Action of Kinetin on Cotyledons of Fenugreek

Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinmtiating 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 (a) knowledge of the time lag between hormone application and the earliest known response (RNA synthesis), (b) analysis of the RNA being synthesized, and (c) a measure of the cytokinin effect on protein synthesis.MATERIALS AND METHODS Segments (I mm) were excised at the 10-11th mm behind the root tip of 66-h-old surface-sterilized, aseptically germinated seeds of Pisum sativum (cv. Little Marvel) as described (20). These segments were cultured as such or converted to cortical explants (segments with the central vascular tissue punched out) and then cultured on standard S2M medium supplemented with kinetin (1 ,ug/ml) as indicated on filter paper wicks in the dark at 25 C (12, 27).After culturing (see figure legends) (New England Nuclear, 53 mCi/mmol, 5 LCi/ml) was added to the medium. The labeling period was ended by transfer to ethanol acetic acid fixative (3:1, v/v) and the segments stored in 70% ethanol for the autoradiographic work. For biochemical work, the tissue was rinsed with ice cold culture medium (3 times) containing unlabeled adenosine (I mM) then drained and frozen in an acetone/dry ice bath and stored at -78 C.RNA extraction was based on a procedure modified from that of McKnight and Schimke (14). Frozen tissue was dropped into sterile SET buffer (10 mm Tris-HCl, pH 7.4, 5 ms EDTA, 1% SDS) containing heparin (I mg/ml) (Riker Laboratories) and 10 pi/ml diethylpyrocarbonate in a ground glass homogenizer. The tissue was ground with several strokes of the pestle until finely dispersed, and the homogenate then transferred to a 15-ml Corex centrifuge tube and centrifuged (2 C, 3,000g max, 15 min) in a Beckman J21B centrifuge with the JA 20 angle rotor. The supernatant was extracted with the phenol chloroform method. Redistilled phenol, saturated with SET buffer (8 ml), and chloroform (8 ml) was added to the low speed supernatant (10 ml) and the mixture shaken at room temperature for 30 min. The phases were separated by centrifugation (21 C, 23,700g max, 30 min). The aqueous phase was extracted in the same way a second time and generally no precipitate was evident at the phase boundary after the second centrifugation. Nucleic acids were precipitated from the aqueous phase at -20 C ovemight by transferring the aqueous phase to a Corex tube containing an equal volume of 0.2 M NaCl in 80%o ethanol. The precipitated nucleic acids were recovered by centrifugation (2 C, 23,700g max, 30 min). The supernatant was discarded, the tube drained, and then rinsed sequentially with 80%o ethanol and anhydrous ethyl ether at -20 C. The samples were lyophylized and stored at -20 C.

supporting

confidence: 73%

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

Shininger

1980

“…Rijven and Parkash (11) reported that sucrose increased expansion of kinetin-treated fenugreek cotyledons when incubated in darkness, but (10 x 20 cm). These trays were then enclosed in polyethylene bags and the seed germinated in darkness at 25°C for 38 h. For the experiments, the inner cotyledon from each seedling was excised and placed adaxial surface down on Whatman No.…”

mentioning

confidence: 99%

Invertase Activity and the Kinetin-Stimulated Enlargement of Detached Radish Cotyledons

Howard¹,

Witham²

1983

Cytokinin treatment is known to promote expansion of light-grown excised radish (Raphanus sativus L. cv Crimson Giant) cotyledons. This expansion, at least in part, seems to be related to an increased accumulation of osmotically active reducing sugars. Kinetin treatment did not cause increased levels of isocitrate lyase activity over the controls, but stimulated increased levels of two invertase forms, designated types I and II. Type I was soluble and type II was insoluble after homogenization in 10 millimolar tris(hydroxymethyl)aminomethane-HCl (pH 7.0). Both types were soluble after homogenization in 300 millimolar NaCI. At low salt concentration, type II was retained on a diethylamioethyl-cellulose column and type I was not. Type II was then eluted from the column at high salt concentration. Types I and II exhibited pH optima of 53 and 43, Michaelis constants of 4.96 and 1.23 millimolar sucrose, and molecular weights of 65,000 and 57,000 daltons, respectively. The kinetin promotion of reducing sugar accumulation may be related to increased levels of the two invertase forms, but is probably not a result of direct cytokinin-stimulated glyoxysomal activity.

“…On the other hand, with 8-azaguanine both net RNA synthesis and expansion are inhibited. These together with other observations suggest that kinetin-induced expansion in cotyledons is mediated by the synthesis of mRNA (8).…”

mentioning

confidence: 71%

“…The use of cotyledons has a number of advantages from the point of simplicity in laboratory practice. Also cotyledons exhibiting cytokinin specific responses may be used to investigate the biochemical mechanism of cytokinin action as we report elsewhere (8). Here we describe the expansion of isolated cotyledons as affected by some growth substances, light, nitrogenous compounds, sucrose, and dormancy status.…”

mentioning

confidence: 99%

Cytokinin-induced Growth Responses by Fenugreek Cotyledons

Rijven

Parkash

1970

Self Cite

As shown below, isolated fenugreek (Trigonella foenum graecum L.) cotyledons respond readily to cytokinins with increased expansion. Similar responses have been described for isolated cotyledons of spinach (4) and radish (5) and for sections of Xanthiwn cotyledons (3). A comparison shows that the fenugreek cotyledons exhibit a higher degree of specificity as they do not respond to GA. The findings may lead to an alternative bioassay of cytokinins. The use of cotyledons has a number of advantages from the point of simplicity in laboratory practice. Also cotyledons exhibiting cytokinin specific responses may be used to investigate the biochemical mechanism of cytokinin action as we report elsewhere (8). Here we describe the expansion of isolated cotyledons as affected by some growth substances, light, nitrogenous compounds, sucrose, and dormancy status. As in other Trifolieae, the attached cotyledons do not merely function as a reserve store, but expand and become green in light.The cotyledons can be separated from the embryonic axis by a cut through the dry seed as the space between these parts is indicated by a groove in the seed exterior. Further manipulationssterilization and washing of cut seeds, their transfer onto wet filter paper in Petri dishes, removal of seed coats 20 hr later, weighing and layering of cotyledons-were done in green safe light. Usually five cotyledons were arranged in a 7-cm Petri dish containing one filter paper disc to which was added 3 ml of test solution containing 1 mg each of mycostatin (nystatin), penicillin, and streptomycin per 50 ml while four to six replicates constituted one treatment. For light treatments the dishes were exposed to continuous incandescent light (30 ft-c near dishes) at 25 C. The dark treatments were enclosed in light tight boxes at the same temperature. CO2 treatment was given by incubating an uncovered Petri dish containing the cut seeds during the initial 20-hr soaking period for 18 hr in a desiccator in which CO2 was released from sodium bicarbonate by hydrochloric acid.To minimize variability due to differences in initial size of cotyledons, results have been expressed as relative increases (%) in fresh weight. Thus, within one experiment equal kinetininduced expansion was observed for samples of seed differing close to 100% in average air dry weight.