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The effects of scale wounding of tulip bulbs on ethylene evolution, plant growth and flowering
Scientia Horticulturae, 53 (1993) 347-359 Elsevier Science Publishers B.V., Amsterdam
347
The effects of scale wounding of tulip bulbs on ethylene evolution, plant growth and flowering L u d w i k a K a w a a, Marcel Le N a r d b and A.A. D e Hertogh c aResearch Institute of Pomology and Floriculture, 96-100 Skierniewice, Poland blNRA, Station d'Ameliorationde la Pomme de Terre et des Plantes a Bulbes, 29260 Ploudaniel, France CDepartment of Horticultural Science, North Carolina State University, Raleigh, NC 27695- 7609, USA. (Received 10 October 1991 )
ABSTRACT Kawa, L., Le Nard, M. and De Hertogh, A.A., 1993.The effects of scale wounding of tulip bulbs on ethylene evolution, plant growth and flowering. Scientia Hortic., 53:347-359 Scale wounding significantly accelerated flowering of Tulipa gesneriana L. bulbs that had been stored for 3,6 and 9 weeks at 2°C for 'Apeldoorn' and for 3 and 6 weeks for 'Oxford'. In addition, final scape length was significantly longer in plants produced from wounded bulbs after 3 and 9 weeks of precooling for 'Apeldoorn' and after 3 weeks for 'Oxford'. Wounding increased ethylene production in all treatments of both cultivars. 'Oxford' produced significantlymore ethylene than 'Apeldoorn', however, the increased ethylene production did not produce floral abortions. Keywords: Bulbs; ethylene; flowering; Tulipa gesneriana; wounding.
INTRODUCTION
Successful forcing of tulips requires the use of bulbs harvested at a mature stage (Le Nard, 1982). After lifting, the bulbs must be stored at warm temperatures ( 1 7 - 3 4 ° C ) until the differentiation of the flower to stage G is achieved. Subsequently, they must be subjected to an extended cold treatment that promotes rapid plant growth and flowering during the greenhouse phase (De Hertogh, 1974). To satisfy the cold requirement, two basic techniques are used: ( 1 ) standard forcing - the bulbs (sometimes after 6 weeks ofprecooling at 9 ° C) are planted at low temperatures where rooting and cooling takes place and only scape growth and flowering occur in the greenhouse; (2) special precooling - the bulbs are dry stored for 9-12 weeks at 2-5 °C and Correspondence to: L. Kawa, Research Institute of Pomology and Floriculture, 96-100, Skierniewice, Poland.
348
L. KAWAET AL.
then planted directly in the greenhouse; where rooting, scape growth, and flowering occur. Exposing tulip bulbs to ethylene, or treating them with ethephon as a source of ethylene, can cause many physiological disorders (Kamerbeek and De Munk, 1976). Some of them are: floral bud necrosis (De Munk, 1972; 1973a), flower malformations and abortions (De Munk, 1973b; De Munk and Hoogeterp, 1975; Moe, 1979 ), inhibition of shoot and root growth (De Munk and De Rooy, 1971; De Munk, 1973b; Nichols and Kofranek, 1982), abnormal leaf formation (De Munk, 1975 ), and gummosis (Kamerbeek et al., 1971; De Munk and Saniewski, 1989). It is well known that wounding is one of the stress factors that can induce increased ethylene production by most plant tissues (Yang and Pratt, 1978 ). In preliminary trials, Le Nard (unpublished data) had shown that wounding of'Apeldoorn' and 'Paul Richter' tulip bulbs, previously stored for 3, 6 and 9 weeks at 5 ° C, led to a faster plant growth when compared with non-wounded bulbs. The objectives of this study were to determine the effects of scale wounding of tulip bulbs after various weeks ofprecooling not only on subsequent plant growth and flowering in the greenhouse but also on ethylene production. MATERIALS AND METHODS
Dutch-grown tulip bulbs ( 12/up cm, in circumference) of'Apeldoorn' and 'Oxford' were used. Prior to precooling, they were stored at 17-20 °C until 26 November 1990, when they were placed at 2°C. After 3, 6, 9 and 12 weeks of cold dry storage, i.e. on 17 December 1990; 8 January 1991; 28 January 1991; and 18 February 1991, 15 bulbs were planted per plastic tray (56.5 cm × 38.5 cm × 17.5 cm). The planting medium was pasteurized and consisted of equal volumes of shredded pine bark, sand, sphagnum peat moss, and sandy loam soil with a pH of 6.8. Immediately before planting, the entire tunic was carefully removed from each bulb. Subsequently, the scales of 30 bulbs per cultivar were cut four times at 90 ° angles (Fig. 1 ). Uncut bulbs were used as the ;ut-1
Cut-4
Cut-2 Cut-3
"Shoot
Flat Side of Tulip Bulb
Fig. l. The techniqueused to wound tulip scalesafter the entire tunic was removed.
GROWTH EFFECTS OF SCALE WOUNDING TULIP BULBS
349
controls. No fungicidal treatments were used before planting. Bulbs were forced in a greenhouse at 13-17 °C under prevailing light conditions. After the bulbs were fully rooted, they were fertilized with 2.4 g 1- 1 C a ( N O 3 ) 2 twice a week. On the date of flowering, the length of all internodes, leaves and the flowers were recorded. The length of the first (basal) internode was measured from the basal plate to the first leaf node after the scales were removed. The length of the fourth internode was measured from the third leaf to the base of the flower. On 26 November 1990, when the bulbs were placed at 2 °C and on each day of planting, 15 intact and 15 cut bulbs per cultivar were used to measure the rate of ethylene production. Each bulb was placed in a separate 490 ml glass container, sealed, and then held at room temperature ( 2 0 - 2 4 ° C ) . Samples ( 1 ml) were taken 18 h after closing. Subsequently, the bulbs were air ventilated for 6 h and the jars were re-closed. Samples were taken daily for 5 days. Ethylene determinations were made on a Varian 1400 gas chromatograph using an alumina column with a flame ionizator detector, RESULTS
Growth and flowering
The growth measurements presented in Tables 1 and 2 were recorded only for the bulbs that rooted and subsequently flowered. The percent of bulbs flowering for the treatments is given in each table and there were no significant differences between wounded and non-wounded bulbs. Those bulbs that failed to flower were mechanically damaged due to excessively deep cutting. A p e l d o o r n . - With control (uncut) bulbs, flowering occurred more quickly as
the duration of precooling at 2°C increased (Table 1 ). Bulb scale wounding significantly reduced the n u m b e r of days to flowering (Table 1, Fig. 2 ) after 3, 6 and 9 weeks of precooling. The wounded bulbs flowered 8, 5 and 2 days earlier than uncut bulbs, respectively. Wounding of bulbs that had been precooled for 3 weeks increased the total length of the plants, but mostly the upper three internodes. After 6 weeks of precooling, the growth differences between cut and uncut bulbs were not statistically significant. It must be pointed out that after 3 and 6 weeks of bulb storage at 2°C, the flowers coloured within the leaves. The greatest~overal ! effect of wounding on plant growth was observed after 9 weeks of bulb precooling. The length of all the internodes as well as the leaves was significantly increased after scale cutting. After 12 weeks of storage at 2 oC, which led to the production of the longest plants, wounding had no significant effect on flowering or total plant height. O x f o r d . - In uncut bulbs, flowering took place more quicldy as the duration of precooling at 2 °C increased (Table 2). After 3 and 6 weeks of precooling,
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Fig. 2. The effects of wounding tulip scales on plant growth and flowering in 'Apeldoorn'. Bulbs were planted on 28 January 1991, after 9 weeks ofprecooling at 2 ° C. The l~hotograph was taken 49 days after planting. Left: control, uncut bulbs. Right: wounded bulbs.
uncut 'Oxford' tulips flowered 10 days earlier than 'Apeldoorn'. Wounding bulbs that had been precooled for 3 and 6 weeks significantly reduced the number of days to flowering (Table 2, Fig. 3 ), but wounding did not affect the earliness of flowering in bulbs that had been precooled for 9 or 12 weeks. Wounding of bulbs that had been precooled for 3 weeks significantly increased the final length of all internodes and leaves. In the bulbs precooled for 6 and 9 weeks, wounded bulbs grew faster than controls, but the stimulatory effect of wounding on the total scape length was not statistically significant. The length of leaves was stimulated by wounding with 9 weeEs of precooling, but not with the 6 weeks precooled bulbs. After 12 weeks ofprecooling, wounding had no effect on the final length of the internodes and leaves.
General growth observationsfor both cultivars Flower length was similar for all treatments and for both cultivars (Tables 1 and 2 ). In the case of'Oxford' bulbs precooled for 12 weeks, a slight partial abortion of petals was observed after wounding. This could have been an ethylene-induced response. After 3, 6 and 9 weeks of precooling, bulbs of 'Apeldoorn' and 'Oxford' showed different growth patterns. However, after 12 weeks of precooling, the
GROWTH EFFECTS OF SCALE WOUNDING TULIP BULBS
353
Fig. 3. The effects of wounding tulip scales on plant growth and flowering in 'Oxford'. Bulbs were planted on 8 January 1991, after 6 weeks of precooling at 2 ° C. The photograph was taken 49 days after planting. Left: control, uncut bulbs. Right: woundedbulbs.
average n u m b e r of days to flowering and the total scape length were similar. In all experiments, w o u n d e d bulbs exhibited a yellow coloration of the outer scales.
Ethyleneproduction The level of ethylene produced by uncut bulbs was very low (0.044-0.210 n l g -1 h -1 ) for both cultivars (Figs. 4 ( a ) , 4 ( b ) , 5(a) and 5 ( b ) ) . The effect of wounding was obvious and it led to an increased ethylene production regardless of the bulb storage treatment. For both cultivars, the largest production took place after 3, 6 and 9 weeks of precooling. 'Oxford' bulbs produced significantly more ethylene than 'Apeldoorn'. The highest level of ethylene evolution by w o u n d e d 'Apeldoorn' bulbs took place after 9 weeks of precooling. In this treatment, the average ethylene production during the 5 days after cutting was 1.479 nl g-~ h -1 and was 4.8 times higher than from wounded uncooled bulbs (0.305 nl g-1 h-~ ) and 1.3 times higher than from 12 weeks precooled bulbs ( 1.170 nl g - ~ h - 1). Wounded 'Oxford' bulbs produced the highest level of ethylene after 6 weeks of precooling. The average ethylene production was 2.288 nl g - 1 h - 1 and this was 5.8 and 1.9 times higher than uncooled (0.397 nl g - 1 h - ~) and 12 weeks precooled ( 1.211 nl g - 1 h - 1) bulbs,
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L. KAWAET AL
4.A. 'Apeldoorn'
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Fig. 4. (a) The rate of ethylene production (ml g-1 h - l ) in tulip bulb scales of 'Apeldoorn' after 0, 3, 6, 9 and 12 weeks of dry storage at 2°C; LSD (0.05) =0.222, (0.01) =0.292. (b) The effect of wounding tulip bulb scales of 'Apeldoorn' on the rate of ethylene production (ml g - ] h - x after 0, 3, 6, 9 and 12 weeks of dry storage at 2 ° C, LSD (0.05) = 0.222, (0.01 ) = 0.292.
GROWTHEFFECTSOF SCALEWOUNDINGTULIPBULBS
355
5.A. 'Oxford'
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Fig. 5. (a) The rate of ethylene production (ml g-1 h - I ) in tulip bulb scales of'Oxford' after 0, 3, 6, 9 and 12 weeks of dry storage at 2°C; LSD (0.05) =0.222, (0.01) =0.292. (b) The effect of wounding tulip bulb scales of 'Oxford' on the rate of ethylene production (ml g-1 h-~ after 0, 3, 6, 9 and 12 weeks of dry storage at 2 ° C; LSD (0.05 ) = 0.222, (0.01 ) = 0.292.
356
L. KAWA ET AL.
respectively. Comparison of ethylene production from 1 to 5 days after wounding showed similar patterns for each cultivar and this was independent of the duration of precooling. The rate of ethylene production greatly increased 2-4 days after wounding of 'Oxford' bulbs and 2-3 days in the case of 'Apeldoorn' bulbs. Subsequently, it remained either at the same level or decreased. DISCUSSION The results (Tables 1 and 2) demonstrate that growth and flowering of partially special precooled tulip bulbs can be accelerated by precise wounding (Fig. 1 ) of the scales before planting. Although the conditions in which the ethylene production by bulbs was measured were not identical to the conditions under which the plants were forced in the greenhouse, it appeared that enhanced ethylene production was associated with the growth stimulation and flowering earliness. There was an apparent correlation between the peaks of ethylene production (Figs. 4(a), 4(b), 5 (a) and 5 (b)) observed for 'Oxford' bulbs stored at 2 °C for 6 weeks and 'Apeldoorn' bulbs stored for 9 weeks and a strong promotion of their subsequent growth and flowering. It is well known that, depending on the species and the precise physiological stage of development, ethylene may inhibit flower induction or promote flower initiation, stimulate flower development, or cause floral abortion of plants. In bulbous plants, ethylene has been shown to be effective in breaking dormancy and accelerating sprouting and flowering of Freesia(Imanishi and Fortanier, 1983; Uyemura and Imanishi, 1984), Gladiolus (Halevy et al., 1970; Ginzburg, 1974), Dutch iris (Imanishi and Yue, 1985), and Triteleia (Brodiaea) (Han et al., 1990). It is also known that exposing tulip bulbs to ethylene or treating them with ethephon can cause many floral disorders (Kamerbeek and De Munk, 1976 ). The direct and delayed effects of exogenously applied ethylene depends on the ethylene concentration, the stage of bulb development, duration of exposure, storage temperature, conditions after the exposure to ethylene (De Munk, 1973a, b). Exposure of'White Sail' tulip bulbs to ethylene for 6 weeks during storage after lifting inhibited the growth of young leaves and stamens within bulbs (De Munk, 1973a). Exposure of bulbs to ethylene at later stages of developmentcaused flower-bud abortion (De Munk, 1972; 1973b). This disorder was strongly influenced by the storage temperature and ethylene concentration. When bulbs were treated with ethylene at a late stage of floral development, both the extent and the percentage of abortion increased during long-term storage and also during growth in the greenhouse (De Munk, 1973b). Lastly, Nichols and Kofranek (19982) showed that ethylene inhibited the extension growth of cut tulips, but this could be reversed by silver
GROWTH EFFECTS OF SCALE WOUNDING TULIP BULBS
357
thiosulphate. It must be pointed out that all these effects of ethylene are highly cultivar dependent (De Munk, 1973b; De Hertogh et al., 1980). In our experiments, we found that bulb scale wounding either increased or had no effect on total length of the flowering plants (Tables 1 and 2). Although the wounding led to increased ethylene production (Fig. 4 (a), 4 (b), 5 (a) and 5 ( b ) ) it did not cause floral abortions and this is highly significant. When floral abortions were observed, they were only caused by very deep cutting of the scales, which was a technique problem. These observations suggest that ethylene at relatively low concentrations and with a duration of only a few days of exposure after planting of the bulbs had a favourable effect on tulip growth and flowering. The results (Figs. 4(a), 4(b), 5(a) and 5 ( b ) ) showed that ethylene production was affected by the duration of bulb cold treatment. It has been found that low-temperature treatments of tulip bulbs decreased the content of endogenous abscisic acid (Aung and De Hertogh, 1979) and increased the endogenous levels of gibberellins and cytokinins (Aung and De Hertogh, 1968 Rakhimbaev et al., 1978; Hanks and Rees, 1980). Thus, it appears that the ability to produce endogenous hormones, including ethylene, is enhanced by low temperature storage. It is important, therefore, to establish the interactions between these hormones in controlling the growth and development of the tulip. Gibberellins and kinetin injected into or near the flower buds can partially or totally prevent ethylene- or ethephon-induced floral abortion (De Munk and Gijzenberg, 1977; Moe, 1979 ). Tulip stem elongation appears to be under the control of endogenous auxins, but it is affected by gibberellins (Saniewski and De Munk, 1981; Okubo and Uemoto, 1985; Okubo et al., 1986). The existence of interactions between auxin and ethylene have been observed (Lieberman, 1979) and it would be valuable to conduct studies with the various PGRs in relation to scale wounding of tulips. It is also probable that wounding may influence other metabolic activities, which can affect plant growth and flowering. Some of these may be related to the storage carbohydrates and others to membrane permeability. When considering the effects of scale bulb wounding on subsequent plant growth, it appears that they show some similarities with those observed when bulbs, after dry storage at 5 ° C, are subjected to a short treatment at 27 ° C (Le Nard, 1980). It is known that while this treatment has a promotive effect on the growth of the aerial parts of the plants, it also exerts a negative effect on rooting (Le Nard, 1982). The effects of scale bulb wounding on the latter process need to be investigated. CONCLUSIONS These studies conducted at Raleigh, NC, completely confirmed the findings of studies previous conducted in Ploudaniel, France. It appears that, ow-
358
L. KAWAETAL.
ing to the positive effects on plant growth, scale wounding of tulip bulbs acts as a partial substitute for cold treatment. This makes it possible to reduce the duration of cold storage required by 'Special Precooled Tulips'. It is clear, however, that many other studies are needed to elucidate the physiological and biochemical responses induced by the wounding and the relations between the increased ethylene .production and promotion of the plant growth. However, when conducting additional studies on the cutting technique one has to consider that this procedure has a potential use for commercial forcing. It could be extremely useful for early forcing and for late bulb maturing years, e.g. the 1991 season. Additional studies will be carried out to determine if the wounding technique is applicable to 'Standard Forced Tulips'. ACKNOWLEDGEMENTS
This research was supported, in part, by the Dutch Bulb Exporters Association, Hillegom, The Netherlands. The authors acknowledge and thank Dr. Sylvia Blankenship for the use of her ethylene analysis equipment and for her inputs into the research. We also acknowledge the technical assistance of Gwendolyn Pemberton and Desire de Vries.
REFERENCES Aung, L.H. and De Hertogh, A.A., 1968. Gibberellin-like substances in non-cold and cold treated tulip bulbs (Tulipa sp.). In: F. Wightman and G. Setterfield (Editors),Biochemistry and Physiology of Plant Growth Substances. Runge Press, Ottawa, pp. 943-956. Aung, L.H. and De Hertogh, A.A., 1979. Temperature regulation of growth and endogenous abscisic acid-like content of Tulipa gesneriana L. Plant Physiol., 63:1111-1116. De Hertogh, A.A., 1974. Principles for forcing tulips, hyacinths, daffodils, Easter lilies and Dutch irises. Scientia Hortic., 2:313-355. De Hertogh, A.A., DiUey, D.R. and Blakely, N., 1980. Response variation of tulip cultivars to exogenous ethylene. Acta Hortic., 109:205-210. De Munk, W.J., 1972. Bud necrosis, a storage disease of tulips, III. The influence of ethylene and mites. Neth. J. Plant Pathol., 78: 168-178. De Munk, W.J., 1973a. Bud necrosis, a storage disease of tulips, IV. The influence of ethylene concentration and storage temperature on bud development. Neth. J. Plant Pathol., 79:1322.
De Munk, W.J., 1973b. Flower-bud blasting in tulips caused by ethylene. Neth. J. Plant Pathol., 79:41-53. De Munk, W.J., 1975. Ethylene disorders in bulbous crops during storage and glasshouse cultivation. Acta Hortic., 51: 321-328. De Munk, W.J. and De Rooy, M., 1971. The influence of ethylene on the development o f 5 ° C precooled 'Apeldoorn' tulips during forcing. HortScience, 6: 40-41. De Munk, W.J. and Gijzenberg, J., 1977. Flower-bud blasting in tulip plants mediated by the hormonal status of the plant. Scientia Hortic., 7: 255-268. De Munk, W.J. and Hoogeterp, P., 1975. Flower-bud blasting in tulip. Acta Hortic., 47: 149155.
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De Munk, W.J. and Saniewski, M., 1989. Gummosis in tulips under the influence of ethephon. Scientia Hortic., 40: 153-162. Ginzburg, C., 1974. Studies on the role of ethylene in gladiolus cormel germination. Plant Sci. Lett., 2: 133-138. Halevy, A.H., Shillo, R. and Simchon, S., 1970. Effect of 2-chloro-ethane phosphonic acid (ethrel) on health, dormancy, and flower and corm yield of gladioli. J. Hortic. Sci., 45: 427-434. Hart, S.S., Halevy, A.H., Sachs, R.M. and Reid, M.S., 1990. Enhancement of growth and flowering of Triteleia laxa by ethylene. J. Am. Soc. Hortic. Sci., 115: 482-486. Hanks, G.R. and Rees, A.R., 1980. Growth substances of tulip: the activity of gibberellin-like substances in field-grown tulips from planting until flowering. Z. Pflanzenphysiol., 98:213223. Imanishi, H. and Fortainier, E.J., 1983. Effects of exposing freesia corms to ethylene or to smoke on dormancy-breaking and flowering. Scientia Hortic., 18:381-389. Imanishi, H. and Yue, D., 1985. Effects of duration of exposure to ethylene on flowering of Dutch iris. Acta Hortic., 177: 141-145. Kamerbeek, G.A. and De Munk, W.J., 1976. A review of ethylene effects in bulbous crops. Scientia Hortie., 4:101-115. Kamerbeek, G.A., Verlind, A.L. and Schipper, J.A., 1971. Gummosis of tulip bulbs caused by ethylene. Acta Hortic., 23:167-172. Le Nard, M., 1980. Influence d'un traitment a temperature elevee applique apres une conservation des bulbes a temperature bassesur l'elongation de la hampe florale de la Tulipe. Ann. Amelior. Plantes, 30: 469-478. Le Nard, M., 1982. Quelques donnees nouvelles pour la preparation thermique et le forcage des bulbes de Tulipe. P.H.M. Rev. Hortic., 229: 43-53. Lieberman, M., 1979. Biosynthesis and action of ethylene. Ann. Rev. Plant Physiol., 30: 533591. Moe, R., 1979. Hormonal control of flower blasting in tulips. Acta Hortic., 91: 221-228. Nichols, R. and Kofranek, A.M., 1982. Reversal of ethylene inhibition of tulip stem elongation by silver thiosulfate. Scientia Hortic., 17:71-79. Okubo, H. and Uemoto, S., 1985. Changes in endogenous gibberellin and auxin activities during first internode elongation in tulip flower stalk. Plant Cell Physiol., 26:709-719. Okubo, H., Shiraishi, S. and Uemoto, S., 1986. Factors controlling elongation of the last internode in tulip flower stalk. J. Jpn. Soc. Hortic. Sci., 55: 320-325. Rakhimbaev, I.R., Syrtanova, G.A. and Solomina, V.F., 1978. The effect of cold treatment on the level of biological activity of endogenous growth regulators of tulip bulbs. Plant Physiol. 25:249-253 (in Russian). Saniewski, M. and De Munk, W.J., 1981. Hormonal control of shoot elongation in tulips. Scientia Hortic., 15: 363-372. Uyemura, S. and Imanishi, H., 1984. Effects of duration of exposure to ethylene on dormancy release in freesia corms. Scientia Hortic., 22: 383-390. Yang, S.F. and Pratt, H.IC, 1978. The physiology of ethylene in wounded plant tissues. In: G. Kahl (Editor), Biochemistry of Wounded Plant Tissues. Walter de Gruyter & Co., Berlin, pp. 595-622.
347
The effects of scale wounding of tulip bulbs on ethylene evolution, plant growth and flowering L u d w i k a K a w a a, Marcel Le N a r d b and A.A. D e Hertogh c aResearch Institute of Pomology and Floriculture, 96-100 Skierniewice, Poland blNRA, Station d'Ameliorationde la Pomme de Terre et des Plantes a Bulbes, 29260 Ploudaniel, France CDepartment of Horticultural Science, North Carolina State University, Raleigh, NC 27695- 7609, USA. (Received 10 October 1991 )
ABSTRACT Kawa, L., Le Nard, M. and De Hertogh, A.A., 1993.The effects of scale wounding of tulip bulbs on ethylene evolution, plant growth and flowering. Scientia Hortic., 53:347-359 Scale wounding significantly accelerated flowering of Tulipa gesneriana L. bulbs that had been stored for 3,6 and 9 weeks at 2°C for 'Apeldoorn' and for 3 and 6 weeks for 'Oxford'. In addition, final scape length was significantly longer in plants produced from wounded bulbs after 3 and 9 weeks of precooling for 'Apeldoorn' and after 3 weeks for 'Oxford'. Wounding increased ethylene production in all treatments of both cultivars. 'Oxford' produced significantlymore ethylene than 'Apeldoorn', however, the increased ethylene production did not produce floral abortions. Keywords: Bulbs; ethylene; flowering; Tulipa gesneriana; wounding.
INTRODUCTION
Successful forcing of tulips requires the use of bulbs harvested at a mature stage (Le Nard, 1982). After lifting, the bulbs must be stored at warm temperatures ( 1 7 - 3 4 ° C ) until the differentiation of the flower to stage G is achieved. Subsequently, they must be subjected to an extended cold treatment that promotes rapid plant growth and flowering during the greenhouse phase (De Hertogh, 1974). To satisfy the cold requirement, two basic techniques are used: ( 1 ) standard forcing - the bulbs (sometimes after 6 weeks ofprecooling at 9 ° C) are planted at low temperatures where rooting and cooling takes place and only scape growth and flowering occur in the greenhouse; (2) special precooling - the bulbs are dry stored for 9-12 weeks at 2-5 °C and Correspondence to: L. Kawa, Research Institute of Pomology and Floriculture, 96-100, Skierniewice, Poland.
348
L. KAWAET AL.
then planted directly in the greenhouse; where rooting, scape growth, and flowering occur. Exposing tulip bulbs to ethylene, or treating them with ethephon as a source of ethylene, can cause many physiological disorders (Kamerbeek and De Munk, 1976). Some of them are: floral bud necrosis (De Munk, 1972; 1973a), flower malformations and abortions (De Munk, 1973b; De Munk and Hoogeterp, 1975; Moe, 1979 ), inhibition of shoot and root growth (De Munk and De Rooy, 1971; De Munk, 1973b; Nichols and Kofranek, 1982), abnormal leaf formation (De Munk, 1975 ), and gummosis (Kamerbeek et al., 1971; De Munk and Saniewski, 1989). It is well known that wounding is one of the stress factors that can induce increased ethylene production by most plant tissues (Yang and Pratt, 1978 ). In preliminary trials, Le Nard (unpublished data) had shown that wounding of'Apeldoorn' and 'Paul Richter' tulip bulbs, previously stored for 3, 6 and 9 weeks at 5 ° C, led to a faster plant growth when compared with non-wounded bulbs. The objectives of this study were to determine the effects of scale wounding of tulip bulbs after various weeks ofprecooling not only on subsequent plant growth and flowering in the greenhouse but also on ethylene production. MATERIALS AND METHODS
Dutch-grown tulip bulbs ( 12/up cm, in circumference) of'Apeldoorn' and 'Oxford' were used. Prior to precooling, they were stored at 17-20 °C until 26 November 1990, when they were placed at 2°C. After 3, 6, 9 and 12 weeks of cold dry storage, i.e. on 17 December 1990; 8 January 1991; 28 January 1991; and 18 February 1991, 15 bulbs were planted per plastic tray (56.5 cm × 38.5 cm × 17.5 cm). The planting medium was pasteurized and consisted of equal volumes of shredded pine bark, sand, sphagnum peat moss, and sandy loam soil with a pH of 6.8. Immediately before planting, the entire tunic was carefully removed from each bulb. Subsequently, the scales of 30 bulbs per cultivar were cut four times at 90 ° angles (Fig. 1 ). Uncut bulbs were used as the ;ut-1
Cut-4
Cut-2 Cut-3
"Shoot
Flat Side of Tulip Bulb
Fig. l. The techniqueused to wound tulip scalesafter the entire tunic was removed.
GROWTH EFFECTS OF SCALE WOUNDING TULIP BULBS
349
controls. No fungicidal treatments were used before planting. Bulbs were forced in a greenhouse at 13-17 °C under prevailing light conditions. After the bulbs were fully rooted, they were fertilized with 2.4 g 1- 1 C a ( N O 3 ) 2 twice a week. On the date of flowering, the length of all internodes, leaves and the flowers were recorded. The length of the first (basal) internode was measured from the basal plate to the first leaf node after the scales were removed. The length of the fourth internode was measured from the third leaf to the base of the flower. On 26 November 1990, when the bulbs were placed at 2 °C and on each day of planting, 15 intact and 15 cut bulbs per cultivar were used to measure the rate of ethylene production. Each bulb was placed in a separate 490 ml glass container, sealed, and then held at room temperature ( 2 0 - 2 4 ° C ) . Samples ( 1 ml) were taken 18 h after closing. Subsequently, the bulbs were air ventilated for 6 h and the jars were re-closed. Samples were taken daily for 5 days. Ethylene determinations were made on a Varian 1400 gas chromatograph using an alumina column with a flame ionizator detector, RESULTS
Growth and flowering
The growth measurements presented in Tables 1 and 2 were recorded only for the bulbs that rooted and subsequently flowered. The percent of bulbs flowering for the treatments is given in each table and there were no significant differences between wounded and non-wounded bulbs. Those bulbs that failed to flower were mechanically damaged due to excessively deep cutting. A p e l d o o r n . - With control (uncut) bulbs, flowering occurred more quickly as
the duration of precooling at 2°C increased (Table 1 ). Bulb scale wounding significantly reduced the n u m b e r of days to flowering (Table 1, Fig. 2 ) after 3, 6 and 9 weeks of precooling. The wounded bulbs flowered 8, 5 and 2 days earlier than uncut bulbs, respectively. Wounding of bulbs that had been precooled for 3 weeks increased the total length of the plants, but mostly the upper three internodes. After 6 weeks of precooling, the growth differences between cut and uncut bulbs were not statistically significant. It must be pointed out that after 3 and 6 weeks of bulb storage at 2°C, the flowers coloured within the leaves. The greatest~overal ! effect of wounding on plant growth was observed after 9 weeks of bulb precooling. The length of all the internodes as well as the leaves was significantly increased after scale cutting. After 12 weeks of storage at 2 oC, which led to the production of the longest plants, wounding had no significant effect on flowering or total plant height. O x f o r d . - In uncut bulbs, flowering took place more quicldy as the duration of precooling at 2 °C increased (Table 2). After 3 and 6 weeks of precooling,
350
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Fig. 2. The effects of wounding tulip scales on plant growth and flowering in 'Apeldoorn'. Bulbs were planted on 28 January 1991, after 9 weeks ofprecooling at 2 ° C. The l~hotograph was taken 49 days after planting. Left: control, uncut bulbs. Right: wounded bulbs.
uncut 'Oxford' tulips flowered 10 days earlier than 'Apeldoorn'. Wounding bulbs that had been precooled for 3 and 6 weeks significantly reduced the number of days to flowering (Table 2, Fig. 3 ), but wounding did not affect the earliness of flowering in bulbs that had been precooled for 9 or 12 weeks. Wounding of bulbs that had been precooled for 3 weeks significantly increased the final length of all internodes and leaves. In the bulbs precooled for 6 and 9 weeks, wounded bulbs grew faster than controls, but the stimulatory effect of wounding on the total scape length was not statistically significant. The length of leaves was stimulated by wounding with 9 weeEs of precooling, but not with the 6 weeks precooled bulbs. After 12 weeks ofprecooling, wounding had no effect on the final length of the internodes and leaves.
General growth observationsfor both cultivars Flower length was similar for all treatments and for both cultivars (Tables 1 and 2 ). In the case of'Oxford' bulbs precooled for 12 weeks, a slight partial abortion of petals was observed after wounding. This could have been an ethylene-induced response. After 3, 6 and 9 weeks of precooling, bulbs of 'Apeldoorn' and 'Oxford' showed different growth patterns. However, after 12 weeks of precooling, the
GROWTH EFFECTS OF SCALE WOUNDING TULIP BULBS
353
Fig. 3. The effects of wounding tulip scales on plant growth and flowering in 'Oxford'. Bulbs were planted on 8 January 1991, after 6 weeks of precooling at 2 ° C. The photograph was taken 49 days after planting. Left: control, uncut bulbs. Right: woundedbulbs.
average n u m b e r of days to flowering and the total scape length were similar. In all experiments, w o u n d e d bulbs exhibited a yellow coloration of the outer scales.
Ethyleneproduction The level of ethylene produced by uncut bulbs was very low (0.044-0.210 n l g -1 h -1 ) for both cultivars (Figs. 4 ( a ) , 4 ( b ) , 5(a) and 5 ( b ) ) . The effect of wounding was obvious and it led to an increased ethylene production regardless of the bulb storage treatment. For both cultivars, the largest production took place after 3, 6 and 9 weeks of precooling. 'Oxford' bulbs produced significantly more ethylene than 'Apeldoorn'. The highest level of ethylene evolution by w o u n d e d 'Apeldoorn' bulbs took place after 9 weeks of precooling. In this treatment, the average ethylene production during the 5 days after cutting was 1.479 nl g-~ h -1 and was 4.8 times higher than from wounded uncooled bulbs (0.305 nl g-1 h-~ ) and 1.3 times higher than from 12 weeks precooled bulbs ( 1.170 nl g - ~ h - 1). Wounded 'Oxford' bulbs produced the highest level of ethylene after 6 weeks of precooling. The average ethylene production was 2.288 nl g - 1 h - 1 and this was 5.8 and 1.9 times higher than uncooled (0.397 nl g - 1 h - ~) and 12 weeks precooled ( 1.211 nl g - 1 h - 1) bulbs,
354
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4.A. 'Apeldoorn'
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Fig. 4. (a) The rate of ethylene production (ml g-1 h - l ) in tulip bulb scales of 'Apeldoorn' after 0, 3, 6, 9 and 12 weeks of dry storage at 2°C; LSD (0.05) =0.222, (0.01) =0.292. (b) The effect of wounding tulip bulb scales of 'Apeldoorn' on the rate of ethylene production (ml g - ] h - x after 0, 3, 6, 9 and 12 weeks of dry storage at 2 ° C, LSD (0.05) = 0.222, (0.01 ) = 0.292.
GROWTHEFFECTSOF SCALEWOUNDINGTULIPBULBS
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Fig. 5. (a) The rate of ethylene production (ml g-1 h - I ) in tulip bulb scales of'Oxford' after 0, 3, 6, 9 and 12 weeks of dry storage at 2°C; LSD (0.05) =0.222, (0.01) =0.292. (b) The effect of wounding tulip bulb scales of 'Oxford' on the rate of ethylene production (ml g-1 h-~ after 0, 3, 6, 9 and 12 weeks of dry storage at 2 ° C; LSD (0.05 ) = 0.222, (0.01 ) = 0.292.
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L. KAWA ET AL.
respectively. Comparison of ethylene production from 1 to 5 days after wounding showed similar patterns for each cultivar and this was independent of the duration of precooling. The rate of ethylene production greatly increased 2-4 days after wounding of 'Oxford' bulbs and 2-3 days in the case of 'Apeldoorn' bulbs. Subsequently, it remained either at the same level or decreased. DISCUSSION The results (Tables 1 and 2) demonstrate that growth and flowering of partially special precooled tulip bulbs can be accelerated by precise wounding (Fig. 1 ) of the scales before planting. Although the conditions in which the ethylene production by bulbs was measured were not identical to the conditions under which the plants were forced in the greenhouse, it appeared that enhanced ethylene production was associated with the growth stimulation and flowering earliness. There was an apparent correlation between the peaks of ethylene production (Figs. 4(a), 4(b), 5 (a) and 5 (b)) observed for 'Oxford' bulbs stored at 2 °C for 6 weeks and 'Apeldoorn' bulbs stored for 9 weeks and a strong promotion of their subsequent growth and flowering. It is well known that, depending on the species and the precise physiological stage of development, ethylene may inhibit flower induction or promote flower initiation, stimulate flower development, or cause floral abortion of plants. In bulbous plants, ethylene has been shown to be effective in breaking dormancy and accelerating sprouting and flowering of Freesia(Imanishi and Fortanier, 1983; Uyemura and Imanishi, 1984), Gladiolus (Halevy et al., 1970; Ginzburg, 1974), Dutch iris (Imanishi and Yue, 1985), and Triteleia (Brodiaea) (Han et al., 1990). It is also known that exposing tulip bulbs to ethylene or treating them with ethephon can cause many floral disorders (Kamerbeek and De Munk, 1976 ). The direct and delayed effects of exogenously applied ethylene depends on the ethylene concentration, the stage of bulb development, duration of exposure, storage temperature, conditions after the exposure to ethylene (De Munk, 1973a, b). Exposure of'White Sail' tulip bulbs to ethylene for 6 weeks during storage after lifting inhibited the growth of young leaves and stamens within bulbs (De Munk, 1973a). Exposure of bulbs to ethylene at later stages of developmentcaused flower-bud abortion (De Munk, 1972; 1973b). This disorder was strongly influenced by the storage temperature and ethylene concentration. When bulbs were treated with ethylene at a late stage of floral development, both the extent and the percentage of abortion increased during long-term storage and also during growth in the greenhouse (De Munk, 1973b). Lastly, Nichols and Kofranek (19982) showed that ethylene inhibited the extension growth of cut tulips, but this could be reversed by silver
GROWTH EFFECTS OF SCALE WOUNDING TULIP BULBS
357
thiosulphate. It must be pointed out that all these effects of ethylene are highly cultivar dependent (De Munk, 1973b; De Hertogh et al., 1980). In our experiments, we found that bulb scale wounding either increased or had no effect on total length of the flowering plants (Tables 1 and 2). Although the wounding led to increased ethylene production (Fig. 4 (a), 4 (b), 5 (a) and 5 ( b ) ) it did not cause floral abortions and this is highly significant. When floral abortions were observed, they were only caused by very deep cutting of the scales, which was a technique problem. These observations suggest that ethylene at relatively low concentrations and with a duration of only a few days of exposure after planting of the bulbs had a favourable effect on tulip growth and flowering. The results (Figs. 4(a), 4(b), 5(a) and 5 ( b ) ) showed that ethylene production was affected by the duration of bulb cold treatment. It has been found that low-temperature treatments of tulip bulbs decreased the content of endogenous abscisic acid (Aung and De Hertogh, 1979) and increased the endogenous levels of gibberellins and cytokinins (Aung and De Hertogh, 1968 Rakhimbaev et al., 1978; Hanks and Rees, 1980). Thus, it appears that the ability to produce endogenous hormones, including ethylene, is enhanced by low temperature storage. It is important, therefore, to establish the interactions between these hormones in controlling the growth and development of the tulip. Gibberellins and kinetin injected into or near the flower buds can partially or totally prevent ethylene- or ethephon-induced floral abortion (De Munk and Gijzenberg, 1977; Moe, 1979 ). Tulip stem elongation appears to be under the control of endogenous auxins, but it is affected by gibberellins (Saniewski and De Munk, 1981; Okubo and Uemoto, 1985; Okubo et al., 1986). The existence of interactions between auxin and ethylene have been observed (Lieberman, 1979) and it would be valuable to conduct studies with the various PGRs in relation to scale wounding of tulips. It is also probable that wounding may influence other metabolic activities, which can affect plant growth and flowering. Some of these may be related to the storage carbohydrates and others to membrane permeability. When considering the effects of scale bulb wounding on subsequent plant growth, it appears that they show some similarities with those observed when bulbs, after dry storage at 5 ° C, are subjected to a short treatment at 27 ° C (Le Nard, 1980). It is known that while this treatment has a promotive effect on the growth of the aerial parts of the plants, it also exerts a negative effect on rooting (Le Nard, 1982). The effects of scale bulb wounding on the latter process need to be investigated. CONCLUSIONS These studies conducted at Raleigh, NC, completely confirmed the findings of studies previous conducted in Ploudaniel, France. It appears that, ow-
358
L. KAWAETAL.
ing to the positive effects on plant growth, scale wounding of tulip bulbs acts as a partial substitute for cold treatment. This makes it possible to reduce the duration of cold storage required by 'Special Precooled Tulips'. It is clear, however, that many other studies are needed to elucidate the physiological and biochemical responses induced by the wounding and the relations between the increased ethylene .production and promotion of the plant growth. However, when conducting additional studies on the cutting technique one has to consider that this procedure has a potential use for commercial forcing. It could be extremely useful for early forcing and for late bulb maturing years, e.g. the 1991 season. Additional studies will be carried out to determine if the wounding technique is applicable to 'Standard Forced Tulips'. ACKNOWLEDGEMENTS
This research was supported, in part, by the Dutch Bulb Exporters Association, Hillegom, The Netherlands. The authors acknowledge and thank Dr. Sylvia Blankenship for the use of her ethylene analysis equipment and for her inputs into the research. We also acknowledge the technical assistance of Gwendolyn Pemberton and Desire de Vries.
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De Munk, W.J., 1973b. Flower-bud blasting in tulips caused by ethylene. Neth. J. Plant Pathol., 79:41-53. De Munk, W.J., 1975. Ethylene disorders in bulbous crops during storage and glasshouse cultivation. Acta Hortic., 51: 321-328. De Munk, W.J. and De Rooy, M., 1971. The influence of ethylene on the development o f 5 ° C precooled 'Apeldoorn' tulips during forcing. HortScience, 6: 40-41. De Munk, W.J. and Gijzenberg, J., 1977. Flower-bud blasting in tulip plants mediated by the hormonal status of the plant. Scientia Hortic., 7: 255-268. De Munk, W.J. and Hoogeterp, P., 1975. Flower-bud blasting in tulip. Acta Hortic., 47: 149155.
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De Munk, W.J. and Saniewski, M., 1989. Gummosis in tulips under the influence of ethephon. Scientia Hortic., 40: 153-162. Ginzburg, C., 1974. Studies on the role of ethylene in gladiolus cormel germination. Plant Sci. Lett., 2: 133-138. Halevy, A.H., Shillo, R. and Simchon, S., 1970. Effect of 2-chloro-ethane phosphonic acid (ethrel) on health, dormancy, and flower and corm yield of gladioli. J. Hortic. Sci., 45: 427-434. Hart, S.S., Halevy, A.H., Sachs, R.M. and Reid, M.S., 1990. Enhancement of growth and flowering of Triteleia laxa by ethylene. J. Am. Soc. Hortic. Sci., 115: 482-486. Hanks, G.R. and Rees, A.R., 1980. Growth substances of tulip: the activity of gibberellin-like substances in field-grown tulips from planting until flowering. Z. Pflanzenphysiol., 98:213223. Imanishi, H. and Fortainier, E.J., 1983. Effects of exposing freesia corms to ethylene or to smoke on dormancy-breaking and flowering. Scientia Hortic., 18:381-389. Imanishi, H. and Yue, D., 1985. Effects of duration of exposure to ethylene on flowering of Dutch iris. Acta Hortic., 177: 141-145. Kamerbeek, G.A. and De Munk, W.J., 1976. A review of ethylene effects in bulbous crops. Scientia Hortie., 4:101-115. Kamerbeek, G.A., Verlind, A.L. and Schipper, J.A., 1971. Gummosis of tulip bulbs caused by ethylene. Acta Hortic., 23:167-172. Le Nard, M., 1980. Influence d'un traitment a temperature elevee applique apres une conservation des bulbes a temperature bassesur l'elongation de la hampe florale de la Tulipe. Ann. Amelior. Plantes, 30: 469-478. Le Nard, M., 1982. Quelques donnees nouvelles pour la preparation thermique et le forcage des bulbes de Tulipe. P.H.M. Rev. Hortic., 229: 43-53. Lieberman, M., 1979. Biosynthesis and action of ethylene. Ann. Rev. Plant Physiol., 30: 533591. Moe, R., 1979. Hormonal control of flower blasting in tulips. Acta Hortic., 91: 221-228. Nichols, R. and Kofranek, A.M., 1982. Reversal of ethylene inhibition of tulip stem elongation by silver thiosulfate. Scientia Hortic., 17:71-79. Okubo, H. and Uemoto, S., 1985. Changes in endogenous gibberellin and auxin activities during first internode elongation in tulip flower stalk. Plant Cell Physiol., 26:709-719. Okubo, H., Shiraishi, S. and Uemoto, S., 1986. Factors controlling elongation of the last internode in tulip flower stalk. J. Jpn. Soc. Hortic. Sci., 55: 320-325. Rakhimbaev, I.R., Syrtanova, G.A. and Solomina, V.F., 1978. The effect of cold treatment on the level of biological activity of endogenous growth regulators of tulip bulbs. Plant Physiol. 25:249-253 (in Russian). Saniewski, M. and De Munk, W.J., 1981. Hormonal control of shoot elongation in tulips. Scientia Hortic., 15: 363-372. Uyemura, S. and Imanishi, H., 1984. Effects of duration of exposure to ethylene on dormancy release in freesia corms. Scientia Hortic., 22: 383-390. Yang, S.F. and Pratt, H.IC, 1978. The physiology of ethylene in wounded plant tissues. In: G. Kahl (Editor), Biochemistry of Wounded Plant Tissues. Walter de Gruyter & Co., Berlin, pp. 595-622.