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Effects of auxin, gibberellin, and cytokinin on petal blackening and flower opening in cut lotus flowers (Nelumbo nucifera)
Postharvest Biology and Technology 75 (2013) 54–57
Contents lists available at SciVerse ScienceDirect
Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Research note
Effects of auxin, gibberellin, and cytokinin on petal blackening and flower opening in cut lotus flowers (Nelumbo nucifera) Wachiraya Imsabai a,∗ , Wouter G. van Doorn b a b
Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
a r t i c l e
i n f o
Article history: Received 22 February 2012 Accepted 20 May 2012 Keywords: Nelumbo nucifera Benzyladenine Blackening Flower opening Gibberellin Naphthylacetic acid Petals Thidiazuron
a b s t r a c t Cut lotus flowers (Nelumbo nucifera Gaertn. cv. Saddabutra), which are sold as closed buds, fail to open and show rapid petal blackening when placed in vase water. We investigated the effect on bud opening and petal blackening of treatments with an auxin, a gibberellic acid, and two cytokinins. Continuous treatment of cut flowers placed in an aqueous solution containing ≥0.1 mM naphthylacetic acid (NAA) hastened petal blackening and resulted in stem curvature, but lower concentrations (0.01–10 M) had no effect. Depending on the experiment, continuous treatment with 0.03–0.45 mM of the gibberellin GA3 delayed petal blackening by 0.5–1.5 d (controls lasted 4 d), but in experiments during the hot/rainy season (May–September) GA3 had no effect. At 25–100 M the cytokinin benzyladenine (6-benzylaminopurine; BA) delayed petal blackening by about 1.0 d. Similarly, the cytokinin thidiazuron (TDZ) delayed petal blackening by about 1.0 d, at 1.25–2.5 M. Pulse treatments had similar or better effects. A 3–12 h pulse treatment with 0.45 mM GA3 or with 10 M TDZ delayed the time to petal blackening by 1.1–2.3 d. However, none of these treatments promoted bud opening. It is concluded, nonetheless, that a pulse treatment with GA3 or TDZ seems promising for practice. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lotus flowers (Nelumbo nucifera spp. nucifera) are a symbol of spiritual transcendence and purity, both in Hinduism and Buddhism. Flowers are cut in the bud stage and used in religious worship. The genus Nelumbo is in the family Nelumbonaceae (APG, 2003, 2009; Chase and Reveal, 2009). There are two extant species (N. lutea, with pale yellow petals, and N. nucifera, with white or pink petals) which have also been regarded to be one species with two subspecies: N. nucifera spp. lutea and N. nucifera spp. nucifera (Hayes et al., 2000). In Thailand one of the two main commercial cultivars of N. nucifera spp. nucifera is Saddabutra, which is probably identical to cv. Album Plenum. The Saddabutra flower has several green outer petals and many white inner petals. It has a long floral stalk, called peduncle. Both the peduncle and the leaves emerge from a rhizome, which grows in the mud of a body of water. As the peduncles are cut at the peduncle–rhizome junction, the cut flowers have no leaves. In the field the full-grown floral bud opens in the morning and closes at night. The end of floral life in the uncut flower is due to petal abscission, which occurs 4–5 d after bud opening. The petals
∗ Corresponding author. Tel.: +66 34 281084/5; fax: +66 34 281086. E-mail address: [email protected] (W. Imsabai). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.05.015
fall off when slightly black or still fully green or white. This is in sharp contrast with flowers that are cut. At the commercial cutting stage the flowers are still in the bud stage, but would open within a day when left uncut. After cutting and placing the peduncles in water the buds fail to open. Additionally, the outer petals show rapid blackening, considerably before they abscise (Pinto et al., 2009; Imsabai et al., 2010). We previously reported that exogenous ethylene had no effect on petal blackening in cut lotus flowers, but that 1methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, delayed petal blackening by about 1.0–1.5 d. However, the 1-MCP treatment did not promote bud opening (Imsabai et al., 2010). Here, we report on experiments with three other hormone classes: auxin, cytokinin and gibberellin. The goal of our experiments was to delay the early petal blackening in cut lotus flowers placed in water, and to promote bud opening. Cytokinins and gibberellins have been reported to delay leaf and petal senescence, and might therefore also delay petal blackening. Their relative effect on leaf senescence depended on the species (Ferrante et al., 2008; van Doorn et al., 2011). Both groups of hormones delayed petal senescence in carnation (Eisinger, 1977; Saks et al., 1992; van Staden, 1995). Cytokinins delayed tulip flower senescence while GA3 had no effect (van Doorn et al., 2011), but neither cytokinins nor a gibberellins had an effect on the senescence of Hemerocallis flowers (Rubinstein, 2000).
W. Imsabai, W.G. van Doorn / Postharvest Biology and Technology 75 (2013) 54–57
55
2. Materials and methods
3. Results
2.1. Plants
3.1. Flower morphology, petal blackening, and vase life
Lotus flower buds (N. nucifera Gaertn. spp. nucifera, cv. Saddabutra, likely the same as cv. Album Plenum) were harvested in the morning. The buds were picked at their normal commercial stage, i.e. with the floral buds still fully closed but, if left uncut, opening within about a day. Workers walked in the water of the lotus pond, which stood about one meter deep, or collected stems by boat. Stems were broken under water, close to their junction with the rhizome. After harvest, the stems were held dry or were placed in purified water (tap water, after passing through reverse osmosis equipment). Stem length at harvest varied from 40 to 60 cm. In some treatments the stems were recut under water in the field (removing about 10 cm) and then immediately placed in purified water. Stems were brought to the laboratory within about 1 h of harvest. In the laboratory the stems were recut in air, to a length of 25 cm, and were then placed individually in glass vials or glass graduated cylinders, containing purified water. The flower stalks were held in a temperature-controlled room at 25 ◦ C, 70% RH, and natural light supplemented with fluorescent light (lights on from about 7 a.m. to 7 p.m., photon flux density about 15 mol m−2 s−1 ).
The perianth of cv. Saddabutra lotus flowers comprises about 28 leaf-like organs. About 10–12 petals have all, or part, of their exterior at the surface of the floral bud. The outer two leafy organs, considered to be sepals, are less than 2 cm long. Often the first petals are barely larger than these sepals. The outer petals increase in size with a more distal position, with a maximum of the length about 8 cm at petal 15–16. The length then decreases again in more distal petals. A sharp distinction is found between the petals and the smaller staminoid petals, which number over 100. The first 12 petals are pale green, with a whitish part at the base. The relative size of the white part increases towards more inward petals. From about petal 13 the petals are only faintly green at the distal part. Petals 17–26 are almost uniformly white. At the time of harvest the two sepals are already uniformly black, while the two smallest petals are usually completely or partially black. The next, relatively small leaf-like organs are blackening already during the first day of vase life. In commercial practice about 4–7 lowermost leaves are removed prior to sales. In our experiments with lotus buds we removed the first 6 leaf-like organs, and determined the time to 50% blackening in the leaf-like organs that were then visible. Blackening in cut lotus buds often starts at the tips of the green outer petals, but can also initially be found at the lateral petal edges. When placed in vase water for one or two days, almost all the visible green petals have started to blacken. Apart from the dark black spots on the petal surface, the remaining of the petals turns grey. The length of vase life was defined as the time until 50% of the surface of the buds is black or grey.
2.2. Petal blackening, flower opening During vase life, petal blackening was assessed visually, at the end of the morning. The length of vase life was defined as the period until half of the visible petals showed black patches. Flower opening was determined visually. The flower was defined to be open if the petals at the tip leave an opening.
2.3. Chemicals Naphthalene acetic acid (NAA) was obtained from Fluka (Buchs, Switzerland), benzyladenine (N6 -benzyladenine; 6benzylaminopurine; BA) from Sigma–Aldrich (St. Louis, MO, USA), thidiazuron ([1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea]; TDZ) and GA3 from Phyto Technology Laboratories (Overland Park, KS, USA). NAA was first dissolved in 100% ethanol (Thomas and Hay, 2011). A control for ethanol as a solvent was included. BA (Mansouri and Preece, 2009), GA3 (Huarte and García, 2009; Mansouri and Preece, 2009) and TDZ (Chamani et al., 2006) were first dissolved in 2 ml 1 N KOH. After adjustment with HCl the pH of the vase solutions containing KOH as a solvent varied between 6.6 and 7.0. Controls for the solvents were included. Two types of experiments were carried out. In the first type the chemicals were included in the vase water at the onset of vase life, and were not replenished. In the second the chemicals were applied as a 3–12 h pulse treatment carried out in the climate-controlled room. After the pulsing the flowers were placed in water in the climate-controlled room.
2.4. Statistics All experiments used 8–10 flowers (replications) per treatment. Where possible, the means between treatments were compared using ANOVA, and calculation of LSD at P ≤ 0.05. All experiments were at least once repeated at a later date.
3.2. Effects on petal blackening of continuous treatments with naphthylacetic acid, GA3 , benzyladenine, or thidiazuron In continuous treatments naphthylacetic acid (NAA) hastened petal blackening at 10 mM, 1 mM and 0.1 mM. At these concentrations it also induced a curvature in the upper part of the peduncle, which is growing during the first day of vase life. NAA had no effect on petal blackening when applied at 0.01 mM (10 M), 1 M, 0.1 M, or 0.01 M. A control for the solvent also had no effect (results not shown). Depending on the experiment GA3 delayed petal blackening at 0.03–0.60 mM or had no effect (Table 1). Petal blackening was delayed by 0.8–1.5 d (average 1.1 d; n = 4) in experiments carried out in November, December and March, which is during the dry and cooler season, and 0.7 d in an experiment carried out in April, which is the beginning of the hot/rainy season. In experiments during the hot/rainy season (May and September) GA3 had no effect (Table 1). A control for the solvent had no effect (not shown) BA delayed petal blackening by about 1.0 d. In one experiment the optimum concentration was 50 M but in another test it was 75–100 M (Table 2). At higher concentrations BA did not further delay petal blackening. A control for the solvent had no effect (Table 2, first experiment; and data not shown). The experiments with BA were carried out in November and December. TDZ delayed petal blackening by about1.0 d, both at 1.25 and 2.5 M, but it had no statistically significant effect at 5 M (Table 3). The experiments were carried out in December, January and March. 3.3. Pulse treatments with GA3 and TDZ GA3 was also applied as a pulse treatment prior to vase life in water. A pulse for 3–12 h at 0.45 mM delayed the time to petal blackening by 1.1–2.0 d (Table 4). No difference was found between
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W. Imsabai, W.G. van Doorn / Postharvest Biology and Technology 75 (2013) 54–57
Table 1 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) continuously placed in aqueous solutions containing gibberellic acid (GA3 ). The solution was prepared at the onset of vase life and was not replenished. Exp. 1 was conducted in March, Exp. 2 in April, Exp. 3 in May, Exp. 4 in September, Exp. 5 and 6 in November, and Exp. 7 in December. The controls were held in distilled water. Treatment
Control GA3 , 0.03 mM GA3 , 0.06 mM GA3 , 0.09 mM GA3 , 0.12 mM GA3 , 0.15 mM GA3 , 0.30 mM GA3 , 0.45 mM
Vase life (d)a Exp. 1
Exp. 2
Exp. 3
Exp. 4
Exp. 5
Exp. 6
Exp. 7
3.6 b – – – – 5.0 a 4.8 a 4.8 a
3.9 bc 4.6 a 4.3 ab 4.2 ab – – – –
4.8 a 4.9 a 4.6 a 4.9 a 4.6 a 4.8 a – –
3.8 a 4.0 a 4.0 a 4.0 a 4.0 a 4.0a – –
4.0 b 4.9 a – – – – – –
4.2 b 4.9 a – – – 5.0 a – –
4.3 b 5.8 a 5.8 a 5.8 a 5.6 a 5.6 a – –
a Data are means of 10 flowers, individually placed in vases. The statistical data refer to the first five treatments. Data in the same column accompanied by the same letter or combination of letters are not statistically different (P ≤ 0.05).
Table 2 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) continuously placed in aqueous benzyladenine (BA) solutions. The solution was prepared at the onset of vase life and was not replenished. Treatment
Control (distilled water) BA, 25 M BA, 50 M BA, 75 M BA, 100 M
Vase life (d)a Exp. 1
Exp. 2
4.8 b 5.4 ab 5.8 a 5.4 ab 5.0 b
4.4 b 5.0 ab 5.2 ab 5.4 a 5.6 a
a Data are means of 10 flowers, individually placed in vases. Data in the same column accompanied by a different letter or a different combination of letters are statistically different (P ≤ 0.05).
Table 3 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) continuously placed in aqueous thidiazuron (TDZ) solutions. The solution was prepared at the onset of vase life and was not replenished. Treatment
Control (distilled water) TDZ, 1.25 M TDZ, 2.5 M TDZ, 5 M
Vase life (d)a Exp. 1
Exp. 2
5.3 b 6.2 a 6.0 a 5.8 ab
5.0 b 6.1 a 6.3 a 5.6 ab
a
Data are means of 10 flowers, individually placed in vases. Data in the same column accompanied by a different letter or a different combination of letters are statistically different (P ≤ 0.05). Table 4 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) after pulsing with aqueous thidiazuron (TDZ) and/or GA3 solutions for various periods, prior to vase life in water. The vase life of each of the treatments excludes the time needed for the pulsing treatment. Treatment
Vase life (d)a Exp. 1
Exp. 2
Exp. 3
Controls (directly in distilled water) Pulsing with 5 M TDZ for 3 h 10 M TDZ for 3 h 10 M TDZ for 6 h 10 M TDZ for 12 h
4.0 b 5.6 a 5.6 a – –
5.1 b – 7.1 a 6.9 a 7.4 a
4.7 c – 6.4 b 7.0 a 6.3 b
Controls (directly in distilled water) 0.45 mM GA3 for 3 h 0.45 mM GA3 for 6 h 0.45 mM GA3 for 12 h
4.0 b 5.6 a 5.1 a 5.3 a
Controls (directly in distilled water) 0.45 mM GA3 for 3 h 5 M TDZ for 3 h 0.45 mM GA3 and 5 M TDZ for 3 h
4.5 c 6.8 a 6.7 a 5.7 b
a Data are means of 10 flowers, individually placed in vases. Data in the same column and same experiment accompanied by a different letter are statistically different (P ≤ 0.05).
the 3 h or longer period of application (Table 4). The experiments were carried out in April and December. Thidiazuron was also used as a pulse treatment. Pulsing for 3 h with an aqueous solution containing 5 or 10 M TDZ delayed petal blackening by about 1.5–2.0 d (Table 4). A 6 or 12 h pulse also delayed the time to petal blackening to 2.0 d (Table 4). Further tests showed a similar 2 d delay of petal blackening after pulsing for 3 h with 20, 30 or 40 M TDZ (data not shown). The experiments in which TDZ was applied as a pulse treatment were carried out in January and March. GA3 at 0.45 mM and TDZ at 5 M were also applied separately and together, as a 3 h pulse treatment. The delay of petal blackening (about 2 d) was the same in the separate and in the combined treatment (Table 4). These experiments were carried out in December. An experiment carried out in August showed a small and identical positive effect of these two compounds, applied separately, as blackening was delayed by 0.5 d. The effect on blackening of the chemicals applied together was the same as that of the compounds given separately (data not shown).
3.4. Effects on flower opening In the controls very little growth occurred in the buds, not enough to result in bud opening. The hormone treatments did not promote bud opening (results not shown).
4. Discussion It is not clear if petal blackening is physiologically similar to leaf or petal senescence. Nonetheless, the effect of GA3 or cytokinins on petal blackening in cut lotus flowers was similar to the effect of these hormones on leaf yellowing in other species. For example, GA3 or cytokinins delayed leaf yellowing in cut stock flowers (Ferrante et al., 2008) and cut tulip flowers (van Doorn et al., 2011), but leaf yellowing in these species was more delayed by cytokinins than by GA3 . The effects of these hormones on petal blackening in lotus are also similar to their effect on Iris tepal senescence. Cytokinins and GA3 delayed Iris tepal senescence by the same amount of time when applied at the time of harvest, i.e. before bud opening (van Doorn, unpublished data). The mechanism of action of cytokinins or gibberellins in plant blackening or senescence is not known (Davies, 2004; Hedden and Thomas, 2006). Tang and Newton (2005) reported that the effect of TDZ on shoot formation was correlated with decreased activity of peroxidase and catalase. This might be due to a decrease in the levels of oxidants. Cell damage by oxidative compounds has been suggested as one possible cause of senescence and death (Jing et al., 2008; Zentgraf and Hemleben, 2008), and may also underlie lotus
W. Imsabai, W.G. van Doorn / Postharvest Biology and Technology 75 (2013) 54–57
petal blackening. The effects of GA3 on senescence have also not as yet been explained in physiological terms (Gan, 2010). The petal blackening found in lotus flowers shows similarity to the leaf blackening in a number of Protea species (Reid et al., 1989; Jones et al., 1995; van Doorn, 2001). The blackening in Protea starts in patches on the leaf, and later on covers the whole leaf area. This is similar to the blackening in lotus petals. The type of black colour was also similar in both genera. The Angiosperm Phylogeny group (APG, 2003, 2009; Chase and Reveal, 2009) placed the genus Nelumbo in the family Nelumbonaceae, in the superfamily Proteales. There is therefore a relatively close taxonomic relationship between lotus and Protea. Leaf blackening in Protea species has been hypothesized to be due to cellular damage, which leads to polymerization of phenolic compounds and possibly tannins (Jones et al., 1995). The effects of GA3 on petal blackening in lotus depended on the time of the year. In tests in November, December, and March a delay of blackening was observed. However, in tests in May and September no effect was found. These data indicate an effect of temperature and rain. In Thailand the dry and relatively cool season occurs from November to February–March. The hot and rainy season starts in April and ends in October. A detailed investigation through the year was not carried out using cytokinins, although one experiment with TDZ pulsing in August showed a delay of blackening by only 0.5 d. It is therefore possible that the effect of cytokinins also depends on the season. The hormone treatments tested had no effect on bud opening. This means that we have at present no solution for the lack of bud opening in cut lotus flowers. It might be argued nonetheless that the closed floral buds, when pulse-treated with TDZ, do have ornamental value, as the time to unacceptable petal blackening was extended to almost one week. It is concluded that although none of the treatments tested promoted flower opening, petal blackening was delayed by about two days after a pulse treatment with GA3 or thidiazuron, at least during the dry season, and that either of these treatment therefore seems promising for the lotus flower trade. References APG, 2003. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141, 399–436. APG, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161, 105–121.
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Chamani, E., Irving, D., Joyce, D.C., 2006. Studies with thidiazuron on the vase life of cut rose flowers. Journal of Applied Horticulture 8, 42–44. Chase, M.W., Reveal, J.L., 2009. A phylogenetic classification of the land plants to accompany APG III. Botanical Journal of the Linnean Society 161, 122–127. Davies, P.J., 2004. Plant Hormones: Biosynthesis, Signal Transduction, Action. Springer Verlag, Berlin. Eisinger, W., 1977. Role of cytokinins in carnation flower senescence. Plant Physiology 59, 707–709. Ferrante, A., Mensuali-Sodi, A., Serra, G., 2008. Effect of thidiazuron and gibberellic acid on leaf yellowing of cut stock flowers. Central European Journal of Biology 4, 461–468. Gan, S., 2010. The hormonal regulation of senescence. In: Davies, P.J. (Ed.), Plant Hormones. Springer, The Netherlands, pp. 597–617. Hayes, V., Schneider, E.L., Carlquist, S., 2000. Floral development of Nelumbo nucifera (Nelumbonaceae). International Journal of Plant Sciences 161, S183–S191. Hedden, P., Thomas, S.G., 2006. Plant Hormone Signalling. Annual Plant Reviews, vol. 24. John Wiley & Sons, New York, NY. Huarte, R., García, M.D., 2009. Tripsacum dactyloides (L.) L. (Poaceae) caryopsis dormancy and germination responses to scarification, hydrogen peroxide and phytohormones. Seed Science and Technology 37, 544–553. Imsabai, W., Ketsa, S., van Doorn, W.G., 2010. Role of ethylene in the lack of floral opening and in petal blackening of cut lotus (Nelumbo nucifera) flowers. Postharvest Biology and Technology 58, 57–64. Jing, H.C., Hebeler, R., Oeljeklaus, S., Sitek, B., Stühler, K., Meyer, H.E., Sturre, M.J.G., Hille, J., Warscheid, B., Dijkwel, P.P., 2008. Early leaf senescence is associated with an altered cellular redox balance in Arabidopsis cpr5/old1 mutants. Plant Biology 10, 85–98. Jones, R.B., McConchie, R., van Doorn, W.G., Reid, M.S., 1995. Leaf blackening in cut Protea flowers. Horticulture Reviews 17, 173–201. Mansouri, K., Preece, J.E., 2009. The influence of plant growth regulators on explant performance, bud break, and shoot growth from large stem segments of Acer saccharinum L. Plant Cell Tissue and Organ Culture 99, 313–318. Pinto, A.C.R., Mello, S.C., Jacomino, A.P., Minami, K., Barbosa, J.C., 2009. Postharvest characteristics and treatments to overcome latex exuded from cut flowers of lotus. Acta Horticulturae 813, 671–677. Reid, M.S., van Doorn, W.G., Newman, J.P., 1989. Leaf blackening in Proteas. Acta Horticulturae 261, 81–84. Rubinstein, B., 2000. Regulation of cell death in flower petals. Plant Molecular Biology 44, 303–318. Saks, Y., van Staden, J., Smith, M.T., 1992. Effect of gibberellic acid on carnation flower senescence: evidence that the delay of carnation flower senescence by gibberellic acid depends on the stage of flower development. Plant Growth Regulation 11, 45–51. Tang, W., Newton, R.J., 2005. Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in Eastern white pine (Pinus strobus L.) zygotic embryos. Plant Physiology and Biochemistry 43, 760–769. Thomas, R.G., Hay, M.J.M., 2011. Existing branches correlatively inhibit further branching in Trifolium repens: possible mechanisms. Journal of Experimental Botany 62, 1027–1036. van Doorn, W.G., 2001. Leaf blackening: recent developments. Acta Horticulturae 545, 197–204. van Doorn, W.G., Perik, R.J.J., Abadie, P., Harkema, H., 2011. A treatment to improve the vase life of cut tulips: effects on tepal senescence, tepal abscission, leaf yellowing and stem elongation. Postharvest Biology and Technology 61, 55–63. van Staden, J., 1995. Hormonal control of carnation flower senescence. Acta Horticulturae 405, 232–239. Zentgraf, U., Hemleben, V., 2008. Molecular cell biology: are reactive oxygen species regulators of leaf senescence? Progress in Botany 69 (Part 2), 117–138.
Contents lists available at SciVerse ScienceDirect
Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Research note
Effects of auxin, gibberellin, and cytokinin on petal blackening and flower opening in cut lotus flowers (Nelumbo nucifera) Wachiraya Imsabai a,∗ , Wouter G. van Doorn b a b
Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
a r t i c l e
i n f o
Article history: Received 22 February 2012 Accepted 20 May 2012 Keywords: Nelumbo nucifera Benzyladenine Blackening Flower opening Gibberellin Naphthylacetic acid Petals Thidiazuron
a b s t r a c t Cut lotus flowers (Nelumbo nucifera Gaertn. cv. Saddabutra), which are sold as closed buds, fail to open and show rapid petal blackening when placed in vase water. We investigated the effect on bud opening and petal blackening of treatments with an auxin, a gibberellic acid, and two cytokinins. Continuous treatment of cut flowers placed in an aqueous solution containing ≥0.1 mM naphthylacetic acid (NAA) hastened petal blackening and resulted in stem curvature, but lower concentrations (0.01–10 M) had no effect. Depending on the experiment, continuous treatment with 0.03–0.45 mM of the gibberellin GA3 delayed petal blackening by 0.5–1.5 d (controls lasted 4 d), but in experiments during the hot/rainy season (May–September) GA3 had no effect. At 25–100 M the cytokinin benzyladenine (6-benzylaminopurine; BA) delayed petal blackening by about 1.0 d. Similarly, the cytokinin thidiazuron (TDZ) delayed petal blackening by about 1.0 d, at 1.25–2.5 M. Pulse treatments had similar or better effects. A 3–12 h pulse treatment with 0.45 mM GA3 or with 10 M TDZ delayed the time to petal blackening by 1.1–2.3 d. However, none of these treatments promoted bud opening. It is concluded, nonetheless, that a pulse treatment with GA3 or TDZ seems promising for practice. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lotus flowers (Nelumbo nucifera spp. nucifera) are a symbol of spiritual transcendence and purity, both in Hinduism and Buddhism. Flowers are cut in the bud stage and used in religious worship. The genus Nelumbo is in the family Nelumbonaceae (APG, 2003, 2009; Chase and Reveal, 2009). There are two extant species (N. lutea, with pale yellow petals, and N. nucifera, with white or pink petals) which have also been regarded to be one species with two subspecies: N. nucifera spp. lutea and N. nucifera spp. nucifera (Hayes et al., 2000). In Thailand one of the two main commercial cultivars of N. nucifera spp. nucifera is Saddabutra, which is probably identical to cv. Album Plenum. The Saddabutra flower has several green outer petals and many white inner petals. It has a long floral stalk, called peduncle. Both the peduncle and the leaves emerge from a rhizome, which grows in the mud of a body of water. As the peduncles are cut at the peduncle–rhizome junction, the cut flowers have no leaves. In the field the full-grown floral bud opens in the morning and closes at night. The end of floral life in the uncut flower is due to petal abscission, which occurs 4–5 d after bud opening. The petals
∗ Corresponding author. Tel.: +66 34 281084/5; fax: +66 34 281086. E-mail address: [email protected] (W. Imsabai). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.05.015
fall off when slightly black or still fully green or white. This is in sharp contrast with flowers that are cut. At the commercial cutting stage the flowers are still in the bud stage, but would open within a day when left uncut. After cutting and placing the peduncles in water the buds fail to open. Additionally, the outer petals show rapid blackening, considerably before they abscise (Pinto et al., 2009; Imsabai et al., 2010). We previously reported that exogenous ethylene had no effect on petal blackening in cut lotus flowers, but that 1methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, delayed petal blackening by about 1.0–1.5 d. However, the 1-MCP treatment did not promote bud opening (Imsabai et al., 2010). Here, we report on experiments with three other hormone classes: auxin, cytokinin and gibberellin. The goal of our experiments was to delay the early petal blackening in cut lotus flowers placed in water, and to promote bud opening. Cytokinins and gibberellins have been reported to delay leaf and petal senescence, and might therefore also delay petal blackening. Their relative effect on leaf senescence depended on the species (Ferrante et al., 2008; van Doorn et al., 2011). Both groups of hormones delayed petal senescence in carnation (Eisinger, 1977; Saks et al., 1992; van Staden, 1995). Cytokinins delayed tulip flower senescence while GA3 had no effect (van Doorn et al., 2011), but neither cytokinins nor a gibberellins had an effect on the senescence of Hemerocallis flowers (Rubinstein, 2000).
W. Imsabai, W.G. van Doorn / Postharvest Biology and Technology 75 (2013) 54–57
55
2. Materials and methods
3. Results
2.1. Plants
3.1. Flower morphology, petal blackening, and vase life
Lotus flower buds (N. nucifera Gaertn. spp. nucifera, cv. Saddabutra, likely the same as cv. Album Plenum) were harvested in the morning. The buds were picked at their normal commercial stage, i.e. with the floral buds still fully closed but, if left uncut, opening within about a day. Workers walked in the water of the lotus pond, which stood about one meter deep, or collected stems by boat. Stems were broken under water, close to their junction with the rhizome. After harvest, the stems were held dry or were placed in purified water (tap water, after passing through reverse osmosis equipment). Stem length at harvest varied from 40 to 60 cm. In some treatments the stems were recut under water in the field (removing about 10 cm) and then immediately placed in purified water. Stems were brought to the laboratory within about 1 h of harvest. In the laboratory the stems were recut in air, to a length of 25 cm, and were then placed individually in glass vials or glass graduated cylinders, containing purified water. The flower stalks were held in a temperature-controlled room at 25 ◦ C, 70% RH, and natural light supplemented with fluorescent light (lights on from about 7 a.m. to 7 p.m., photon flux density about 15 mol m−2 s−1 ).
The perianth of cv. Saddabutra lotus flowers comprises about 28 leaf-like organs. About 10–12 petals have all, or part, of their exterior at the surface of the floral bud. The outer two leafy organs, considered to be sepals, are less than 2 cm long. Often the first petals are barely larger than these sepals. The outer petals increase in size with a more distal position, with a maximum of the length about 8 cm at petal 15–16. The length then decreases again in more distal petals. A sharp distinction is found between the petals and the smaller staminoid petals, which number over 100. The first 12 petals are pale green, with a whitish part at the base. The relative size of the white part increases towards more inward petals. From about petal 13 the petals are only faintly green at the distal part. Petals 17–26 are almost uniformly white. At the time of harvest the two sepals are already uniformly black, while the two smallest petals are usually completely or partially black. The next, relatively small leaf-like organs are blackening already during the first day of vase life. In commercial practice about 4–7 lowermost leaves are removed prior to sales. In our experiments with lotus buds we removed the first 6 leaf-like organs, and determined the time to 50% blackening in the leaf-like organs that were then visible. Blackening in cut lotus buds often starts at the tips of the green outer petals, but can also initially be found at the lateral petal edges. When placed in vase water for one or two days, almost all the visible green petals have started to blacken. Apart from the dark black spots on the petal surface, the remaining of the petals turns grey. The length of vase life was defined as the time until 50% of the surface of the buds is black or grey.
2.2. Petal blackening, flower opening During vase life, petal blackening was assessed visually, at the end of the morning. The length of vase life was defined as the period until half of the visible petals showed black patches. Flower opening was determined visually. The flower was defined to be open if the petals at the tip leave an opening.
2.3. Chemicals Naphthalene acetic acid (NAA) was obtained from Fluka (Buchs, Switzerland), benzyladenine (N6 -benzyladenine; 6benzylaminopurine; BA) from Sigma–Aldrich (St. Louis, MO, USA), thidiazuron ([1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea]; TDZ) and GA3 from Phyto Technology Laboratories (Overland Park, KS, USA). NAA was first dissolved in 100% ethanol (Thomas and Hay, 2011). A control for ethanol as a solvent was included. BA (Mansouri and Preece, 2009), GA3 (Huarte and García, 2009; Mansouri and Preece, 2009) and TDZ (Chamani et al., 2006) were first dissolved in 2 ml 1 N KOH. After adjustment with HCl the pH of the vase solutions containing KOH as a solvent varied between 6.6 and 7.0. Controls for the solvents were included. Two types of experiments were carried out. In the first type the chemicals were included in the vase water at the onset of vase life, and were not replenished. In the second the chemicals were applied as a 3–12 h pulse treatment carried out in the climate-controlled room. After the pulsing the flowers were placed in water in the climate-controlled room.
2.4. Statistics All experiments used 8–10 flowers (replications) per treatment. Where possible, the means between treatments were compared using ANOVA, and calculation of LSD at P ≤ 0.05. All experiments were at least once repeated at a later date.
3.2. Effects on petal blackening of continuous treatments with naphthylacetic acid, GA3 , benzyladenine, or thidiazuron In continuous treatments naphthylacetic acid (NAA) hastened petal blackening at 10 mM, 1 mM and 0.1 mM. At these concentrations it also induced a curvature in the upper part of the peduncle, which is growing during the first day of vase life. NAA had no effect on petal blackening when applied at 0.01 mM (10 M), 1 M, 0.1 M, or 0.01 M. A control for the solvent also had no effect (results not shown). Depending on the experiment GA3 delayed petal blackening at 0.03–0.60 mM or had no effect (Table 1). Petal blackening was delayed by 0.8–1.5 d (average 1.1 d; n = 4) in experiments carried out in November, December and March, which is during the dry and cooler season, and 0.7 d in an experiment carried out in April, which is the beginning of the hot/rainy season. In experiments during the hot/rainy season (May and September) GA3 had no effect (Table 1). A control for the solvent had no effect (not shown) BA delayed petal blackening by about 1.0 d. In one experiment the optimum concentration was 50 M but in another test it was 75–100 M (Table 2). At higher concentrations BA did not further delay petal blackening. A control for the solvent had no effect (Table 2, first experiment; and data not shown). The experiments with BA were carried out in November and December. TDZ delayed petal blackening by about1.0 d, both at 1.25 and 2.5 M, but it had no statistically significant effect at 5 M (Table 3). The experiments were carried out in December, January and March. 3.3. Pulse treatments with GA3 and TDZ GA3 was also applied as a pulse treatment prior to vase life in water. A pulse for 3–12 h at 0.45 mM delayed the time to petal blackening by 1.1–2.0 d (Table 4). No difference was found between
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Table 1 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) continuously placed in aqueous solutions containing gibberellic acid (GA3 ). The solution was prepared at the onset of vase life and was not replenished. Exp. 1 was conducted in March, Exp. 2 in April, Exp. 3 in May, Exp. 4 in September, Exp. 5 and 6 in November, and Exp. 7 in December. The controls were held in distilled water. Treatment
Control GA3 , 0.03 mM GA3 , 0.06 mM GA3 , 0.09 mM GA3 , 0.12 mM GA3 , 0.15 mM GA3 , 0.30 mM GA3 , 0.45 mM
Vase life (d)a Exp. 1
Exp. 2
Exp. 3
Exp. 4
Exp. 5
Exp. 6
Exp. 7
3.6 b – – – – 5.0 a 4.8 a 4.8 a
3.9 bc 4.6 a 4.3 ab 4.2 ab – – – –
4.8 a 4.9 a 4.6 a 4.9 a 4.6 a 4.8 a – –
3.8 a 4.0 a 4.0 a 4.0 a 4.0 a 4.0a – –
4.0 b 4.9 a – – – – – –
4.2 b 4.9 a – – – 5.0 a – –
4.3 b 5.8 a 5.8 a 5.8 a 5.6 a 5.6 a – –
a Data are means of 10 flowers, individually placed in vases. The statistical data refer to the first five treatments. Data in the same column accompanied by the same letter or combination of letters are not statistically different (P ≤ 0.05).
Table 2 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) continuously placed in aqueous benzyladenine (BA) solutions. The solution was prepared at the onset of vase life and was not replenished. Treatment
Control (distilled water) BA, 25 M BA, 50 M BA, 75 M BA, 100 M
Vase life (d)a Exp. 1
Exp. 2
4.8 b 5.4 ab 5.8 a 5.4 ab 5.0 b
4.4 b 5.0 ab 5.2 ab 5.4 a 5.6 a
a Data are means of 10 flowers, individually placed in vases. Data in the same column accompanied by a different letter or a different combination of letters are statistically different (P ≤ 0.05).
Table 3 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) continuously placed in aqueous thidiazuron (TDZ) solutions. The solution was prepared at the onset of vase life and was not replenished. Treatment
Control (distilled water) TDZ, 1.25 M TDZ, 2.5 M TDZ, 5 M
Vase life (d)a Exp. 1
Exp. 2
5.3 b 6.2 a 6.0 a 5.8 ab
5.0 b 6.1 a 6.3 a 5.6 ab
a
Data are means of 10 flowers, individually placed in vases. Data in the same column accompanied by a different letter or a different combination of letters are statistically different (P ≤ 0.05). Table 4 Vase life of cut lotus flowers (Nelumbo nucifera cv. Saddabutra) after pulsing with aqueous thidiazuron (TDZ) and/or GA3 solutions for various periods, prior to vase life in water. The vase life of each of the treatments excludes the time needed for the pulsing treatment. Treatment
Vase life (d)a Exp. 1
Exp. 2
Exp. 3
Controls (directly in distilled water) Pulsing with 5 M TDZ for 3 h 10 M TDZ for 3 h 10 M TDZ for 6 h 10 M TDZ for 12 h
4.0 b 5.6 a 5.6 a – –
5.1 b – 7.1 a 6.9 a 7.4 a
4.7 c – 6.4 b 7.0 a 6.3 b
Controls (directly in distilled water) 0.45 mM GA3 for 3 h 0.45 mM GA3 for 6 h 0.45 mM GA3 for 12 h
4.0 b 5.6 a 5.1 a 5.3 a
Controls (directly in distilled water) 0.45 mM GA3 for 3 h 5 M TDZ for 3 h 0.45 mM GA3 and 5 M TDZ for 3 h
4.5 c 6.8 a 6.7 a 5.7 b
a Data are means of 10 flowers, individually placed in vases. Data in the same column and same experiment accompanied by a different letter are statistically different (P ≤ 0.05).
the 3 h or longer period of application (Table 4). The experiments were carried out in April and December. Thidiazuron was also used as a pulse treatment. Pulsing for 3 h with an aqueous solution containing 5 or 10 M TDZ delayed petal blackening by about 1.5–2.0 d (Table 4). A 6 or 12 h pulse also delayed the time to petal blackening to 2.0 d (Table 4). Further tests showed a similar 2 d delay of petal blackening after pulsing for 3 h with 20, 30 or 40 M TDZ (data not shown). The experiments in which TDZ was applied as a pulse treatment were carried out in January and March. GA3 at 0.45 mM and TDZ at 5 M were also applied separately and together, as a 3 h pulse treatment. The delay of petal blackening (about 2 d) was the same in the separate and in the combined treatment (Table 4). These experiments were carried out in December. An experiment carried out in August showed a small and identical positive effect of these two compounds, applied separately, as blackening was delayed by 0.5 d. The effect on blackening of the chemicals applied together was the same as that of the compounds given separately (data not shown).
3.4. Effects on flower opening In the controls very little growth occurred in the buds, not enough to result in bud opening. The hormone treatments did not promote bud opening (results not shown).
4. Discussion It is not clear if petal blackening is physiologically similar to leaf or petal senescence. Nonetheless, the effect of GA3 or cytokinins on petal blackening in cut lotus flowers was similar to the effect of these hormones on leaf yellowing in other species. For example, GA3 or cytokinins delayed leaf yellowing in cut stock flowers (Ferrante et al., 2008) and cut tulip flowers (van Doorn et al., 2011), but leaf yellowing in these species was more delayed by cytokinins than by GA3 . The effects of these hormones on petal blackening in lotus are also similar to their effect on Iris tepal senescence. Cytokinins and GA3 delayed Iris tepal senescence by the same amount of time when applied at the time of harvest, i.e. before bud opening (van Doorn, unpublished data). The mechanism of action of cytokinins or gibberellins in plant blackening or senescence is not known (Davies, 2004; Hedden and Thomas, 2006). Tang and Newton (2005) reported that the effect of TDZ on shoot formation was correlated with decreased activity of peroxidase and catalase. This might be due to a decrease in the levels of oxidants. Cell damage by oxidative compounds has been suggested as one possible cause of senescence and death (Jing et al., 2008; Zentgraf and Hemleben, 2008), and may also underlie lotus
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petal blackening. The effects of GA3 on senescence have also not as yet been explained in physiological terms (Gan, 2010). The petal blackening found in lotus flowers shows similarity to the leaf blackening in a number of Protea species (Reid et al., 1989; Jones et al., 1995; van Doorn, 2001). The blackening in Protea starts in patches on the leaf, and later on covers the whole leaf area. This is similar to the blackening in lotus petals. The type of black colour was also similar in both genera. The Angiosperm Phylogeny group (APG, 2003, 2009; Chase and Reveal, 2009) placed the genus Nelumbo in the family Nelumbonaceae, in the superfamily Proteales. There is therefore a relatively close taxonomic relationship between lotus and Protea. Leaf blackening in Protea species has been hypothesized to be due to cellular damage, which leads to polymerization of phenolic compounds and possibly tannins (Jones et al., 1995). The effects of GA3 on petal blackening in lotus depended on the time of the year. In tests in November, December, and March a delay of blackening was observed. However, in tests in May and September no effect was found. These data indicate an effect of temperature and rain. In Thailand the dry and relatively cool season occurs from November to February–March. The hot and rainy season starts in April and ends in October. A detailed investigation through the year was not carried out using cytokinins, although one experiment with TDZ pulsing in August showed a delay of blackening by only 0.5 d. It is therefore possible that the effect of cytokinins also depends on the season. The hormone treatments tested had no effect on bud opening. This means that we have at present no solution for the lack of bud opening in cut lotus flowers. It might be argued nonetheless that the closed floral buds, when pulse-treated with TDZ, do have ornamental value, as the time to unacceptable petal blackening was extended to almost one week. It is concluded that although none of the treatments tested promoted flower opening, petal blackening was delayed by about two days after a pulse treatment with GA3 or thidiazuron, at least during the dry season, and that either of these treatment therefore seems promising for the lotus flower trade. References APG, 2003. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141, 399–436. APG, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161, 105–121.
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