Shoot apices of grapevine (Vitis vinifera L.) were cuUured on a nutrient medium containing differerut concentrations singly and in combination of 6-benzylaminopurine (BAP) and zeatin riboside (ZR). lf supplied separately, maximum elonga0tion and shoot proliferation resulted with BAP at 2 mg/l or with ZR at 10 mg/l. Both processes were inhibited by BAP at 5 mg/l. Combined applications of ZR (even at 10 mg/l failed in cancelling this inhibition. Optimum shoot proliferation resulted with the cytokinins in combination, provided the concentration of BAP did not exceed 2 mg/l.Der Einfluß von Cytokininen auf Längenwachstum, Vermehrung und Gesamtmasse der aus Rebentriebspitzen bei in-vitro-Kultur entstandenen TriebeTriebspitzen der Rebe (Vitis vinifera L.) wurden auf einem Nährmedium kultiviert, das - einzeln und kombiniert - unterschiedliche Konzentrationen von 6-Benzylaminopurin (BAP) und Zeatinribosid (ZR) enthielt. Bei getrennter Anwendung der Wachstumsregulatoren förderten 2 mg BAP/l oder 10 mg ZR/l das Längenwachstum und die Triebvermehrung . am meisten. Beide Vorgänge wurden durch 5 mg BAP/l unterdrückt. Diese Hemmung konnte auch nicht durch zusätzliche Anwendung von ZR, selbst nicht in der Konzentration von 10 mg/l, aufgehoben werden. Eine optimale Triebvermehrung wurde durch ZR in Verbindung mit BAP erzielt, falls die BAP-Konzentration 2 mg/l nicht überschritt

Goussard, P. G.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 20, 228-234 (1981) Department of Vltlculture, Unlversity of Stellenbos.ch, Republic of South Afrlca Effects of cytokinins on elongation, proliferation and total mass of shoots derived from shoot apices of grapevine cultured in vitro1) by P. G. GoussARD Der Einfluß von Cytokininen auf Längenwachstum, Vermehrung und Gesamtmasse der aus Rebentriebspitzen bei in-vitro-Kultur entstandenen Triebe Z u s am m e n f a s s u n g . - Triebspitzen der Rebe (Vitis vinifera L.) wurden auf einem Nährmedium kultiviert, das - einzeln und kombiniert - unterschiedliche Konzentrationen von 6-Benzylaminopurin (BAP) und Zeatinribosid (ZR) enthielt. Bei getrennter Anwendung der Wachstumsregulatoren förderten 2 mg BAP/l oder 10 mg ZR/l das Längenwachstum und die Triebvermehrung . am meisten. Beide Vorgänge wurden durch 5 mg BAP/l unterdrückt. Diese Hemmung konnte auch nicht durch zu-sätzliche Anwendung von ZR, selbst nicht in der Konzentration von 10 mg/l, aufgeho-ben werden. Eine optimale Triebvermehrung wurde durch ZR in Verbindung mit BAP erzielt, falls die BAP-Konzentration 2 mg/l nicht überschritt. Introduction Clonal production of plants has become a major economic application of in vitro techniques (MuRAsHIGE 1974, 1977, SAGAWA 1976, ABBOTT 1978). Shoot apices of herbaceous plants are widely used for in vitro propagation, but those of woody species to a lesser degree (MuRAsHIGE 1974, 1977, Assorr 1978). BARLAss and SKENE (1978, 1979) reported that prolüeration of advenititious buds from fragmented shoot apices of grapevine was greatly stimulated by 6-benzylamino-purdne (BAP). PooL (1975) stated .that zeatin riboside (ZR) is föe main cytokinin in grapevine xylem exudate. He also found that ZR favoured the in vitro develop-ment of ovaries and filaments of excised Concord flowers (prior to bloom). Budburst and shoot elongation of Vitis labruscana B. (cv. Concord) cultured in vitro from nonflowering unrooted pieces of shoots increased dramatically in response to ZR treatments (PooL 1974). Zeatin riboside apparently plays an important role in the physiology of the grapevine. The successful in vitro culture of small apices and apical meristems of shoots of grapevines have major advantages, hence the need to study .füe effects of ZR and BAP singly andin combination. 1) Part of a Ph. D. (Agrlc.) thesls to be submltted to the Unlverslty of Stellenbosch. Promotor: Prof. C. J. Oa••BR. Effects of cytokinins on growth of shoots from apices 229 Material and methods 1. Plant material Dormant canes of V. vinifera L. (cv. Chenin blanc) were collected during winter from selected vines. Only vines visually free of symptoms induced by virus and virus-like diseases were used. The canes were cut into lengths of ca. 40 cm, treated with Captan (2 °/o) and stored in sealed plastic bags at 2-3 °c until use. Upon removal from cold storage, the canes were sectioned into segments of 12-15 cm comprising 2 •internodes with 3 buds. These segments were placed in 250 ml beakers With the bases in ca. 80 ml water. Activation of shoot primordia was en-hanced at an ambient temperature of 28---30 °c under constant illumination. Bud-burst, . followed by rapid shoot elongation, occurred after 5-8 d. Preliminary ex-periments indicated that most of the young shoot tips were free of fungi, which are 1the main contaminants in culture. Large quantities of elongating shoots could hereby be procured on a small bench area within a short per.iod and virtually at any time of the year. Shoot tips (15 mm) were removed from elongating shoots exceeding 40 mm in length. Following removal of some of the outer leaves, the tips were surface-ster-ilized in a 0,5 % sodium hypochlorite solution for 10 min and rinsed 1thrice in sterile distilled water. Shoot apices (0,75-1 mm) containing 2-3 leaf primordia were ex-cised aseptically under a dissecting microscope fitte<! with an ocular micrometer. The explants were individually cultured in test tubes (25 X 250 mm) containing 20 ml of ithe test medium. The tubes were stoppered with cotton wool and covered with tin foil caps. 2 . Culture medium Explants were cultured on the full-strength medium of MuRASHIGE and SKooG (1962) supplemented w.iith 30 g sucrose/1 and 6,8 g Difco Bacto agar/1. The concen-trations of BAP and ZR added are indicated in the fable and in Fig. 1. The pH of the medium was adjusted to 5,7 with n NaOH prior to autoclaving. 3. Culture conditions Explants were incubated in a walk-in temperature-controlled ctilture room, maintained at 27 °c during a 16 h light period and at 24-25 °c during ·an 8 h dark period. The light source consisted of "Atlas" cool-white fluorescent tubes providing ca. 3 000 lux at culture •level. 4. Experimentaldesign A two-dimensional grid experiment was established with each treatment con-sisting of two replicates of 10 explants each (Fig. 1) with a culture period of 42 d. Recordings were made of <a) shoot elongation (length of primary shoot), (b) mass of shoots, (c) number of proliferated shoots exceeding 7 mm in length and (d) percen-tages of explants with proliferated shoots. Data were subjected to analysis of vari-ance followed by the Student Newmann-Keuls plural comparison test (SNEDECOR and CocuaAN (1967). 230 P. G. GovssAno Fig. 1: Effects of 6-benzylaminopurine (BAP) and zeatin riboside (ZR) on elongation and shoot proliferation of shoot apices of Vitis vinifera L. Fig. 2: Shoot elongation and proliferation. Fig. 3: Shoot proliferation in a submerged culture. Effects of cytokinins on growth of shoots from apices 231 Results 1. Elongation Elongation increased considerably with concomitant leaf expansion in response to single ZR treatments (Table, Fig. 1). Maximum elongation was obtained with 10 mg ZR/l, which was signüicantly higher (5 % level) ithan that obtained with any other treatment (Fig. 2). With BAP used singly, maximum elongation was recorded at 2 mg/l; however, at 5 mg/l elongation was severely inhibited. Combined applica- _ tions of BAP (0,5-2 mg/l and ZR (1-5 mg/l) resulted in a slight increase in elonga-tion. In comparison with elongation obtained with ZR singly at 10 mg/l, a decrease was observed with ZR (10 mg/l) in combination with BAP (0,5-2 mg/1). The inhibi-tory effect of 5 mg BAP/l on elongation could not be cancelled by adding ZR (1-10 mg/l). There were no significant differences (5 % level) between elongation with BAP (5 mg/l) combined with ZR (1-10 mg/l) or the control. 2. Shoot mass Maximum increase in mass was achieved with a combination of BAP (2 mg/1) and ZR (2 mg/l) (Table, Fig. 1). Although elongation was inhibited with BAP (5 mg/1) singly and in combination with ZR (1-10 mg/l), a small increase in mass was recorded. 3 . Shoot proliferaition Singly ZR induced maximum shoot proliferation_at the highest concentration tested (10 mg/l) (Table, Figs. 1-2). Although a higher level of proliferation was achieved with ZR (10 mg/) compared wilth BAP (2 mg/), the results do not differ significantly (5 % level). At 5 mg/l, BAP resulted in a complete <inhibition of shoot proliferation. More shoot proliferation occurred with a combination of cytokinins (Table, Fig. 1). A combination of BAP and ZR (both at 2 mg/l) proved the most ef-fective (significant at 5 % level). It was also evident thait the agar medium (Fig. 3) did not inhibit submerged growth i.e. elongation and proliferation. 4. Percentages of explants with proliferated shoots Only those explanits treated with the highest concentrations (5 and 10 mg/l) of ZR singly reached the prolüeration stage (Table, Fig. 1). BAP by itself was most effective at 2 mg/l. Proliferation could be induced with lower concentrations of cytokinins in combination rather than with each cytokinin applied separately. Discussion Elongation and proliferation of detached shoot apices of Vitis are dependant on the presence of an external source of cytokinin. This is in agreement with re-ports for oither plant species (Boxus 1974, ADAMS 1975, AseoTT and WHITELEY 1976, HussEY 1977, MA and WANG 1977, MEKERS 1977, PREIL and ENGELHARDT 1977). Abb. 1: Einfluß von 6-Benzylaminopurin (BAP) und Zeatinribosid (ZR) auf Längen-wachstum und Vermehrung der Triebe aus Triebspitzenkulturen der Rebe (Vitis vinifera L.). Abb. 2: Längenwachstum und Vermehrung von Trieben. Abb. 3: Triebvermehrung in einer Submerskultur. 232 P. G. GoussARD The effect of different concentration ratios of 6-benzylaminopurine (BAP) and zeatin riboside (ZR) an elongation, proliferation and total mass of shoots derived from shoot apices of grapevine (Vitis vinifera L.) cultured in vitro. Der Einfluß unterschiedlicher Konzentrationsverhältnisse von 6-Benzylaminopurin (BAP) und Zeatinribosid (ZR) auf Längenwachstum, Vermehrung und Gesamtmasse der Triebe, die aus in vitro kultivierten Triebspitzen der Rebe (Vitis vinifera L .) her-Treatment (mg/l) BAP: ZR 0 0 2 5 10 0,5 0 2 5 10 0 2 5 10 2 0 2 5 10 5 0 2 5 10 Elongation (mm) 3,22 f 14,27 d 22,72 bc 23 ,02 bc 36,24 a · 8,95 e 21,15 bc 23,83 bc 24,55 bc 26 ,08 b 12,11 de 21,85 bc 23,95 bc 25,00 bc 23,85 bc 19,14 c 22,00 bc 25,13 bc 23,95 bc 13,33 de 3,29 f 3,33 f 3,60 f 3,70 f 3,80 f vorgingen Shoot mass (mg) 10,60 k 185,50 ijk 245,80 hijk 701,00 defg 853 ,90 cde 77,80 jk 344,20 hijk 433,00 ghij 881 ,70 cde 1 107,00 abc 170,20 ijk 382,60 ghijk 587,40 efgh 1 012,50 bcd 1 248,70 ab 430,50 ghij 501,30 fghi 1 343,50 a 781,50 def 248,30 hijk 25,20 k 27,90 k 30 ,50 k 32,10 k 32,62 k Number of proliferated shoots 0,00 f 0,00 f 0,00 f 1,20 ef 3,40 def 0,00 f 2,00 def 2,10 def 3,30 def 6,20 cd 0,00 f 2,30 def 4,40 cdef 7,65 bc 9,65 b 2,30 def 3,00 def 14,30 a 5,30 cde 2,55 def 0,00 f 0,00 f 0,00 f 0,00 f 0,00 f ' /o explants with proliferated shoots 0 b 0 b 0 b 100 a 100 a 0 b 90 a 100 a 100 a 100 a 0 b 100 a 100 a 100 a 100 a 90 a 100 a 100 a 100 a 100 a 0 b 0 b 0 b 0 b 0 b Means accompanied by the same letter in a column do not differ statistically at the 5 ' /o level of significance. Low concentrations of BAP (0,5-2 mg/l) stimulated elongation of explants, whilst higher concentrations (5 mg/l) inhibited elongation; this agrees with the findings of ADAMS (1975) for hops, JoNEs (1967) for apple and MEKERs (1977) for ornamental bromeliad culitivars. The results also indicate that optimal single con-centrations of BAP and ZR differ considerably. Stimulation of elongation obtained with combinations of BAP (0,5-2 mg/l) and ZR (1-5 mg/l) is probably due to the favourable effect of ZR at relatively high concentrations. However, this response was reduced in combinations of ZR (10 mg/l) with BAP (0,5-2 mg/l). Although elon-gation is reduced with combinations of BAP (0,5-1 mg/l) and ZR (10 mg/l). mass in-creased. This may be attributed to an increase in leaf expansion, proliferation of shoots and diameter increase of stem parts. Effects of cytokinins on growth of shoots from apices 233 Siimulatory effects of BAP on shoot proliferation are in agreement with re-ports on other plant species (Boxus 1974, AneoTT and WmTELEY 1976, HussEY 1977, MEKERs 1977, Yrn and L1Aw 1977). Although PnEIL and ENGELHARDT (1977) reported that shoot proliferaiion of azaleas was siimulated wiih increasing concentrations of BAP (2-5 mg/l), lower concentrations of BAP (0,1-1 mg/l) seem ito be optimal for most species. The results of the present study clearly indicate that, although shoot proliferation was achieved with single applications of BAP (2 mg/l) and ZR.(5 and 10 mg/l), interaotions between the cytokinins exist, especially perceptible with a combination of BAP (2 mg/l) and ZR (2 mg/1) (Fig. 1). The inhibitory effect of BAP (5 mg/1) on shoot proliferation could however not be eliminated with combined ZR treatments (1-10 mg/1). Although single applications of BAP (2 mg/l) and ZR (5 and 10 mg/l), as well as in combination (0,5-2 mg/l and 1-10 mg/l), induced a 90-100 % proliferation of shoots the results varied considerably between different treatments. Summary Shoot apices of grapevine (Vitis vinifera L.) were cuUured on a nutrient medium containing differerut concentrations singly and in combination of 6-benzylamino-purine (BAP) and zeatin riboside (ZR). lf supplied separately, maximum elonga0tion and shoot proliferation resulted with BAP at 2 mg/1 or with ZR at 10 mg/l. Both processes were inhibited by BAP at 5 mg/1. Combined applications of ZR (even at 10 mg/1 failed in cancelling this inhibition. Optimum shoot proHferation resulted with the cytokinins in combination, provided the concentration of BAP did not exceed 2 mg/l. Acknowledgement The author wishes to thank Proff. J . A. De BRuYN and M. J. HATTJNGH for thelr critical review of the manuscript. Literature cited Aeeorr, A. J., 1978: Practice and promise of mlcropropagatlon of woody species. Acta Hort. 79 113-127. - - and WmTELBY, E., 1976: Culture of Malus tlssues in vltro. 1. Multiplicatlon of apple plants from lsolated shoot apices. Scientia Hort. 4, 183-189. ADAMS, A. N., 1975: Elimination of viruses from the hop (Humu!us !upu!us) by heat therapy and merlstem culture. J . Hort. Sei. 50, 151-160. BARLAss, M. and SKENE, K. G. M., 1978: In v!tro propagation of grapevine (Vitis vinifera L .) from fragmented shoot apices. Vitis 17, 335-340. - - and - - , 1979: Clonal propagation through tissue culture. Austral. Grapegrower and Winemaker 16 (191), 12-13. Boxus, P., 1974: The production of strawberry plants by in vltro micro-propagatlon, J. Hort. Sei. 49, 209-210. Huss•Y, G., 1977: In vitro propagation of some members of the Liliaceae, Irldaceae and Amaryl-lidaceae. Acta Hort. 78, 303-309. JoNes, O. P., 1967: Effect of benzyl adenine on lsolated apple shoots. Nature 215, 1514-1515. MA, s. s. and WANG, S. o., 1977: Clonal multlplication of Azaleas through tlssue culture. Acta Hort. 78, 209-215. 234 P. G. GoussARD M•K•Rs, o., 1977: In vttro propagatlon of some Tlllandsloldeae (Bromellaceae). Acta Hort. 78, 311-317. MuRASHIGE, T., 1974: . Plant propagatlon through tlssue cultures. Ann. Rev. Plant Physlol. 25, 135-166. - - , 1977: Plant cell and organ cultures as hortlcultural practlces. Acta Hort. 78, 17-30. - - and SKooc, F„ 1962: A revlsed medium for rapid growth and bloassays wlth tobacco tlssue cultures. Physlol. Plant. 15, 473-497. Poot, R. M., 1974: Relation of cytokinlns, abscislc acld and metabolltes of absclslc acld to vlne growth and leaf function In Vttls. Ph. D Thesis, Cornell Universlty, Ithaca, New York. - - , 1975: Effect of cytoklnin on tn vttro development of Concord flowers. Amer. J. Enol. Vltlcult. 26, 43-46. PREIL, W. and ENGELHARDT, MARGARETE, 1977: Meristem culture of Azaleas (Rhododendron Stmsli). Acta Hort. 78, 203-208. SAGAWA, Y., 1976: Potential of in vttro culture techniques for lmprovement of florlcultural crops. Acta Hort. 63, 61-66. SNEDBCOR, G . w. and COCHRAN, w. G„ 1967: Statistlcal Methods. 6th Ed. The Iowa State Universlty Press, Ames, Iowa. Y1e, S . and LrAw, 1., 1977: Plant regeneratlon from shoot tlps and callus of papaya. In vttro 13, 564-568. Eingegangen am 7. 4. 1981 P. G. GoussARD Department of Viticulture University of Stellenbosch Stellenbosch 7600 Republic of South Africa 

Effects of cytokinins on elongation, proliferation and total mass of shoots derived from shoot apices of grapevine cultured in vitro

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Effects of cytokinins on elongation, proliferation and total mass of shoots derived from shoot apices of grapevine cultured in vitro

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