Basic Heller’s medium is not suitable for the growth and development of fragments of pumpkin hypocotyls. On this medium the fragments hardly survive a culture period of one month. The addition of growth regulators (IAA, 2,4-D or kinetin) to Heller’s medium delays the senescence of the tissue and its decay. Moreover, it provokes cell divisions resulting in an increase of fresh and dry weight

Sibila Jelaska

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Acta Bot. Croat. 32 (1973) 95—100T H E  E F F E C T  O F  G R O W T H  R E G U L A T O R S  O N  T H E  S U R V I V A L  O F  E X C I S E D  P U M P K I N  H Y P O C O T Y L - T I S S U ESIBILA JELASKA(Institute o l  Botany. U niversity o f  Zagreb)R eceived  January 24, 1973I n t r o d u c t i o nC h i b  n a i l  (1939) has suggested that protein synthesis in leaves depends on some cooperation on the part of the root, and 1954 he proved that rooting of leaf cuttings prevents the yellowing of the blade. M o t h e s  and E n g e l b r e c h t  (1956) confirmed this finding and showed that root formation can in fact cause resumption of chlorophyll and protein formation. Those detached, rooted leaves can be kept alive much longer than if left on the plant ( M a t h u s e  1906). Recently, it has been found possible to replace at least a part of the rooting effect by certain chemicals. R i c h m o n d  and L a n g  (1957) showed that the chlorophyll and protein loss of detached Xanthium pennsylvanicum leaves was considerably slowed down, if the leaves were supplied with kinetin. O s b o r n e  (1959) observed similar effects of 2,4-D and 2,4,5-T on detarhed as well as attached leaves of Primus serrulata.The effect of rooted leaves as well as experiments with added chemicals indicated that growth regulators are undoubtedly impor­tant in the process of senescence.After the investigations on the pumpkin-tissue (J e 1 a s k a 1972), similar effects of growth regulators on the survival of fragments of hypocotyls have been observed.M a t e r i a l s  a n d  M e t h o d sExperiments were made with the species Cucurbita pepo L. Seeds of pumpkin were sterilized with 3% calcium hypochloride and germi­nated in test tubes in distilled water under light for 10 days under95sterile conditions. Fragments of hypocotyls, 1 cm long, were cut and cultivated individually in test tubes (23 X 200 mm) on 25 ml of cul­ture medium for one month. These fragments were implanted hori­zontally on the medium and finally the tubes were capped with thin aluminium foil. The basal medium was Heller’s solution of mineral salts ( H e l l e r  1953) or Murashige-Skoog’s solution ( M u r a s h i g e  and S k o o g 1962) +  3°/o glucose +  0,9% Difco Bacto agar +  one of the growth substances or some combinations of them. Before auto­claving, the pH of the medium was 5,5—5,6. The media were auto­claved at 120 °C for 20 minutes. The cultures were maintained at 26 ±  1 °C under artificial light (485 ±  45 lux, fluorescent lamps IPR, 40 W, 220 V, 6500 °K, 16 h light and 8 h darkness daily). Each expe­riment was made with 20 explants.A b b r e v i a t i o n s  : IAA — indoleacetic acid, 2,4-D — 2,4-dichlorophen- oxyacetic acid, MS — Murashige and Skoog’s medium.R e s u l t sSystematic investigations (J e 1 a s k a 1972), which had been car­ried out on fragments of pumpkin hypocotyls (in vitro), have shown that Heller’s basal medium without any addition of growth substances has no favourable influence to tissues. The fragments of hypocotyls hardly survived the culture period and decayed during the first month of culturing in nearly 100% of specimens. The combination of salts used by Murashige-Skoog, also without growth regulators, was much more suitable (Table 1). The tissue fragments survived on the MS-medium the culturing of one month, they retained their green colour and showed even the phenomenon of rhizogenesis, which was especially vigorous when the complete MS-medium was used.By the addition of auxin and kinetin to Heller’s medium the decay pf the tissue was delayed and it showed even an increase of dry and fresh weight, which indicated that in spite of the unsuitable nutrient medium growth regulators activated the tissue. Table 2 shows the in­crease of the fresh and dry weight dependent on the concentrations of IAA and kinetin. This increase was more dependent on the in­creasing concentration of auxin than on that of kinetin (Table 2).Table 3 shows the results obtained with 2,4-D and kinetin. One can easily see how the addition of 2,4-D causes, by its increase in con­centration, the raise of the percentage of survived explants. The ad­dition of kinetin made this numbers even higher. In this way 20% of explants survived on a medium with 2,4-D (10~8). The addition of kinetin to this medium increased the survival to 40—50% of fragments, or to 70—75% when 2,4-D had been added in concentrations 10-7 or 10~®. The addition of kinetin, thus, raised the percentage of survived explants still for about 20% in comparison to that of survived explants when cultivated only with 2,4-D.96T a b l e  1. Survival of excised pumpkin hypocotyl-tissue and increase of fresh and dry weight after 1 month on Heller’s and MS-medium.Medium Survival of cultures (%)Fresh weight (mg)Dry weight (mg)HellerHeller +  KT« IAA +20 90.5 5.5+  3 X 1(T7 kinetin 70 188.1 15.5MS- salts 70 140.2 7.2MS- completeMS- complete +  10~6 IAA +85 207.8 17.5+  3 X KT7 kinetin 90 259.0 21.6T a b l e  2. Effect of IAA and kinetin on fresh and dry weight of pumpkin hypocotyl-tissue after 1 month (Heller’s medium).IAA Kinetin Fresh weight (mg)Dry weight (mg)90.5 5.5io-8 10‘7 83.9 6.310~8 3 X IO- 7 94.5 7.3IO“8 IO-8 65.1 8.510“7 10'7 188.3 13.110 -7 3 X 10~7 172.3 15.01 0 7 10-8 186.4 14.010 8 lO“7 225.5 18.710-6 3 X lO"7 188.1 15.510-8 KT« 239.0 20.2T a b l e  3. Effect of 2,4-D and kinetin on fresh and dry weight and sur­vival of excised pumpkin hypocotyl-tissue after 1 month (Heller’s medium).Survival Fresh weight Dry weightof cultures (°/o) (mg) (mg)0 90.0 5.2lO“8 — 20 103.5 8.210~7 — 50 155.6 15.51CT6 — 50 208.6 18.62 X 10-* — 30 135.1 1.6lO-8 lO-7 43 52.1 3.710-8 3 X lO“7 55 79.8 4.710-8 lO-» 42 121.0 8.510~7 10~7 62 184.2 14.410~7 3 X lO^7 70 202.4 14.2lO"7 lO-« 60 199.5 13.5KT* lO-7 75 330.6 29.610-« 3 X lO“7 76 237.9 19.110 8 10-8 76 326.3 22.77 97D i s c u s s i o nFragments of pumpkin hypocotyls, planted on Heller’s medium with 3°/o glucose and 0,9% agar, were not able to survive the culturing period of one month. They lost they colour at a very high percen­tage in this period of one month and decayed without any consi­derable increase in weight. By adding growth regulators (2,4-D, IAA and kinetin) to Heller’s medium, the senescence of the tissue was delayed for a longer time. The fragments showed even growth and cell divisions, which was evident from the increase of fresh and dry weight. P a u p a r d i n  (1964a, b) has, however, proved by her ana­tomic investigations, that a dediferentiation of tissues such as collen- chym|a, vessels and parenchyma, occurs under the influence of IAA and kinetin or 2,4-D. In the experiments presented here the addition of 2,4-D to Heller’s medium induced even a weak differentiation of roots.The senescence of plant tissue may be considered as a problem of phytopathology ( C o m f o r t  1956). It is certainly a result of a se­quence of processes, which can be hardly analyzed separately, but which cause senescence and finally cell death. Auxins should also be responsible for that to a certain extent.By investigating the quantities of growth hormones in young and senescent tissue ( We nt  and T h i m a n n  1937, S h o j i  et al. 1951, G a l s t o n  and D a l b  e r g  1954, P i l e t  and G a l s t o n  1955, P i l e  t 1956) it could be stated that the concentration of auxin is lowered in senescent cells. According to these findings the senescence of tissues could be partly explained by hypoauxiny, which would increase with progressing cell senescence. The experiments carried out by O s b o r n e  (1959) would confirm this statement. He treated leaves of Prunus ser- rulata with different auxin substances (2,4-D, 2,4,5-T) and got very clear symptoms of delayed senescence.However, a whole sequence of other processes ( P i l e t  1959) com­plicates this relatively simple scheme of senescence stated for a cer­tain tissue. The autor’s experiments on pumpkin hypocotyls also con­firm, to a certain extent, the influence of growth regulators on the processes of senescence.S u m m a r yBasic Heller’s medium is not suitable for the growth and deve­lopment of fragments of pumpkin hypocotyls. On this medium the frag­ments hardly survive a culture period of one month. The addition of growth regulators (IAA, 2,4-D or kinetin) to Heller’s medium delays the senescence of the tissue and its decay. Moreover, it provokes cell divisions resulting in an increase of fresh and dry weight.A c k n o w l e d g e m e n t .  I express my thanks to the Head of the De­partment of Plant Physiology Professor Z. Dévidé for the facilities pro- weed during the course of this work and for his interest in these investigations.98R e f e r e n c e sChibnall, A. C. 1939. Protein metabolism in the plant. New Haven, Conn., Yale University Press.Comfort, A. 1956. The biology of senescence. Rontledge & Kegan, London.Galston, A. W. and Dalberg, Y. L. 1954. The adaptive formation and phy­siological significance of indoleacetic acid oxidase. Amer. J. Bot. 41, 373—380.Heller, R. 1953. Recherches sur la nutrition minérale de tissus végétaux cul­tivés in vitro. Ann. Sc. Nat. Bot. Biol. Veg. 14, 1—223.Jelaska, S. 1972. Morphogenesis of pumpkin expiants cultivated in vitro. Ph. D.-Thesis. Faculty of Sciences, University of Zagreb, 118 p.Mathuse, O. 1906. Über abnormales sekundäres Wachstum von Laubblättern, insbesondere von Blattstecklingen dikotyler Pflanzen. Beih. bot. Zbl. 20, I, 174/1—46.Mothes, K. and Engelbrecht, L. 1956. Uber den Stickstoffumsatz in Blattsteck­lingen. Flora (Jena) 143, 428— 472.Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473—497.Osborne, D. J. 1959. Control of leaf senescence by auxins. Nature 183, 1459.Paupardin, C. 1964a. Sur les modifications de la structure histologique de fragments d’hypocotyles de Courge (Cucurbita maxima var. rouge vif d’Étampes) cultivés in vitro. C. R. Acad. Sc. Paris 258, 1024— 1027.Paupardin, C. 1964b, Sur la dédifferéntiation partielle des vaisseaux jeunes dans l’hypocotyle de Courge (Cucurbita maxima var. rouge vif d’Étampes) cultivé in vitro. C. R. Acad. Sc. Paris 259, 3345—3347.Filet, P. E. 1956. Activité des auxine-oxydases dans les fragments de Carotte cultivés in vitro. C. R. Acad. Sc. 243, 1141.Pilet, P. E. 1959. Un cas d’adaption auxines-oxydasique (racine). Rev. Gén. Bot. 66, 450.Pilet, P. E. and Galston, A. W. 1955. Auxin destruction, peroxidase activity and peroxide genesis in the roots of Lens culinaris. Physiol. Plant. 8, 888—898.Richmond, A. E. and Lang, A. 1957. Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125, 650— 651.Shoji, K., Addicott, F. T. and Swets, W. A. 1951. Auxin in relation to leaf blade abscission. Plant Physiol. 26, 189— 191.Went, F. W. and Thimann, K. V. 1937. Phytohormones. McMillan Co. New York.99S A D R Ž A JDJELOVANJE REGULATORA R A ST A  N A PRE2IVLJENJE FRAGM ENATA H IFOKOTILA BUNDEVESibila Jelaska(Institut za botaniku Sveučilišta u Zagrebu)Fragmenti hipokotila bundeve veličine 1 cm zasijani na Hellerovom mediju s 3% glukoze i 0,9°/o agara nisu mogli preživjeti period kulti- viranja od mjesec dana. Oni su u vrlo velikom postotku, u tom vremen­skom razdoblju propadali, izgubivši svoju zelenu boju i pokazujući ne­znatni prirast težine. Nasuprot tome oni su na MS-mediju u velikom broju preživjeli i znatno povećali svoju težinu (tabela 1).Dodavanjem nekih regulatora rasta kao npr. IAA, 2,4-D i kinetina (tabela 2 i 3) Hellerovom mediju uočeno je da je postotak preživjelih kultura rastao s porastom koncentracija dodanih stimulatora. Na Helle­rovom mediju s 2,4-D u koncentraciji IO-8 preživjelo je 20% eksplantata. Dodatak kinetina istoj koncentraciji 2,4-D povećao je preživljenje eks­plantata na 40—50%. Kod koncentracije 10“ 7 i 10“° 2,4-D preživjelo je 50% eksplantata, a dodatkom kinetina taj se postotak povisio još za daljnjih 20%. Dodatak stimulatora rastenja, osim na preživljenje eks­plantata, djelovao je i na znatan porast svježe i suhe tvari.Dr Sibila Jelaska O djel za fiz io log iju  Instituta za botaniku Sveučilišta R ooseveltov  trg 6/III, p. p. 933 41001 Zagreb  (Jugoslavija)100

Djelovanje regulatora rasta na preživljenje fragmenata hipokotila bundeve

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