Increases in the number of adventitious roots caused by 2-thiouracil and 5-bromodeoxyuridine in Phaseolus mungo cuttings

A cutting of Phaseolus mungoproduced about 4 adventitious rootsat the basal 1 mm region when the basal part of the cuttingwas dipped in water. Rootlets became visible after a 36 hr lagperiod in untreated cuttings. Treatment with 2-thiouracil or5-bromodeoxyuridine increased the number of roots formed onthe cutting and prolonged the lag period. Effects of 2-thiouraciland 5-bromodeoxyuridine were reversed by simultaneously applieduracil and thymidine. The number of roots decreased and thelength of lag period was shortened. The increases in the numberof roots by 2-thiouracil or 5-bromodeoxyuridine was reducedby gibberellic acid, which did not cause a decrease in the numberof roots to be formed on control cuttings. Roots formed at thebasal region seem to suppress further root formation at theupper part of the hypocotyl. Inhibitors used here probably workby blocking the formation of these bottommost roots. (Received April 30, 1971; )     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

PROMOTING ACTIVITY OF TERPENIC LACTONES IN PHASEOLUS ROOTING AND THEIR REACTIVITY TOWARD CYSTEINE

Sesquiterpenoids which contain exomethylenes conjugated to -lactonecarbonyl-heliangine, helianginol, pyrethrosin and cyclopyrethrosinacetate—promoted the adventitious root formation on hypocotylsof cuttings taken from light-grown (1900 lux) 6-day old Phaseolusmungo seedlings, but their derivatives in which the methylene-lactone systems are reduced-to the saturated lactones—dihydroheliangine,hexahydro-heliangine, dihydrohelianginol, dihydrocyclopyrethrosinacetate and tetrahydrocyclopyrethrosin acetate—showedno effect on the root formation. As far as the present experiment is concerned, every substancewhich showed promoting activity in Phaseolus rooting reactedwith cysteine and formed an adduct, but any of the substancesincapable of promoting root formation did not react with cysteine.The correlation of promoting activity of terpenic lactones inPhaseolus rooting with their reactivity toward SH groups maythus be demonstrated. ¹ Contribution No. 14 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo.     

Effects of indoleacetic, p-chlorophenoxyisobutyric and 2,4,6-trichlorophenoxyacetic acids on three phases of rooting in Azukia cuttings

In adventitious root formation of disbudded epicotyl cuttingstaken from light-grown, 5-day-old Azukia angularis seedlings,indoleacetic acid (IAA), 1 x 10^–4 M, applied during thefirst day showed no effect, but enhanced the effect of IAA,1 x 10^–4 M, applied during the second day. Treatment duringthe second day promoted rooting by about 70%, and a combinationof treatments for the first and second days promoted rootingsome 200%. p-Chlorophenoxyisobutyric acid (PCIB), 3 x 10^–4M, and2,4,6-trichlorophenoxyacetic acid (2,4,6-T), 2 x 10^–44M, applied the first day also enhanced the effect of IAA, 2x 10^–4 M, applied the second day. When applied the second day, PCIB, 2 x 10^–4M, increasedthe number of root primordia or clusters of small cells, butnot die number of protruded roots. Formation of the cell clusterwas inhibited by 2,4,6-T, 3 x 10^–4M, applied the secondday. Rooting processes in Azukia cuttings seem to include at leastthree phases: the first phase is induced not only by IAA butalso by PCIB or 2,4,6-T, the second phase is induced by IAAor PCIB and the diird phase depends specifically on IAA. (Received October 28, 1970; )     

STUDIES ON THE MECHANISM OF GROWTH INHIBITING EFFECT OF LIGHT

1. A substance which inhibits indoleacetic acid (IAA)-and naphthaleneaceticacid (NAA)-induced elongation of Avena coleoptile section andIAA-induced Avena coleoptile curvature was found in an ethersoluble neutral fraction of water extract of sunflower leavesand in agar blocks containing the diffusate from young sunflowerleaves.
2. This substance also inhibits the growth of isolatedsunflowerepicotyl.
3. The R_f value (0.9) of the substance ona paper chromatogramdeveloped with ammoniacal iso-propanolindicates that it isidentical with the inhibitor reported byAUDUS et al. (1956),but not with inhibitor-ß.
4. Theinhibitor can be transported from leaf to stem, and thetransportseems to be accelerated by illuminating the leaf.
5. The auxindiffused from sunflower leaf into agar block may beidenticalwith IAA.
6. A substance, which has the same properties as theinhibitorfrom sunflower leaf, was obtained in crystalline formfrom theleaf of Jerusalem artichoke.
7. The mechanism of growthinhibition caused by this crystallinesubstance seems to involveinactivation of a sulfhydryl group.
8. The reason why the stemgrowth of sunflower seedlings is reducedby strong light isdiscussed: the amount of the inhibitor transportedfrom leafto stem is increased under strong light, and in thestem, growthinhibition is caused by a direct effect of thisinhibitor ongrowth and by its inhibiting effect on the transportof IAAfrom leaf to stem.

¹ Present address: Botanical Garden, Faculty of Science, Universityof Tokyo, Tokyo (Received February 15, 1961; )     

Portulal: a rooting promoting substance in Portulaca leaves

A new bioassay which employs disbudded epicotyl cuttings takenfrom light grown Azukia seedlings (A. angularis) was devisedfor testing rooting promotion activity. By use of this method,the rooting promoting principle in leaves of Portulaca grandiflorawas isolated and identified with portulal which had been previouslyobtained. Portulal was reported as an inhibitor of rooting inetiolated Raphanus cuttings and has recently been determinedto be a bicyclic diterpene containing a perhydroazulene nucleus.Portulal promoted the adventitious root formation in severalkinds of plants, i.e. Azukia angularis, Vigna Catiang var. sinensis,Phaseolus Mungo and Raphanus sativus var. acanthiformis ‘Risodaikon’. The rooting process in Azukia and Raphanus cuttings seems toinclude at least two phases; a "preparatory phase" or a portulal-and gibberellin-sensitive phase and a "main phase" or an auxin-sensitiveand portulal-insensitive phase. ¹Contribution No. 16 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received April 3, 1969; )     

INTERACTION BETWEEN HELIANGINE AND PYRIMIDINES IN ADVENTITIOUS ROOT FORMATION OF PHASEOLUS CUTTINGS

1. Heliangine at 110^–4 M promoted the adventitious rootformation in hypocotyls of cuttings taken from light-grown (1,900lux) Phaseolus mungo seedlings. The promotion was almost completelyreversed by 310^–4 M uracil, uridine, cytidine, oroticacid or 610^–4 M carbamoyl DL-aspartic acid, and partlyby 310^–4 M thymine or thymidine. Neither 310^–4M cytosine, adenine, adenosine, guanine, guanosine nor a combinationof 310^–4 M carbamoyl phosphate and 310^–4 M L-asparticacid reduced the promotion by heliangine.
2. Uracil did not reducethe inhibiting effect of heliangine onthe indoleacetic acidinduced elongation of etiolated Avenacoleoptile sections.
3. Helianginein an aqueous uracil solution was recovered unchangedafter24-hr incubation at room temperature.
4. The root formation ofPhaseolus cuttings was promoted also by2-thiouracil and 5-fluorouracil.The effect was reversed byorotic acid or carbamoyl asparticacid, but not by carbamoylphosphate plus aspartic acid.
5. Ribonucleaseat 100 µg/ml increased the number of rootsprotruded fromhypocotyls of cuttings by about 260%.
6. A possible interpretationfor the promotion of root formationby heliangine is offered.

¹ Contribution No. 15 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. ² Dedicated to Prof. Dr. H. SODING in commemoration of the 70thbirthday.     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

ISOLATION OF AN INHIBITOR OF GROWTH AND ROOT FORMATION FROM PORTULACA GRANDIFLORA LEAVES

1. From leaves of Portulaca grandiflora, a substance which inhibitedthe IAA-induced elongation of Avena coleoptile sections andthe adventitious root formation of Raphanus hypocotyl cuttingswas separated by means of thin layer chromatography. It wasisolated and crystallized. 2. On paper chromatograms, this substance gave the same R_f valuesas the inhibitor from leaves of Xanthium strumarium and thatfrom leaves of Helianthus tuberosus ("heliangine"), namely,R_f0.9 in ammoniacal isopropanol, R_f 0.85 in methanol-water andR_f 0.0 in n-hexane-water. On thin layer chromatograms, however,these inhibitors were clearly separated from each other. 3. Infra-red absorption spectrum also indicated that this substanceis identical with neither xanthinin nor heliangine. ¹ Contribution No. 8 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo     

EFFECT OF MANGANESE IONS ON THE IAA-INDUCED ELONGATION OF AVENA COLEOPTILE SECTIONS INHIBITED BY SOME ORGANIC ACIDS

1.Organic acids, such as citric, -ketoglutaric, succinic, fumaricand L-malic acids, inhibit the IAA-induced growth of Avena coleoptilesections. But pyruvic acid has no effect on the growth. 2.High concentrations of MnCl₂ (for example 10^–3 m) alleviatethe inhibition due to L-malic, -ketoglutaric, succinic and fumaricacids, but not that due to D-malic, tartaric and malonic acids. 3.A mechanism of the alleviating effect of Mn⁺⁺ on the inhibitiondue to the organic acids is discussed with the reference tothe activating effect of Mn⁺⁺ on "malic" enzyme. ¹Contribution No. 6 from the Botanical Gardens. Faculty of Science,University of Tokyo, Koishikawa, Tokyo.