Catabolism of glucose in pea stem section during root formation and its inhibition by kinetin and ethionine

The catabolism of specifically¹⁴C-labelled glucose during the root formation and its inhibition by kinetin and ethionine in the etiolated pea stem sections were studied. The formation of root meristematic foci in the pericycle region of sections was accompanied by the decrease of the C₆/C₁ ratio. Such a result and activation of pentose phosphate cycle, which was also checked by another method, suggested increased participation of pentose phosphate cycle in glucose oxidation. The above mentioned changes were also found after the prevention of root formation by ethionine and, therefore, do not seem to be specific for the meristematic foci formation. The growth of newly formed roots was closely connected with the rise of C₆/C₁ values. The increase of CO₂ release from the first carbon atom of glucose molecule was recorded after the inhibition of root formation by ethionine. The rise of C₆/C₁ values and decrease of pentose phosphate cycle activity was observed after the treatment of pea stem sections by kinetin in the first 64 hours after sectioning. In this case root formation was prevented and the growth of lateral buds was stimulated. The secondary xylem formation which took place later was accompanied by the activation of the pentose phosphate cycle. These phenomena are discussed in relation to cell division and biosynthesis of lignin-precursors.     

Dynamics of amino acids in pea stem sections during root formation and its inhibition by kinetin and ethionine

An investigation was conducted to study the interrelation of free amino acid metabolism and root formation in etiolated pea stem sections as dependent on time and on inhibition of root formation by kinetin and ethionine. The rise in the level of aspartic acid and increase in the rate of conversion of¹⁴C-labeled glucose to free amino acids were found to be characteristic features of the formation of foci of meristematic cells in pericyclo region. The formation of roots was reflected, in general, much more in the rate of conversion of labeled glucose to free amino acids than in the levels of corresponding amino acids. The total amount of free amino acids was not significantly changed during incubation of stem sections in a solution of kinetin (5×10^?5 m). A rapid fall in their level was recorded in the next 24 hours. The incorporation of¹⁴C from glucose into a precursor of lignin, phenylalanine, was completely inhibited by kinetin which stimulated simultanously the growth of adjacent buds. Stimulation of secondary xylem formation, which appeared later, was accompanied by the resumption of¹⁴C-incorporation into phenylalanine. Inhibition of root formation by ethionine resulted in the rapid fall of the level of most amino acids and in a significant decrease in the rate of incorporation of¹⁴C from glucose into amino acids. A decreasing level of ethionine in tissues during cultivation of ethionine-treated stem sections was accompanied by a gradual rise in the individual amino acids and in the rate of conversion of glucose into free amino acids.     

Fractionation of proteins of pea stem sections during root formation and its inhibition by kinetin and ethionine

Changes in the protein constituents in pea stem sections during root formation and its inhibition by kinetin and ethionine were studied. Only quantitative differences in the protein fractions separated on DEAE-cellulose column were noted. The formation of foci of meristematic cells in pericycle was accompanied by an increase in the amount of fraction “l”. This fraction disappeared rapidly in sections where root formation either did not occur (internodial sections without nodes) or was inhibited by ethionine (stem sections with basal and apical nodes). Incubation of stem sections in a kinetin solution for 16 hours after cutting of stems partially preserved fraction “l”. The increase in the amount of fraction “l” was one of the first metabolic changes in root-forming pea stem sections after cutting of stems.     

Interaction of Growth Retardants and Temperature in Growth, Flowering, Regeneration, and Auxin Activity of Begonia × cheimantha Everett

Soil application of CCC reduced stem and leaf growth in Begonia plants. This effect was evident with all concentrations tested at 18°C, whereas at 21 and 24°C no growth–retarding effect was observed with 2 × 10^?2 M CCC, and with 5 × 10^?3 M growth was even stimulated. Flowering was promoted by CCC in long day and neur–critical temperature, particularly under low light intensity in the winter. The formation of adventitious buds in leaves of plants grown at 21 and 24°C was stimulated when the plants received 5 × 10^?2 and 2 × 10^?2 M CCC, while 8 7times; 10^?2 M was inhibitory. In plants grown at 18°C bud formation was inhibited by all CCC concentrations. Root formation in the the leaves was usually stimulated by high CCC concentrations, while root elongation was reduced. The level of ether–extractable. acidic auxin (presumably IAA) in the leaves was lowered by CCC treatment of the plants, hut this required higher CCC concentrations at higt than at low temperature. When applied to detached leaves CCC stimulated bud formation at concentrations ranging from 10^?4 to 10^?2 M in leaves planted at 18 and 21°C. At 24°C budding was inhibited by 10^?2 M CCC, the lower concentrations being stimulatory also at this temperature. Root formation and growth were not much affected by CCC treatment of the leaves, but increased with the temperature. Soil application of Phosfon (4 × 10^?4 M) had no effect on growth and flowering, nov did it affect the subsequent regeneration of buds and roots in the leaves. In detached leaves Phosfon stimulated bud formation with au optimum at 10^?6 M. Root formation was stimulated by Phosfon at all temperatures, the optimal concentration being 10^?5 M, whereas root length was conversely affected. Foliar application of B-995 to intact plants and treatment of detached leaves greatly inhibited the formation of buds and had little effect on root formation. B-99D reduced the growth and delayed flowering in the plants.     

In vitro plant regeneration of Hedychium roxburghii blume through rhizome-meristem culture

Full grown plants have been raised successfully in vitro by culturing meristems excised from rhizome of Hedychium roxburghii Blume on Murashige and Skoog's medium supplemented with NAA and kinetin. Zeatin associated with IAA stimulated the development of buds on the basal region of the stem. A low concentration of NAA (0.2 mg.1^-1) enhanced the root formation on young excised buds.     

Patterns of protein synthesis in dormant and growing vegetative buds of pea

Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silver-staining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N⁶-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.Abbreviations IAA indole-3-acetic acid - IEF isoelectric focusing - KIN kinetin (N⁶-furfurylaminopurine) - SDS sodium dodecylsulfate - TCA trichloroacetic acid - 2D-PAGE two-dimensional polyacrylamide gel electrophoresis     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Biochemical and Cytological Analyses of RNA Synthesis in Kinetin-treated Pea Root Parenchyma

Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinitiating DNA synthesis. Few cells synthesize RNA in the 1st hour of culture. In the presence of kinetin/auxin, the nuclear labeling index increases 2.5-fold as compared to control cultures. The RNA synthesis response has an apparent lag period of 2-4 hours as shown by double label ([³H]adenosine/[¹⁴C]adenosine) experiments. Qualitatively, the RNA synthesized at 4-6 hours sediments between 18S and 5S. The RNA synthesized at 14-16 hours and 24-26 hours is primarily ribosomal RNA when kinetin is present. In the absence of kinetin, no clear pattern of RNA synthesis emerges.     

Biochemical responses of pea root tissue to cytokinin: enhanced rates of RNA synthesis

Rates of RNA synthesis were studied in cultured pea (Pisum sativum) root segments and cortical explants which require the hormone cytokinin for DNA replication and cell proliferation. Rate calculations were based on the specific radioactivity of the extracted RNA and the specific radioactivity of the extracted ATP pool after a pulse with ³H-adenosine. The kinetics of RNA synthesis was studied after 24 hours of culture with or without kinetin. We found that kinetin stimulated a 2- to 4-fold enhancement in the rate of RNA synthesis after 24 hours of culture as compared to controls. A similar order of magnitude of stimulation of RNA synthesis was found when RNA was isolated by cesium chloride centrifugation. Pulses during the first 24 hours indicate that kinetin stimulates the rate of RNA synthesis as early as 9 hours after treatment has begun. During the first 24 hours of culture, kinetin did not affect the specific radioactivity of the ATP pool. The ATP pool equilibrated slowly with the exogenous label (³H-adenosine) in the presence or absence of kinetin. After 3 days in culture, we found kinetin to cause an expansion of the extractable ATP pool and a corresponding reduction in the ATP pool specific radioactivity. We interpret these results to indicate a stimulation in the rate of RNA synthesis due to kinetin treatment prior to any other known response.     

Effects of kinetin and root tip removal on exudation and potassium (rubidium) transport in roots of honey locust

Exudation, (86)Rb transport, and water permeability were examined in excised roots of honey locust (Gleditsia triacanthos L.) treated by removing the tip 2 mm (tip-cut 2 mm) or tip 8 mm of the root, or by adding kinetin, or by both treatments. Tip removal increased the rate of exudation. Kinetin, 5 x 10(-6)m, inhibited exudation and Rb transport in tip-cut 2-mm roots; the inhibition was reversible. Kinetin inhibition of exudation was initially associated with lower K(Rb) transport and later with decreases in both ion transport and water permeability. Exudation was also inhibited at 10(-10) to 10(-7)m kinetin. Exudation from roots with intact tips was not altered by kinetin until after about 24 hours. Light during the exudation period had no significant (95%) influence on rate of exudation during the first 24 hours whether root tips were cut or kinetin applied.The results suggest the involvement of the root tip in regulating exudation in other parts of the root. This regulation might occur through cytokinin control of water permeability and the rate of ion transport.     

Growth and Organogenesis in Tissue Cultures of Allium cepa var. proliferum

Callus isolated from aerial bulbs of Allium cepa var. proliferum was grown in agar and liquid cultures on a synthetic medium containing 5 × 10^?6M 2,4-D. Root formation occurred in the absence of 2,4-D and was highly stimulated by 5 × 10^?6M NAA. Cytokinin was not necessary for growth and organ formation but slightly stimulated the formation of leafy buds. Combinations of NAA or IAA and cytokinin stimulated growth and root formation to a greater extent than anyone of these substances added alone. Pieces of callus in liquid culture developed roots in one week in root-inducing medium, but bud or embryo formation was not observed in liquid cultures.     

In vitro formation and development of floral buds on tobacco stem explants: effects of kinetin and other factors

Stem segments were excised from plants of Wisconsin 38 tobacco (Nicotiana tabacum L.) in three regions differing in their distance below the inflorescence. They were cultured in vitro in 8- or 16-hr days. After 8 weeks, floral and vegetative buds were counted, and extent of floral development was assessed. Kinetin at 10(-5)m inhibited formation and development of floral buds regardless of indoleacetic acid concentration. Supplied at this concentration with adequate auxin, kinetin stimulated vegetative bud formation and may have caused floral bud abortion. Indoleacetic acid (>/= 10(-6)m) inhibited vegetative and floral bud formation when supplied with low kinetin concentration (/= 10(-6)m), it inhibited floral bud formation and stimulated vegetative bud formation. More floral buds were formed in 16-hr days than in 8-hr days. Few formed on explants other than those derived from the region nearest the inflorescence regardless of other treatment.     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

On ethylene and stem elongation in green pea seedlings

Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10⁻⁵ molar indoleacetic acid (IAA), with submaximal responses occurring at 10⁻⁴ and 10⁻³ molar. Accompanying elongation at concentrations of IAA of 10⁻⁶ to 10⁻³ molar was production of ethylene, with the amount increasing up to 10⁻⁴ molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10⁻⁵ molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable.     

Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls

The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca²⁺, kinetin, or ethylene. Both Ca²⁺ and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca²⁺, kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls.     

Effects of Kinetin on Tiller Bud Elongation in Wheat (Triticum aestivum L.)

Experiments are described in which at four stages of developmentmain tillers of wheat (Triticum aestivum L.) were isolated andinserted in test-tubes containing distilled water or kinetin.Leaves and leaf sheaths were split off to expose a lateral budapproximately 3 mm long whose growth was measured during a periodof 8 days. Kinetin at 6 ppm promoted bud elongation and dry-weightgain at all stages of development and in different light intensitiesand photoperiods. Reducing assimilate supply either by partialdefoliation of the main tiller or treatment with DCMU inhibitedbud growth during the early part of development, an effect whichwas relieved by kinetin. TIBA applied to prevent auxin transportincreased bud growth at the first two but not later stages ofdevelopment. Kinetin also inhibited nodal root growth consistently. At least two phases of bud control in grasses have to be considered,the first being the imposition of apical dominance after initiationof floral organs and stem elongation, the second being the releaseof buds from inhibition around ear emergence. Imposition ofbud inhibition appears to be a function of auxin and assimilatesupply, and at this stage the promoting effects of kinetin maybe through its action of mobilizing flow of assimilates. Therelease of buds at a later stage is apparently not associatedwith auxin, and kinetin may no longer be concerned with assimilateflow but rather with overcoming an inhibitory effect on budgrowth emanating from the leaves.     

Kinetin and naphtaleneacetic acid controlled starch formation in isolated roots ofPisunt sativum

The formation of starch in excised 3-days-old pea roots was enhanced up to three fold after cultivation for five days in liquid media containing 3% glucose, microelements, and 10^-6 M kinetin or 10^-7 M naphtaleneacetic acid (NAA). A synergistic effect of kinetin and NAA on starch formation was observed when both hormones were applied simultaneously over a wide range of concentrations.     

Effects of ethylene, kinetin, and calcium on growth and wall composition of pea epicotyls

Ethylene supplied with indoleacetic acid at 0.1 and 1 mum inhibited elongation and enhanced swelling in epicotyls of decapitated and derooted pea seedlings (Pisum sativum L., var. Alaska). These growth responses were correlated with the development of cell walls rich in weak acid-extractable materials and pectic uronic acids. Ethylene had no effect on the formation of hemicellulose, or hemicellulosic uronic acid. Ethylene stimulated the formation of residual materials at 0.1 mum indoleacetic acid but had little effect at 1 mum. With indoleacetic acid at 10 mum, ethylene modified neither the growth or wall composition appreciably. Growth and wall composition in intact seedlings were modified in similar fashion by ethylene. In intact seedlings ethylene promoted the development of walls high in weak acid-extractable materials and pectic uronic acid. These effects were less impressive in the first 24 hours than in the second 24 hours when the control plants suffered a net loss of these constituents. Ethylene considerably inhibited the formation of hemicellulose and residual wall materials in the apical sections but promoted it in the basal sections of the intact seedlings.Measurements of ethylene production by decapitated and derooted pea seedlings suggest that Ca(2+) and kinetin do not promote swelling through an effect on the formation of ethylene.We propose that cells of ethylene-treated pea epicotyls lack polarity because their walls are abnormally rich in pectic substances.     

Coumarin effects on Glycine max hypocotyl explants

Coumarin induced root formation and stimulated fresh weight production in hypocotyl explants of Glycine max L. cultured in vitro. All stimulatory effects caused by coumarin were induced within a relatively narrow range of concentrations between 1–500 μM, yielding optimum dose response curves. When coumarin was combined with kinetin fresh weight increased considerably, at optimum concentrations to a level almost as high as that obtained with NAA (10 μM) and kinetin (10 μM). Root formation was almost completely inhibited when kinetin was added in combination with coumarin. NAA + coumarin had small stimulatory effects on fresh weight. but were inhibitory in root formation. The frequency of rooting per explant, texture and pigmentation were also affected by different treatments.