Cytokinins in Seeds of Pumpkin

Abstract

Extracts of seeds of pumpkin (Cucurbita pepo Linn.) contain three chromatographically distinguishable cytokinins which are held on Dowex 50-W and are extractable by ethanol and n-butanol. Two of the active factors are precipitable by silver nitrate at acidic pH. The chromatographic behavior and the spectral characteristics of one of these cytokinins are similar to those of zeatin. However, the RF values of the other two active compounds do not match with those of any of the known natural cytokinins.In recent years, considerable interest has been shown in the isolation and characterization of natural cytokinins. Several free cytokinins have been identified in higher plants. They are: zeatin or 6-(4-hydroxy-3-methylbut-trans-2-enyl)aminopurine (14,18), 9-,3-D-ribofuranosylzeatin (16), 9-13-D-ribofuranosylzeatin-5'-phosphate (17), all first isolated from immature corn kernels, and dihydrozeatin, reported to occur in lupin seeds (11,12). Two other cytokinins, namely a cis isomer of zeatin riboside and 6-('y ,y-dimethylallyl)adenosine, have been found as constituents of soluble RNA of plant tissues (6, 7). From this laboratory, two preliminary reports were communicated dealing with the occurrence of cytokinins in developing seeds of watermelon and pumpkin (21,22). This paper gives results of further work on cytokinins in seeds of pumpkin and our attempts to identify these factors. MATERIAL AND METHODSFruits of pumpkin (Cucurbita pepo Linn.) were collected from plants growing on the bank of river Jamuna, Delhi. Seeds were separated from the pulp, fixed in 95% ethanol (300 ml for 100 g of seeds) and stored at 4°. When required, the material was homogenized in a Waring Blendor and filtered, and the residue was twice extracted with 2 volumes of 95% ethanol. The ethanol extracts were pooled and evaporated in vacuo at about 45°. The crude extract thus obtained was partitioned thrice with 2 volumes of petroleum ether each time to remove pigments and fatty substances.The aqueous extract, equivalent to 250 g, fresh weight, of seeds, was reduced to 62.5 ml by evaporation in vacuo, and its pH was adjusted to 5.5. It was then percolated through a 2.5 X 40 cm column of Dowex 50W-X8 (20-50 mesh, H+ form); elution was done with 1 liter of 2 N ammonia. The eluate was evaporated in vacuo to remove ammonia and to reduce its volume further. The final drying was done in a VirTis lyophilizer, and the freezedried eluate was dissolved in a small quantity of distilled water.Descending paper chromatography was mostly done on Whatman No. 3MM chromatography paper which had previously been washed with 1 N formic acid by allowing the acid to run down the papers for 2 to 3 days. The fractions were applied as streaks on the side of the paper. The following solvent systems were used: (a) water-saturated n-butanol; (b) n-butanol-N ammonia, 10:7; (c) n-butanol-acetic acid-water, 12:3:5; (d) isopropanol-water, 4: 1; (e) isopropanol-water, 4:1 (0.1 ml of concentrated ammonia per liter of the tank volume was kept at the bottom of the chamber); (...

Cytokinins in Seedling Roots of Pea

“…It is also extracted into 1-butanol from aqueous solutions at pH 8. The properties of factor II are similar to those of an unidentified cytokinin isolated from sweet corn (17), apple fruitlets (18), and seeds of pumpkin (9) and watermelon (27). We have found a compound with similar properties in pea root callus tissue cultures.…”

“…Based on chromatographic evidence described above, precipitation of 9-substituted purines seems to have occurred in these experiments. Such precipitation of ribonucleosides by silver at an acid pH has been reported before (9,15). It is assumed that under the conditions employed in these experiments, precipitation of 9-substituted purine derivatives was probably complete, since no cytokinin activity was detected in extracts which were made of the soluble fraction in acidic silver nitrate solutions.…”

Cytokinins in Populus×robusta: Changes during Chilling and Bud Burst

“…When the active component from fraction 13 is chromatographed on paper in butan-2-ol: 25 »/o NH4OH (4 :1 v/v) it runs to Rf 0.2. Various other properties (unpublished results) indicate that it is similar, or identical, to an unidentified cytokinin present in a range of plants, including fruitlets of apple (16), seeds of sweet corn (15), pumpkin (11), and water melon (25), pea roots and pea root callus (31), leaves of Begonia (12) and poplar, and buds of sycamore (unpublished results). The nature of the two remaining components has not yet been determined, but they are consistently present in extracts from poplar leaves, and no doubt are of physiological impor-tance.…”

Endogenous Auxin and Cytokinin Changes Dxjring Embryoid Development in Celery Tissue Cultures

“…For instance, in the cycle of leaf expansion and maturation (Henson and Wareing, 1976;Van Staden, 1976) the cytokinins changed both quantitatively and qualitatively during development. W^here cytokinins have been measured in seeds, the total cytokinin level is at a maximum in the early stages (Gupta and Maheshwari, 1970;Wheeler, 1972;Abdel Raman et al., 1975) when the endosperm must make a considerable contribution as with the auxin. No measurements of the cytokinin in the developing embryo were available to enable a direct comparison to be made with the cytokinins in the embryoid.…”