Research Report

Effect of Different Chemical Compounds on Leaf Chlorophyll Content and Postharvest Quality of Lilium  

Dhiman M.R. , Guleria M.S. , Chander Parkash , Raj Kumar
ICAR-Indian Agricultural Research Institute, Regional Station, Katrain, Kullu Valley, Himachal Pradesh- 175129, India
Author    Correspondence author
International Journal of Horticulture, 2015, Vol. 5, No. 18   doi: 10.5376/ijh.2015.05.0018
Received: 16 Oct., 2015    Accepted: 01 Dec., 2015    Published: 29 Dec., 2015
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Dhiman M.R., Guleria M.S., Chander Parkash, and Raj Kumar, 2015, Effect of different chemical compounds on leaf chlorophyll content and postharvest quality of Lilium, International Journal of Horticulture, 5(18): 1-6 (doi:  10.5376/ijh.2015.05.0018)


The effects of different chemicals treatment on postharvest cut flower quality of hybrid lily cultivar ‘Eyeliner’ were investigated. Lily stems were harvested when the first flower bud showed full colour. A whole stem (leaf and bud) sprayed with water (control) and with a growth regulator solutions containing TDZ (1 & 2 ppm), BA (25 ppm), GA3 (25 ppm), BA (50 ppm), GA3 (50 ppm), BA + GA3 (25 ppm + 25 ppm), BA + GA3 (50 ppm + 50 ppm), SAMRAS (2&4 ml·L-1), Agri-Herbo-99 (1 & 2 ml·L-1) and Super Gibbre (1 & 2 ml·L-1). After the treatments, each stem was then maintained for postharvest evaluation in an individual 500 ml conical flask with 300 ml of normal tap water.  Within each treatment, the numbers of buds per stem were kept constant as possible for ease of data recording. In general, BA alone or in combination with GA3 treatments significantly delayed the flower bud opening. Leaf yellowing and flower senescence was substantially delayed up to one week when cut flowers were sprayed with a solution containing TDZ (2 ppm). Application of BA @ 50 ppm significantly delayed total chlorophyll (a + b) degradation after 6 and 9 days. After 3 days sharp increase in total chlorophyll content was found when cut stems were sprayed with 1 ppm TDZ and slightly declined after 6 and 9 days. In conclusion, TDZ @ 2 ppm not only delayed leaf yellowing but also reduced onset of petal senescence in Lilium cut flowers. BA was able to delay the chlorophyll degradation.

GA3; TDZ; BA; Leaf yellowing; Chlorophyll; Flower senescence; Lilium; Postharvest

Quality loss of cut flowers during the postharvest stage can be due to petal wilting, abscission, colour changes, leaf yellowing or weight loss. Leaf yellowing is a form of leaf senescence that is both highly programmed and genetically regulated (Reid and Kofranek, 1980). Some cut flowers are sensitive to leaf yellowing which occurs before petal wilting and lose their commercial value. The physiological disorders responsible for chlorophyll loss are mainly driven by hormonal and environmental factors (Reid and Kofranek, 1980; Ferrante et al., 2005). Several cut flowers show chlorophyll loss before petal wilting viz., Alstroemeria spp., Lilium, Mathiola incana, Pelargonium, Euphorbia pulcherrima. One of the main factors involved in the induction of chlorophyll degradation is the lack of cytokinins. This plant hormone is preferentially biosynthesized in roots (Sakakibara, 2006). Therefore, after harvest, cut flowers do not receive cytokinins and leaf yellowing takes place.
Among various cut flowers, Lilium has just opened its way in floriculture industry of our country due to its immense potential as cut flower. One of the major post-production disorders is the occurrence of leaf yellowing or browning, which usually starts on the lower leaves and moves progressively upward. The vase life of cut lilies depends on leaf yellowing or tepal wilting and abscission. Some investigations suggested that exposure to ethylene promotes leaf, bud and flower abscission and leaf yellowing (Celikel et al., 2002) and that treatment with ethylene inhibitors increase flower longevity (Swart, 1980). Lily flowers were initially reported to have very less sensitivity to ethylene levels in the environment (Woltering et al., 1988), although variability was found to exist across and within different groups. Post harvest leaf yellowing in Easter lily is disastrous and termed as catastrophic ‘yellowing’. This disorder strikes quickly, causing a normal looking plant turn almost entirely yellow within a few days after cold storage (Emonger and Tshwenyane, 2004). Cultural factors such as growth regulators, low phosphorous, poor root-rot controls, high temperature during forcing, shipping delays and cold storage have been attributed to be the major causes (Miller, 1997).
Several commercial formulations are available for preventing leaf yellowing of cut flowers (Celikel et al., 2002). Usually, they contain cytokinins (6-benzyladenine) or gibberellic acids (GA3, GA4 and GA4+7). Pre-harvest application of gibberellic acids (GA 4+7) and benzyladenine (BA) reduces leaf yellowing in Easter lilies (Whitman et al., 2001; Ranwala et al., 2003). Han (Han, 1995) first reported that a combination of gibberellins (GAs) and benzyladenine (BA) reduced yellowing in excised Easter lily leaves and later reported that one application of Promalin, which contains both GA4+7 and BA, completely prevented postproduction leaf yellowing (Han, 1997).
Thiadiazuron (TDZ, N-phenyl-N’-1, 2, 3- thiadiazol-5-ylurea) registered as a plant growth regulator is commonly used in tissue culture for its cytokinin like activity. It is about 50-100 times more active than common cytokinins as observed in in-vitro experiments (Celikel et al., 2002). Thidiazuron treated lily cut stems has higher chlorophyll content as compared with control and benzyladenine treatments (Ferrante et al., 2011). TDZ has been successfully used for inhibiting leaf yellowing in a wide range of cut flowers such as Alstroemeria (Ferrante et al., 2002), tulips, chrysanthemum (Ferrante et al., 2003; Ferrante et al., 2005) and Iris (Macnish et al., 2010). To date, no systematic investigations has been conducted to study the use of TDZ, GA3 , BA and other GA rich compounds to prevent leaf yellowing in cut hybrid lilies. Thus, the present study was conceived with the objectives to investigate the optimum concentration and means of application of TDZ, GA3, BA and other chemicals such as SAMRAS multiplex (mixture of seventeen natural amino acids, rich in glutamic acid (3.50), leucine (2.00) and proline (2.00) on %age basis), Agri-Herbo-99 (herbal growth hormone rich in amino acids (aspartic acid + glutamic acid), indole acetic acid, gibberellin, humic acid and iron, zinc, manganese and copper) , Super Gibbre (water soluble gibberellin) alone or in combinations  for improving postharvest leaf and flower quality of cut lilium.
1 Results
1.1 Chemical effect on bud opening
The results showed that BA alone or in combination with GA3 treatments significantly delayed the flower bud opening. Cut stems sprayed with 50 ppm BA resulted in greater extension of 4.3 days to opening of flower buds followed by TDZ (2 ppm), BA (50 ppm) + GA3 (50 ppm) and GA3 (50 ppm) (Figure 1).



Figure 1 Effect of different chemicals on days to bed opening in Lilium cv. Eyeliner


1.2 Chemical effect on leaf yellowing

The striking effect of TDZ in preventing leaf yellowing was found most outstanding among all the other chemicals tested as spray treatments (Figure 2). Besides this, BA plus GA3 was the most effective, particularly at 50 ppm concentration; all the other chemicals had modest but non-significant preventative effects on leaf yellowing at all the concentrations tested. Spray  treatments with solutions containing GA3 (50 ppm) significantly delayed the initiation of leaf yellowing up to one day as compared to control. Leaf yellowing was substantially delayed if the cut flowers were sprayed with TDZ, and was delayed up to one week, when cut flowers were sprayed with a solution containing TDZ (2 ppm) (17.8 days) as compared to control (10.8 days) (Figure 2).



Figure 2  Effect of different chemicals on days to initiation of leaf yellowing and days to complete leaf yellowing in lilium cv. Eyeliner


1.3 Chemical effect on flower senescence
TDZ had no apparent effect on days to initiation of flower senescence. However, days to complete senescence of flowers were significantly improved with spray application of TDZ (2 ppml·L-1) and extended up to 7.1 days compared to non treated flowers (Figure 3). Spray treatments with BA (50 ppm) plus GA3 (50 ppm) delayed the days to initiation of flower senescence (2.0 days) during all the experimental period.



Figure 3 Effect of different chemicals on days to initiation of flower senescence and days to complete flower senescence in lilium cv. Eyeliner


1.4 Flower size
Spray applications significantly affected the flower diameter over control. BA(25 ppm), SAMRAS (4 ml·L-1) and BA (25 ppm) plus GA3(25 ppm) treatments had played an important role in increasing the flower diameter with a maximum 18.7 cm and 18.6 cm, respectively. However, statistically difference was non-significant (Figure 4).



Figure 4 Effect of different chemicals on flower Diameter (cm) in Lilium cv. Eyeliner


1.5 Changes in Chlorophyll content
Chlorophyll a (Chl a), b (Chl b) and total (Chl a+b) was progressively affected by the chemical spray treatments. At the beginning of the experiment there were 0.265, 0.765 and 1.054 mg g-1 f.w of Chl a, Chl b and Chl a+b, respectively (Figure 5, Figure 6, & Figure 7). The degradation rate of Chl b was faster compared to Chl a. After 9 days, the Chl a and b were reduced by 78 and 86%, respectively. The two chlorophyll pigments a and b had different behaviour in all the treated cut flowers as compared to control. The chlorophyll a (Figure 5) after 3 days slightly declined and increased again after 6 days and again declined after 9 days. Opposite trend was observed for Chl b (Figure 6) that sharply increases after 3 days and slightly declined after 6 and 9 days. The application of BA concentrations delayed the chlorophyll degradation after 9 days in comparison to other chemical treatments. The best treatments in this regards was BA @ 25 ppm for Chl a and BA @ 50 ppm for Chl b. Similarly, application of BA @ 50 ppm significantly delayed total chlorophyll (a+b) degradation after 6 and 9 days (Figure 7). After 3 days sharp increase in total chlorophyll content was observed when cut stems were sprayed with 1 ppm TDZ and slightly declined after 6 and 9 days (Figure 7).



Figure 5 Effect of chemicals on change in leaf chlorophyll 'a' content after 3,6 and 9 days in lilium cv. Eyeliner



Figure 6 Effect of different chemicals of Chlorophyll'b' content afer 3, 6 and 9 days in Lilium cv. Eyeliner



Figure 7 Effect of different chemicals on change in total chlorophy (a+b) content after 3, 6 and 9 days in Lilium cv. Eyeliner


2 Conclusion
Results of this study suggested that, TDZ @ 2 ppm not only delayed leaf yellowing but also reduced onset of petal senescence in lilium cut flowers. BA was able to delay the chlorophyll degradation. However, more studies are necessary to determine the effect of TDZ in combination with other materials.
3 Discussion
The global transport of cut flowers requires storage at cool temperatures in order to sustain the quality of buds and flowers, but this practice may lead to an increase in leaf yellowing in lilies. Spraying leaves either before or after cold storage with a solution containing TDZ 2 ppm and 50 ppm BA plus 50 ppm GA3, however, counteracted the detrimental effects of cold storage. The flexibility in the timing of application indicates that treatments can be applied either by growers prior to shipping or by the retail florists after cold storage. Leaf yellowing was prevented by growth regulators only on leaves that had been in direct contact with the hormones, indicating that the growth regulators were not mobilized in the plant. In potted lilies also reported that spraying with 100 mg·L–1 each of BA and GA4+7 before cold storage completely prevented leaf yellowing in ‘Stargazer’ (Ranwala and Miller, 1998). Application of GA3 or GA4+7 alone or in combination with benzyladenine were able to reduce leaf yellowing in lilies and Alstroemeria (Ferrante et al., 2002; Whitman et al., 2001). The physiological effect of TDZ is due to its cytokinin-like activity that is higher than benzyladenine. TDZ is not metabolized by the plants therefore its activity lasts longer than that of other cytokinins (Genkov and Ivanova, 1995). Moreover, it has been found that TDZ might promote the conversion of cytokinin ribonucleotides to more biologically active ribonucleosides (Capelle et al., 1983). It was demonstrated that spray application of TDZ and BAP increased the photosynthetic rate and activities of key photosynthetic enzymes in sugar beet, pea, meadow fescue and red fescue (Chernyad’ev, 1994). He also reported that TDZ also retards leaf yellowing in a range of other cut flowers (lilies, stock, tulip, and iris) and potted flowering plants (poinsettia and miniature roses).
Petal senescence is a key factor affecting vase life and quality of cut flowers. In flowers of many plants, petal senescence is often accompanied by decline in the endogenous cytokinin levels (Borochov and Woodson, 1989). In a variety of systems TDZ was much more effective than purine type cytokinins in influencing typical cytokinins responses, including induction of somatic embryogenesis and organogenesis and prevention of leaf yellowing and senescence (Murthy et al., 1998; Mok et al., 2000). TDZ prevented flower abscission and delayed leaf senescence in phlox and Lupinus densiflorus (Sankhla et al., 2003, 2005). In our study, spraying lilium cut flowers with TDZ considerably delayed flower senescence. Most significant results were obtained when cut stems were sprayed with TDZ (2 ppm·L-1).
Application of BA (50 ppm) delayed bud opening significantly compared to other growth regulators treatments and control. BA application has also shown to delay the bud opening in lilium cv. Stargazer (Han and Miller, 2003). The increase in flower diameter in presence of BA, GA3 and SAMRAS treated cut flowers may be due to acceleration of cell elongation and maintenance of structural integrity of chloroplast membrane system as well as stimulate photosynthesis (Salisbury and Ross, 1985).
In general, BA treated flower had the highest chlorophyll content during whole the experimental period except TDZ in the 3rd day. Surprisingly BA treatment did not prevent leaf yellowing and flower senescence in this lily cultivar, but even hastened. Cytokinins, in particular BA, usually inhibit leaf yellowing in many sensitive cut flower species (Ferrante et al., 2011). The TDZ is a substituted phenylurea that induces cytokinin-like responses. The efficacy of TDZ in cut flower species depends on the cultivar as observed in alstroemeria (Ferrante et al., 2002). TDZ in dark condition is less effective in inhibiting chlorophyll degradation. The reduced effect of TDZ might be due to its inactivation or by the lack of light in the chlorophyll turnover. In fact, in the biosynthesis of chlorophyll, the last step is mediated by NADH protochlorophyllide reductase, which converts the protochlorophyllide to chlorophyll and this process requires light (Kraepiel and Miginiac, 1997). Some previous studies have suggested that light regulates chlorophyll biosynthesis at transcriptional level while the cytokinins act at the post-transcriptional level (Genkov and Ivanova, 1995; Celikel et al., 2002; Flores and Tobin, 1986).
4 Materials and Methods
4.1 Experimental site
The present investigations were carried out at Experimental Research Farm and laboratory of ICAR-Indian Agricultural Research Institute, Regional Station, Katrain, Kullu-Valley (Himachal Pradesh), India, during 2014-15.
4.2 Plant material
Cut Lily flowers of cv. Eyeliner were harvested from the research farm, when first flower bud of the inflorescence showed full colour and were taken to the laboratory within an hour. Stems were placed in clean tap water immediately upon arrival at the laboratory. Stems were selected for uniformity; leaves on the lower third portion of the stems were stripped.
4.3 Standard treatments
Unless otherwise stated, leaves were sprayed with water (control) and with a growth regulator solutions containing TDZ (1&2ppm), BA (25 ppm), GA3 (25 ppm), BA (50 ppm) , GA3  (50 ppm), BA +GA3 (25 ppm +25 ppm), BA+GA3 (50 ppm +50 ppm), SAMRAS (2&4 ml·L-1), Agri-Herbo-99 (1&2 ml·L-1) and Super Gibbre (1&2 ml·L-1). After the treatments, each stem was then maintained for postharvest evaluation in an individual 500 ml conical flask with 300 ml of normal tap water. Within each treatment, the numbers of buds per stem were kept constant as possible for ease of data recording.
4.4 Quality assessment
Data on days to floret opening, days to initiation of leaf yellowing, days to complete leaf yellowing, days to initiation of floret senescence, days to complete floret senescence and flower diameter were observed daily. Leaves were considered senescent when >50% of the leaf area had become chlorotic or necrotic.
4.5 Chlorophyll estimation
Chlorophyll a, b and total chlorophyll (a +b) in the sample were extracted according to the standard protocol (Ranganna, 2008) and quantified using spectrophotometer. 2 gm of sample was crushed using 80% Acetone in pestle and mortar until the sample became colourless and then filtered through Whatmann filter paper no. 1. Volume was made to 100 ml and then it was read on a UV-Vis spectrophotometer at 645 and 663 nm wavelength using 80% acetone as blank. Chlorophyll a, b and total chlorophyll were calculated on fresh weight basis using the following formulas:




4.6 Statistical analysis
This experiment was conducted in Completely Randomized Design (CRD) with 3 replications. Three stems were used for each replication in different treatment combinations. Results were analysed by using COSTAT software. Mean comparison to identify significant difference between treatments were performed using least significant difference (LSD) (Gomez and Gomez, 1984).
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