investigation the existenceof a direct correlation between ethylene evolution and the directionof gravistimulus is demonstrated as well as an inverse correlationbetween ethylene production by the inverted upper shoot andits elongation. An inverse correlation also exists between elongationof the inverted upper shoot and the outgrowth of the highestlateral bud if the lower portion of the shoot (below the bend)is oriented in an upright position. The latent period for shoot–inversioninduction of ethylene production is about 2 h. These resultssupport the hypothesis of indirect ethylene control of apicaldominance release by retardation of elongation of the invertedshoot. Key words: Shoot inversion, gravistimulus, ethylene, latent period, bud outgrowth, pharbitis nil     

An assessment of the role of ethylene in mediating lettuce (Lactuca sativa) root growth at high temperatures

Growth of temperate lettuce (Lactuca sativa) plants aeroponically in tropical greenhouses under ambient root-zone temperatures (A-RZTs) exposes roots to temperatures of up to 40 degrees C during the middle of the day, and severely limits root and shoot growth. The role of ethylene in inhibiting growth was investigated with just-germinated (24-h-old) seedlings in vitro, and 10-d-old plants grown aeroponically. Compared with seedlings maintained at 20 degrees C, root elongation in vitro was inhibited by 39% and root diameter increased by 25% under a temperature regime (38 degrees C/24 degrees C for 7 h/17 h) that simulated A-RZT in the greenhouse. The effects on root elongation were partially alleviated by supplying the ethylene biosynthesis inhibitors aminooxyacetic acid (100-500 microM) or aminoisobutyric acid (5-100 microM) to the seedlings. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to seedlings grown at 20 degrees C mimicked the high temperature effects on root elongation (1 microM) and root diameter (1 mM). Compared with plants grown at a constant 20 degrees C root-zone temperature, A-RZT plants showed decreased stomatal conductance, leaf relative water content, photosynthetic CO(2) assimilation, shoot and root biomass, total root length, the number of root tips, and root surface area, but increased average root diameter. Addition of 10 microM ACC to the nutrient solution of plants grown at a constant 20 degrees C root-zone temperature mimicked the effects of A-RZT on these parameters but did not influence relative water content. Addition of 30 microM aminoisobutyric acid or 100 microM aminooxyacetic acid to the nutrient solution of A-RZT plants increased stomatal conductance and relative water content and decreased average root diameter, but had no effect on other root parameters or root and shoot biomass or photosynthetic CO(2) assimilation. Although ethylene is important in regulating root morphology and elongation at A-RZT, the failure of ethylene biosynthesis inhibitors to influence shoot carbon gain limits their use in ameliorating the growth inhibition induced by A-RZT.     

Role of ethylene and cytokinins in the initiation of lateral shoot growth in bromeliads

Aechmea victoriana var discolor L. B. Foster and Aechmea dactylina Bal. are commercially propagated in vitro through lateral shoot growth. A modified Murashige and Skoog medium is used which contains both BA and IAA. These growth substances were shown in the present study to synergistically stimulate the production of ethylene by the cultured plants. The stimulation of ethylene production is correlated with the outgrowth of the lateral buds. The rise in ethylene production was concluded to induce lateral shoot growth, because: (a) outgrowth of the shoots was blocked by preventing an increase in ethylene production, (b) 1-aminocyclopropane-1-carboxylic acid (ACC), the natural precursor of ethylene biosynthesis, substituted for IAA in the promotion of ethylene production and lateral bud outgrowth. Although ACC could substitute for IAA, it could not substitute for BA; therefore, cytokinins are concluded to be essential for lateral bud outgrowth in vitro in Aechmea. These results suggest that cytokinins and ethylene both play roles in natural lateral bud initiation and that the cytokinin function involves two stages of the process.     

Gravistimulus direction, ethylene production and shoot elongation in the release of apical dominance in Pharbitis nil

Release of apical dominance can be induced in Pharbitis nil by the inversion of the upper shoot. This promotion of outgrowth of the highest lateral bud adjacent to the bend of the stem appears to be mediated by ethylene inhibition of growth of the inverted main shoot. In the present investigation the existence of a direct correlation between ethylene evolution and the direction of gravistimulus is demonstrated as well as an inverse correlation between ethylene production by the inverted upper shoot and its elongation. An inverse correlation also exists between elongation of the inverted upper shoot and the outgrowth of the highest lateral bud if the lower portion of the shoot (below the bend) is oriented in an upright position. The patent period for shoot-inversion induction of ethylene production is about 2 h. These results support the hypothesis of indirect ethylene control of apical dominance release by retardation of elongation of the inverted shoot.     

Response of spring rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant

Responses of rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria, Pseudomonas putida Am2, Pseudomonas putida Bm3, Alcaligenes xylosoxidans Cm4, and Pseudomonas sp. Dp2, containing 1-aminocyclopropane-l-carboxylate (ACC) deaminase were studied using growth pouch and soil cultures. In growth pouch culture, the bacteria significantly increased root elongation of phosphorus-sufficient seedlings, whereas root elongation of phosphorus-deficient seedlings was not affected or was even inhibited by the bacteria. Bacterial stimulation of root elongation of phosphorus-sufficient seedlings was eliminated in the presence of a high ammonia concentration (1 mM) in the nutrient solution. Bacterial effects on root elongation of potassium-deficient and potassium-sufficient seedlings were similar. The bacteria also decreased inorganic phosphate content in shoots of potassium- and phosphorus-sufficient seedlings, reduced ethylene production by phosphorus-sufficient seedlings, and inhibited development of root hairs. The effects of treatment with Ag+, a chemical inhibitor of plant ethylene production, on root elongation, ethylene evolution, and root hair formation were similar to bacterial treatments. The number of bacteria on the roots of phosphorus-deficient seedlings was not limited by phosphorus deficiency. In pot experiments with soil culture, inoculation of seeds with bacteria and treatment with aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis in plants, increased root and (or) shoot biomass of rape plants. Stimulation of plant growth caused by the bacteria was often associated with a decrease in the content of nutrients, such as P, K, S, Mo, and Ba, in shoots, depending on the strain used. The results obtained show that the growth-promoting effects of ACC-utilizing rhizobacteria depend significantly on the nutrient status of the plant.     

Lateral Bud Growth in Phaseolus vulgaris L. and the Levels of Ethylene in the Bud and Adjacent Tissue

Ethephon and the ethylene inhibitors Ag⁺ and aminoethoxyvinylglycine (AVG) inhibited outgrowth of the axillary bud of thefirst trifoliate leaf in decapitated plants of Phaseolus vulgaris.Endogenous ethylene levels decreased in the stem upon decapitationalthough it is not conclusive that a causal relationship existsbetween this decrease and the release of axillary buds frominhibition. The proposition that auxin-induced ethylene is responsiblefor the suppression of axillary bud growth in the decapitatedplant when the apical shoot is replaced by auxin is not borneout in this study. Application of IAA directly to the axillarybud of intact plants gave rise to a transient increase in budgrowth. This growth increment was annulled when AVG was suppliedwith IAA to the bud despite the fact that the dosage of AVGused did not affect the normal slow growth rate of the bud ofthe intact plant or bud outgrowth resulting from shoot decapitation.     

RAPD,ISSR, and SCoT markers based genetic stability assessment of micropropagated Dendrobium fimbriatum Lindl. var. oculatum Hk. f.- an important endangered orchid

Dendrobium fimbriatum is an ornamental and medicinal orchid listed in the Red data book of IUCN. Phytohormones’ effect on the in vitro regeneration of the orchid was studied using Mitra medium supplemented with different growth regulators. KN produced effective shoot formation when present alone or in combination with IBA or NAA. The shooting was gradually increased when KN concentration was increased from 0.8 to 4.8 mg L⁻¹, but the opposite response was observed with BAP at higher concentration (4.8 mg L⁻¹). IBA either in combination with BAP or KN promoted effective root development and multiplication. Micropropagated orchids grown in the basal medium devoid of any phytohormone showed 100% monomorphism, while low genetic polymorphism of 1.52% (RAPD—Random Amplification of Polymorphic DNA), 1.19% (ISSR-Inter Simple Sequence Repeat) and 3.97% (SCoT—Start Codon Targeted) was exhibited among the regenerants propagated in the hormone enriched medium. UPGMA (Unweighted pair group method using arithmetic averages) dendrograms showed the grouping of mother plant (MP) with the in vitro regenerants. The principal coordinate analysis (PCoA) further confirmed the clustering patterns as determined by the cluster analysis. The study reported for the first time the successful in vitro propagation of Dendrobium fimbriatum and their genetic stability assessment using molecular markers.     

Flooding effects on rapid responses of the invasive plant Alternanthera philoxeroides to defoliation

In experiments under controlled growth conditions it was examined how flooding affected the responses of the invasive plant Alternanthera philoxeroides to defoliation. In drained and flooded conditions, plants were subjected to five defoliation levels: 0, 10, 50, 90% removal of leaf tissue and apex removal (90% leaf tissue plus apical bud removal). Plants were harvested weekly for five weeks. In drained conditions, plant biomasses including total biomass, shoot biomass and root biomass after 50% defoliation rapidly recovered to the control plant level. They were significantly lower for the 90% defoliation and apex removal treatments compared to control plants throughout the experiment. In flooded conditions, total biomass and shoot biomass after 50% defoliation, 90% defoliation, and apex removal treatments could return to control plant levels before the end of the experiment. In 90% defoliation and apex removal treatments root to shoot biomass ratios of both drained and flooded plants were initially much higher than in control plants, but the difference disappeared rapidly. The final biomasses decreased with increased defoliation intensity in drained conditions, but no significant difference was generally found in any of the defoliation treatments in flooded conditions. The rapid re-growth of A. philoxeroides plants after defoliation may partly be responsible for its invasion success. However, defoliation capable of removing 90% of the leaf tissue may be desirable in restricting the growth of this invasive species in drained conditions.     

Long-term copper (Cu2+) exposure impacts on auxin, nitric oxide (NO) metabolism and morphology of Arabidopsis thaliana L.

Plants are able to dynamically adapt to their environment by reprogramming of their growth and development. Copper (Cu²⁺) excess modifies shoot and root architecture of plants by a lesser known mechanism, therefore the involvement of a major hormone component (auxin) and a signal molecule (nitric oxide) in Cu²⁺-induced morphological responses were studied in Arabidopsis using microscopic methods. Auxin-inducible gene expression was visualized in DR5::GUS Arabidopsis and nitric oxide (NO) levels were detected by DAF-FM fluorophore in the stem and root system. Copper excess caused the inhibition of stem and root growth of Arabidopsis, during which cell elongation, division and expansion were also affected. The symptoms of stress-induced morphogenic response were found in the root system of 25???M Cu²⁺-treated plants. In both organs, the decrease of auxin-dependent gene expression was found, which can partly explain the growth inhibitions. Besides hormonal system, nitric oxide metabolism was also affected by Cu²⁺. In root tips, copper excess induced NO generation, while NO content in lateral roots was not affected by the treatments. Using nia1nia2 mutants, nitrate reductase enzyme as a putative source of Cu²⁺-induced NO was identified in Arabidopsis primary roots.