AGRICULTURAL COMMUNICATIONS, 2014, 2(1):

Transcription

1 AGRICULTURAL COMMUNICATIONS, 2014, 2(1): The Effect of Gibberellic Acid Treatment at Different Stages of Inflorescence Development on Anthocyanin Synthesis in Oriental Hybrid Lily var. Sorbbone. NASRIN TAHERI-SHIVA 1 *, ABDOLLAH HATAMZADE 1, DAVOOD BAKHSHI 1, MOUSA RASOULI 2 AND MAHMOOD GHASEMNEZHAD 1 1 Department of Horticultural Science, Faculty of Agriculture, Guilan University. 2 Faculty of Agriculture, Malayer University, Malayer, Iran. *Corresponding Author: taherishiva60@gmail.com (Accepted: 1 Nov. 2013) ABSTRACT This study was conducted to investigate the effects of gibberellic acid spraying on anthocyanin synthesis in florets of oriental hybrid lily var. Sorbbone and its relationship with other qualified traits of lily cut flowers. Two concentration of gibberellic acid (GA3)(100 and 200 mgl -1 ) were separately sprayed on plants at three different stages of inflorescence development (flower bud initiation, 5 cm long florets, and 3 days before harvest). All treatments were arranged in a factorial complete randomized design with two factor (GA3 concentration and inflorescence development stage) in three replications. Evaluated traits were anthocyanin content of petals, antioxidant activity in petals, vase life, fresh weight, total soluble solids (TSS) and leaf chlorophyll content. Although GA3 application improved postharvest performance, but there was not a significant difference between applied concentrations considering most postharvest characteristics. There was also a significant difference between spraying at three different stages of inflorescence development. Spraying with 200 mgl -1 GA3, at the third stage, retained lily cut flower s fresh weight, leaf chlorophyll content and greenness during long storage periods. Other traits such as vase life, petal anthocyanin content, TSS, and antioxidant activity also recorded the highest amount in this treatment. Results indicate that GA3 spraying 3 days before harvest may enhance flowers vase life by increasing antioxidant activity and anthocyanin content of petals. Keywords: Antioxidant activity, chlorophyll content, postharvest, TSS, vase life. Abbreviations: chs: chalcone synthase; dfr: dihydroflavonol reductase; EBG: early biosynthetic gene; GA3: gibberellic acid; LBG: late biosynthetic gene; chi: chalcone flavanone isomerase; TA: total anthocyanin; TSS: total soluble solid. INTRODUCTION Being one of the major cut flowers in the global ornamental market and having a great number of bred varieties, lilies are mainly grown under protected environment. The genus Lilium is classified into six sections. The Oriental hybrid lily, one of the most important ornamental plants world-wide, is derived from inter-specific crosses of species of the section Sinomartagon (Leslie, 1982). Most Oriental hybrid lily cultivars have pink flowers due to anthocyanin pigmentation. They contain cyanidin 3-O-b-rutinoside as a major anthocyanin (Norbak and Kondo, 1999). Biochemical and enzymatic studies have clarified the anthocyanin biosynthesis pathway, and many genes encoding anthocyanin biosynthetic enzymes have been isolated (Dooner et al. 1991; Holton and Cornish 1995). The hormonal regulation of anthocyanin synthesis has been investigated by Kwack et al. and Chung et al. Gibberellins play a vital role in enhancing phenylalanine ammonia layase (PAL) synthesis and consequently increase anthocyanin contents. Hee- Ock et al. postulated that there was a close physiological relationship between them. Montero et al. stated that GA3 treatment increased anthocyanin content in addition to the enhancement of PAL activity in Fragaria ananossa fruits. Raifa et al. (2005), also showed that GA3 spraying (at 200 mgl -1 concentration) on Hibiscus sabdariffa plants (60 days after planting) enhances anthocyanin content of flowers due to increase in PAL activity. Flowering buds of Protea Pink Ice,

2 AGRICULTURAL COMMUNICATIONS. about 40 mm long, sprayed with aqueous solution of 1000 mgl -1 gibberellic acid, were more attractive than the untreated control flowers with more vase life and intense color (Ben-Jaacov, 2006). Generally, corolla elongation is accompanied by the accumulation of anthocyanin pigments, which suggests that corolla elongation and anthocyanin accumulation processes have common steps in their signal transduction pathways (Weiss and Halevy, 1989). In petunia (Petunia hybrida), the effect of GA3 on elongation and pigmentation has been studied in an in vitro flower bud culture system in which young detached corollas were grown in a sucrose medium. The corollas continue to elongate and become pigmented only in the presence of GA3 (Weiss and Halevy, 1989; Weiss et al., 1992). The synthesis of anthocyanins involves many enzymatic steps (Heller and Forkmann, 1988) and requires the coordinate expression of all the biosynthetic genes involved. In the in vitro culture system, GA3, induces expression of EBGs, such as chs and chi and LBGs such as dfr (Weiss et al., 1992). It was demonstrated that GA activates transcription and it is assumed that expression of other anthocyanin biosynthetic genes is similarly induced (Weiss et al., 1992). Anthocyanin pigments show potent antioxidant properties in vitro. In a recent analysis of red and green leaf extracts from Elatostema rugosum it was proven that anthocyanic leaves had a significantly greater antioxidant potential (Neill et al., 2002). Along with other flavonoids, the anthocyanins can directly scavenge molecular species of active oxygen, including hydrogen peroxide, singlet oxygen, and the superoxide, hydroxyl, and peroxyl radicals (Bors et al., 1994; Yamasaki et al., 1996). Thus, anthocyanins have an inherent potential to protect cell membranes from the effects of oxidative damage and prevent plant tissue's disruption and senescence. Our objective in the present research was to evaluate the effect of GA3 spraying on anthocyanin content of petals, flower's vase life and other qualified characteristics and also to find out the most suitable stage of spraying and optimum GA concentration in order to enhance better marketable flowers. MATERIALS AND METHODS Plant Material and Treatments: Oriental hybrid lily (cv. Sorbbone ) bulbs (14-16 cm circumference) were obtained from a commercial grower and planted in 1.4 L plastic containers. Plants were spaced cm and grown in a glass greenhouse. Soil mixture was a combination of washed sand, leaf mold and garden soil which was disinfected in an autoclave with 120 C over 15 minutes. Plants were fertigated with water containing 20N-10P-8K soluble fertilizer with trace elements (Melsspray) at 0.2 gl -1 and they occasionally were flushed with water. A set point temperature of C was used during the experiment with day time temperatures occasionally rising to 26 C. Plants were separately sprayed at three stages of inflorescence development (flower buds initiation, 5 cm long florets, and 3 days before cut flower harvest) with growth regulator solutions containing various concentrations of GA3 (100 and 200 mgl -1 ) to run off. In all the treatments, spray solutions contained 0.2 % Tween 20 as a surfactant. Plants remained into the greenhouse until harvesting time. Flower stems in all treatments were cut when the first flower was fully colored but not open. After harvest, they were taken to a postharvest evaluation room (20±2 C, 70% relative humidity, 950 Lux light for 12 h) and placed into jars contained distilled water before initial weighing. Postharvest traits such as anthocyianin content of petals, antioxidant activity, TSS, fresh weight, and vase life and leaf chlorophyll content (greenness) were measured at determined intervals as follow: Anthocyanin Extraction of Petals: Petal slices of first, second and third florets (0.4 g of each treatment) were macerated in 1 ml of acidic methanol solution (1% HCL in MeOH) in a test tube and allowed to equilibrate overnight at 4 C. These samples were centrifuged at g for 10 minutes at 4 C. Two stages of opening (entire opening stage and 7 th day after opening) for first and second florets were considered and for the third floret, anthocyanin content was only measured at entire opening stage. Determination of Total Anthocyanin Content: Total anthocyanin (TA) content of the petal extract was measured using the ph differential method described by Wrolstad and Giusti (2001). A crude petal extract were separately dissolved in potassium chloride buffer (KCl, M, ph 1.0) and sodium acetate (CH3CO2Na3H2O, 0.4 M, ph 4.5) with a pre-determined dilution factor. Sample measurement absorbencies were read at 541 and 700 nm against a blank cell containing distilled water by a spectrophotometer (6405 UV-Vis, Jenway, England). The absorbance (A) of the diluted sample was then calculated as follow: (A)=(A541pH1-A700pH1)-(A541pH4.5-A700pH4.5) The total anthocyanin (TA) pigment (mgl -1 ) was calculated as follow: Total anthocyanin(ta)(mgl -1 )=(A/a)(10 3 )(b)(c) In which: A: Absorbance; a: molar extinction coefficient of Cyanidin-3-rutinoside (=28800); b: Cyanidin-3-rutinoside molecular weight (=595.2); c: dilution factor (=1); 10 3 : conversion factor. 50

3 TAHERI-SHIVA ET AL. Antioxidant Activity: Petal antioxidant activity was measured on 7 th day according to Ramandeep et al. (2005). The antioxidant activity of the petal extracts was evaluated by DPPH free radical-scavenging methods. 100 μl of various concentrations of the extracts in methanol was added to 2900 μl of a 0.1 mm DPPH methanol solution. The mixture was shaken immediately after adding DPPH solution and allowed to stand at room temperature in the dark. The decrease in absorbance at 517 nm was measured after 25 min until the reaction reach a plateau. These experiments were run in triplicate at 25 C. The inhibitory percentage of DPPH was calculated as follows: Scavenging effect (%) [(Ao (A Ab))/Ao] 100 In which: Ao: A517 of DPPH without sample (control); A: A517 of sample and DPPH; Ab: A517 of sample without DPPH (blank). Vase Life: Vase life of cut flowers were recorded (in days) until more than 50% of petals started wilting and became discolored. The laboratory room was illuminated at 950 Lux at flowers level by Sylvania cool white fluorescent lamps (12h on off cycle). Fresh Weight: Flower fresh weight was immediately measured with a digital balance after cutting and it considered as an initial weight. This trait was also measured every third day over the storage period. Data are presented as proportional (%) change relative to the initial fresh weight (Han, 2001). Total Soluble Solids: TSS is presented as percentage and includes a collection of different sugars, proteins, organic acids and, etc. in an extracted tissue. In order to determine TSS content, a refractometer (CETI, Belgium) was used. A small piece of petal in each treatment was squeezed then a drop of obtained extract was used to measure refractive index by the refractometer. This trait was measured on 3 rd and 7 th days of postharvest life. Chlorophyll Content: Initially, leaves on a cut flower stem were divided into three imaginary parts (upper, middle and lower part) and their leaf greenness was estimated using a portable SPAD-502 meter (Minolta, Osaka, Japan). The final data was the average of leaf greenness in three mentioned parts. This trait was measured on 2 nd, 5 th, 9 th and 14 th days over the storage period. Statistical Analysis: Data were subjected to analysis of variance in order to determine treatment effects. Treatment means were compared using Tukey s multiple range test (General Linear Models Procedure, SAS software). RESULTS AND DISCUTIONS Anthocyanin Content: Results showed that there was no significant difference between two used concentrations at each stage on anthocyanin content but three stages were significantly different with each other. Third stage of spraying (3 days before harvest) caused significantly more anthocyanin in the petals of the first, second and third florets (p < 0.05) (Table 1). Source of Variation df Table1. Variance analysis of evaluated postharvest traits in Sorbbone lily cut flowers. Vase Life Leaf Greenness (SPAD Value) Fresh Weight (%) (Day) Day-2 Day-5 Day-9 Day-14 Day-3 Day-6 Day-9 Day-12 GA3 1 2 ns 0.05 ns 0.22 ns 3.92 ns 12 ** ns ns ns ** Spraying Stage ** 1.48 ns 1.39 ns 7.76 * ** ns ns ns ** Interaction ns ns 0.16 ns 1.71 ns 1.80 * ns 3.12 ns 8.63 ns 5.18 ns Error % C.V ns = non-significant,** = significant at the 99% level= p<0.01, *=significant at the 95% level= p<0.05. Table1. (Continue). Variance analysis of evaluated postharvest traits in Sorbbone lily cut flowers. Total Soluble Anthocyanin Content Source of Solid df first floret first floret on second floret second floret Variation third floret at Day-3 Day-7 at full day-7 after at full on day-7 Day-7 full anthesis anthesis opening anthesis after opening GA ns 0.88 ns 0.37 ns 3.73 ns 0.88 ns 3.73 ns 0.76 ns 0.68 ns Spraying Stage ns 4.51 ** 4.04 * * 3.40 ns 6.80 * 7.54 * 5.07 ** Interaction ns Antioxidant Activity 0.25 ns 0.01 ns 2.06 ns 0.09 ns 2.17 ns 0.09 ns 0.01 ns Error % C.V ns = non-significant, ** = significant at the 99% level= p<0.01, *=significant at the 95% level= p<

4 AGRICULTURAL COMMUNICATIONS. Table 2. Effect of Gibberellic acid spraying stages on qualified traits of Sorbbone lily cut flowers. Spraying Stages Antioxidant Activity (%) Vase Life (day) Leaf Greenness on Day-9 Total Soluble Solids (%) T1 (flower bud initiation) 2.7 b 17 b b 3.2 b T2 (5 cm long florets) 3.2 b b b 3.7 b T3 (3 days before harvest) 4.5 a 19.8 a 54.5 a 4.9 a Means within a column followed by the same letters are not significantly different at P Various researches have showed that GA3 is one of the most important plant growth regulators involved in anthocyanin synthesis. It induces expression of some genes such as chi, chs and dfr and enhances enzymes activity (Weiss et al., 1992). Montero et al. (1998), showed that exogenous application of GA3 increases anthocyanin content in addition to the enhancement of PAL activity in Fragaria ananossa fruits. In most plants, anthocyanin synthesis in the flowers is under developmental regulation and its accumulation coincides with petal growth (Mol et al. 1996). Anthocyanin accumulation usually occurs at later stages of petal development (Martin and Gerats, 1993). The third stage of gibberellic acid spraying in our experiment was along with the initial stage of pigmentation process and expression of related genes. That is probably why exogenous application of GA3 at mentioned stage resulted in the most intense color of petals. Elatostema rugosum leaves had significantly greater antioxidant potential. Thus higher antioxidant activity in flowers at third stage spraying is probably related to higher anthocyanin content. Vase Life: Vase life of flowers treated at third stage, regardless of GA applied concentrations was significantly more than the other two stages (p<0.01) (Table 1 and 2). This may be related to more anthocyanins content and their role in delaying plant senescence due to scavenging free radicals and protecting cell membranes from the effects of oxidative damage. Fresh Weight: Fresh weights of cut flower stems didn t show any difference on 3rd, 6 th and 9 th days of experiment, but significant variation (p<0.01) was seen among spraying stages and also between used concentrations in long storage period on day-12 at laboratory (Table 1). At each stage of spraying, 200 mgl -1 gibberellic acid resulted in higher fresh weight. Among the three mentioned stages, the 3 rd stage had the highest fresh weight (Fig. 2 and 3). Higher fresh weight could be due to more vase life and preservation of leaf greenness in the late postharvest period. Fig. 1. Anthocyanin content of Sorbbone lily florets at different stages of evaluation. (T1, T2 and T3 are the stages of spraying of GA3; 1: First floret at full anthesis; 2: First floret on day-7 after opening; 3: Second floret on day-7 after opening; 4: Third floret at entire opening stage.) Antioxidant Activity: Spraying stages showed significant difference in antioxidant activity of petals (p<0.01, Table 1). Third stage treatment had higher amount of antioxidants (Table 2). No significant difference was seen between used concentrations. This treatment also caused more anthocyanin content of petals (Fig. 1). Many researchers have showed that anthocyanins can directly scavenge active oxygen molecules including hydrogen peroxide, singlet oxygen, superoxide, hydroxyl, and peroxyl radicals (Bors et al., 1994; Yamasaki et al., 1996). Also Neill et al. (2002) stated that anthocyanic content of Fig. 2. Effect of applied GA concentrations on fresh weight of Sorbbone lily on day-12. Total Soluble Solids: Results showed that there was no significant difference between GA concentrations but there was a significantly difference between spraying stages (p<0.01) (Table 1). Total soluble solids in petals of flowers sprayed at third stage were higher than two other stages on day-7 (Table 2). Leaves on flower stems are the main source of photosynthesis and production of essential 52

5 TAHERI-SHIVA ET AL. carbohydrates like sucrose for preparing nutritional substances in non-photosynthetic tissues like petals of flowers (van Doorn, 2004). Gibberellic acid can enhances hydration of starch to glucose and fructose. Combination of these sugars results in production of more sucrose and strength of cell walls. Having more sugar in tissues preserves them from early disruption and besides that increases their longevity (Halevy and Mayak, 1981). period has an important role in senescence acceleration and ending of lilie s vase life. Using GA3 as different treatments like pulsing or spraying on flower leaves avoid chlorophyll disruption and preserves more nitrogen supply (Emongor, 2004). GA has a structural role in chloroplast membrane and conserves more chlorophyll due to enhancing photosynthesis. Fig. 3. Effect of spraying stages on fresh weight of Sorbbone lily on day-12. (T1: flower buds initiation, T2: 5 cm long florets, and T3: 3 days before cut flower harvest). Chlorophyll Content: Leaf greenness were significantly higher on days-9 and 14 in leaves of cut flowers sprayed at third stage (3 days before harvest). On day-9, difference was just among spraying stages (p<0.05) (Table 1). On day-14, applied concentrations showed significant variation and cut flowers sprayed with 200 mgl -1 GA3 at third stage preserved their leaf chlorophyll content (greenness) more than other treatments (p<0.05, Table 1) (Fig. 4). Leaf yellowing over postharvest Fig. 4. Interaction of spraying stages and GA3 concentrations on Sorbbone lily leaf greenness at day-14. (T1: flower buds initiation, T2: 5 cm long florets, and T3: 3 days before cut flower harvest). CONCLUSION Considering all studied aspects, our results suggest that spraying Sorbbone lily flowers with GA3 three days before harvest will enhances flower vase life and quality. This could be a good and economical way to improve flower s visual and marketable quality. Ben-Jaacov, J Gibberellic acid spray increased size and quality of Protea Pink Ice Flowers. Acta Horticulturea. 716: Bors, W., C. Michel and M. Saran Flavonoid antioxidants: rate constants for reactions with oxygen radicals. Methods in Enzymology. 234: Chung, B., S. Lee, B.N. Chung and S. Lee Effect of plant growth regulators, nitrogen and sucrose on anthocyanin formation by callus culture of Euphorbia milii var. Splendens. Journal of Horticultural Science. 40: Dooner, H.K., T.P. Robbins and R.A. Jorgensen Genetic and developmental control of anthocyanin biosynthesis. Annual Review of Genetics. 25: Emongor, V.E Effect of Gibberellic acid on postharvest quality and vase life of Gerbera cut flowers (Gerbera jamesonii). Journal of Agronomy. 3: REFERENCES Halevy A.H. and S. Mayak Senescence and postharvest physiology of cut flowers. Part 2. Horticultural Reviews. 3: Hee-Ock B., T. Saito, Y. Tomitaka and H.O. Boo Effect of plant growth regulators on the anthocyanin synthesis of Perilla ocymoides L. Journal of the Korean Society for Horticultural Science. 38: Heller W. and G. Forkmann Biosynthesis. In: J.B. Harborn (Ed.). The Flavonoids. Chapman and Hall, London, UK. pp: Holton, T.A. and E.C. Cornish Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell. 7: Kwack, H., J. Lee, H. Kwack and J.S. Lee Effects of uniconazole and gibberellin on leaf variegation of ornamental plants under different light conditions. Journal of the Korean Society for Horticultural Science. 38: Leslie, A.C The International Lily Register, third ed., The Royal Horticultural Society, London, UK. 53

6 AGRICULTURAL COMMUNICATIONS. Martin, C. and T. Gerats Control of pigment biosynthesis genes during petal development. Plant Cell. 5: Mol, J., G. Jenkins, E. Schafer and D. Weiss Signal perception, transduction, and gene expression involved in anthocyanin biosynthesis. Critical Review on Plant Science. 15: Montero, T., E. Molla, M. Martin-Cabrejas and F.J. Lopez Andreu Effect of gibberellic acid (GA3) on strawberry PAL (Phenylalanine Ammonia Lyase) and TAL (Tyrosine Ammonia Lyase) enzyme activities. Journal of Science, Food and Agriculture. 77: Neill, S.O., K.S. Gould, P.A. Kilmartin, K.A. Mitchell and K.R. Markham Antioxidant activities of red versus green leaves in Elatostema rugosum. Plant, Cell and Environment. 25: Norbak, R. and T. Kondo Anthocyanin from flowers of Lilium (Liliaceae), Phytochemistry. 50: Olszewski, N.T., P. Sun and F. Gubler Gibberellin signaling: biosynthesis, catabolism and response pathways. Plant Cell. 14: Ramandeep, K.T. and P. Geoffrey Savage Antioxidant activity in different fractions of tomatoes. Food Research International. 38: Van Doorn, W.G Is petal senescence due to sugar starvation? Plant Physiology. 134: Weiss, D. and A.H. Halevy Stamens and gibberellin in the regulation of corolla pigmentation and growth in Petunia hybrida. Planta. 179: Weiss, D., R. Van Blokland, J.M. Kooter and A.J. Vim- Tunen Gibberellic acid regulates chalcone synthesis gene transcription in the corolla of Petunia hybrida. Plant Physiology. 98: Wrolstad, R.E Color and pigment analyses in fruit products. In: Agricultural Station Bulletin 624, Oregon State University, Oregon, USA. Yamasaki, H., H. Uefuji and Y. Sakihama Bleaching of the red anthocyanin induced by superoxide radical. Archives of Biochemistry and Biophysics. 332: