MATERIALS AND METHODS

Study area: Completely Randomized Design (CRD) was applied in the methodology. The experiment was carried out in a garden in the Department of Biological Science, University of Malaya located in Kuala Lumpur, Malaysia (April-September, 2015) and laboratory analysis was done in the Department of Biological Science, University of Hail, KSA (October-January, 2016).

Plant materials: Hibiscus rosa-sinensis plant was used in this experiment.

Treatment setting: The treatments were control, thinning, phloemic stress and hormone gibberellic acid (GA₃). Thinning was done by removing excess leaves on the branches. A partial ring was made by removing a 3 cm long strip of bark from around the circumference of the trunk with a knife (thin razor blade type) leaving a connecting band of bark of 4 mm wide. Hormone gibberellic acid (GA₃ of 20 mg L^–1 concentration) was applied twice a week by dipping. The experiment was conducted in a completely randomized design. There were three replications and 4 treatments (including control) used in the experiment. A total of 12 branches were used in the experiment from 4 trees.

Data collection: Number of buds, leaves, flower size, petal diameter and flower weight were counted at 7 days interval for 4 weeks. Flower buds and leaves were measured per 20 cm of shoot.

Longevity: Stage of flowering was observed everyday to determine its longevity. There were four stages of flowering; (i) Bud stage, (ii) Partially bloom stage, (iii) Full bloom stage and (iv) Senescence stage. Vase life of the flower begins with the bud stage until it was dropped from the plant. The start of senescence was indicated by initial browning and discoloration of the petals.

In vivo measurement of leaf chlorophyll: The chlorophyll meter SPAD-502 (Minolta Co. Japan) was used for determination of chlorophyll in leaves. The SPAD-502 determines the relative amount of chlorophyll present by measuring the absorbance of the leaf in two wavelength regions.

Determination of chlorophyll a, b and total carotenoid content: Total carotenoids and chlorophylls were determined according to the methods¹². The method consisted of repeated acetone extraction, until obtained colorless residue with a pestle and mortar and filtered over filter paper (Whatman No. 1). The extracts were made up to 50 mL with acetone. The concentration of carotenoids was measured at 470 nm, chlorophyll a at 666 mm and chlorophyll b at 653 nm in a Shimadzu UV 160A spectrophotometer. The amount of chlorophyll a, b and total carotenoid content were calculated¹².

Statistical analysis: Least Significant Different Test (LSDT) was used in this study.

RESULTS

All treatments showed an increase in the number of buds (Fig. 1), but subsequently remained unchanged at 3rd and 4th week. In fact, both control and hormone application treatments remained unchanged since, the 2nd week of treatment. Branches treated with GA₃ induced the highest number of buds (6.00±2.65) followed by thinning (5.00±2.00) and phloemic stress (4.00±0.58). Control exhibited the least number of buds among the four treatments. Figure 2 showed that initially, the number of leaves for all treatments were lower than that of control, however, they increased after 2nd week of treatments.

Fig. 1:	Number of buds for Hibiscus rosa-sinensis at 1-4 week after treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Fig. 2:	Number of leaves for Hibiscus rosa-sinensis at 1-4 week after treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Fig. 3:	Leaf size of Hibiscus rosa-sinensis at 1-4 week after treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Branches treated with GA₃ application showed an obvious increase in the number of leaves. This means that GA₃ was capable of inducing leaves formation. However, not much difference could be seen in the number of leaves between the treatment of thinning and phloemic stress. As shown in Fig. 3, leaf size with the treatment of hormone was much higher compared to other treatments.

Fig. 4:	Flower size (cm2) of Hibiscus rosa-sinensis after treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Fig. 5:	Petal diameter (cm) of Hibiscus rosa-sinensis flower for treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Fig. 6:	Flower weight (g) of Hibiscus rosa-sinensis after treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

However, this was due to its initial leaf size which is the biggest of all. Its increment in leaf size was not so obvious in the first two weeks, but subsequently increased afterward, thus, resulting in the highest leaf size among all treatments (38.00±19.15).

Fig. 7:	Longevity (days) of Hibiscus rosa-sinensis after treatments
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Leaves with no treatment (control) showed a drop in leaf size at week 3 (27.45±8.63), but slightly increased at week 4 (28.20±13.88).

Based on Fig. 4, the flower size of Hibiscus rosa-sinensis was more or less the same for all treatments. However, the biggest size of flower can be seen in the flower sprayed by hormone GA₃ (176±2.46), whilst the smallest was shown in control. This is meant that GA₃ application was effective in flower growth and development. Petal diameter was the highest in the flower treated with hormone GA₃ (7.13±0.31) and the least in the flower with no treatment (control) (Fig. 5). However, petal diameter for both phloemic stress and thinning treatment were only slightly lower than that of hormone application. This showed that all three treatments managed to induce flowers with high petal diameter.

As shown in Fig. 6, all treated flowers exhibited higher flower weight compared to control. The highest flower weight could be seen in the flowers applied with hormone GA₃, phloemic stress and thinning. This result showed that flowers treated with GA₃ contained highest water content and thus, it is believed that it is to be able to delay the onset of senescence. The longevity of Hibiscus rosa-sinensis flowers were determined by petal discoloration and senescence. As shown in Fig. 7, hormone GA₃ was most effective in prolonging the longevity of the flowers from 14-15 days. The average longevity of phloemic stress was slightly higher than thinning.

From the results shown in Fig. 8, it could be seen that there were no significant differences between control and thinning and also between phloemic stress and hormone application. Chlorophyll content value for Hibiscus rosa-sinensis leaves were the highest in leaves treated with hormone GA₃.

Fig. 8:	Chlorophyll content of Hibiscus rosa-sinensis (SPAD value) for control, phloemic stress, thinning and hormone application
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

Fig. 9:	Chlorophyll a, b and content of Hibiscus rosa-sinensis (SPAD value) for control, phloemic stress, thinning and hormone application
	Means followed by the common letters are not significantly different at 5% level by Least Significant Different Test (LSDT)

The absorbance properties of photosynthetic pigments allowed the amounts of chlorophyll a, b and carotenoid to be determined spectrometrically. As shown in Fig. 9, the amount of chlorophyll b was higher than chlorophyll a in Hibiscus rosa-sinensis flowers. This was followed by carotenoid contents, which showed the lowest content. From the result, the control treatment showed the highest content of chlorophyll a, while, GA₃ treated flowers contained highest amount of chlorophyll b and carotenoid.

DISCUSSION

Finding of this study showed that GA₃ application induced the highest number of buds and leaves in Hibiscus rosa-sinensis plant. These results showed that GA₃ was capable of breaking the dormancy in plants. It was reported by Szalai et al.¹³ that the exogenous application of Gibberellic Acid (GA) enhanced dormancy breakage, despite the fact that the levels of endogenous gibberellins did not increase prior to visible spouting of the tubers in tuber plants. This was further supported by Hossain et al.¹⁴, including the correlation between gibberellins and germination. Gibberellins encouraged the germination by breaking the dormancy. During flower bud opening, various events takes place in a well defined sequences representing all aspects of plant development, such as; cell division, cellular differentiation, cell elongation or expansion and a wide spectrum of gene expression¹⁵. It is reported that GA₃ involved in the stimulation of cell division at the shoot apex by shortening the cell cycle¹⁶. Gibberellins stimulate cell division and extension in buds and the synthesis of α-amylase in the aleurone layer of wheat¹⁷.

Thinning induced the second highest number of buds in this study. However, phloem cut stress produced higher number of leaves compared to thinning. Carbohydrates are necessary for the growth of any plant art as carbohydrates provide energy and the building blocks for growth processes. Flowers usually do not have chlorophyll and therefore, cannot carry out photosynthesis to produce carbohydrates for their needs. In addition, flowers have very rapid growth rates that require large amounts of carbohydrates. For these reasons, flowers are dependent upon other parts, especially leaves, for their carbohydrate supply¹⁸.

Gibberellins have been shown to stimulate cell division by shortening the cell cycle. As for stimulation of cell growth, it is done by increasing the hydrolysis of starch and other food materials. Exogenous application of GA induced an increase in the concentration of reducing sugars¹⁹, while it was observed¹⁶ an increase in glucose concentration in the tissues near the buds of GA-treated tubers prior to visible sprouting. The increase in starch hydrolysis and the concentration of soluble carbohydrates can also be seen. In this study, phloem cut stress was done via partial ringing in which part of the bark was removed from around its periphery, thereby blocking the downward translocation of photosynthates and metabolites through the phloem^17,20. It was reported by Janssen²¹ that when bark ringing was done, the flow of manufactured food in plants was stopped. The roots starved for lack of food and the plant eventually died.

Thinning (pruning) is the judicious removal of leaves, flowers, branches, twigs and shoots of roots to increase the usefulness of plants¹⁰. The principal mode of action for thinning is to reduce the competition between plants. It was reported by Janssen²¹ and Jones and Woodson²² that the ones that remain have room to grow and access to the sunlight, water and nutrients they need to grow and produce to their full potential. As was shown in the results, GA₃ treated flowers were the heaviest among all treatments and also was effective in prolonging the longevity of Hibiscus rosa-sinensis up to 14-15 days compared to 9 days in control.

Petal wilting is due to loss of turgor which is mainly caused by the degradation of carbohydrates, proteins, lipids and nucleic acids in petal tissues. On the other hand, withering is a color change resulting from degradation of anthocyanin pigments and lowered water uptake²³. It was reported by Gomes et al.⁹ that water deficit also led to accumulation of abscisic acid (ABA), another of type growth inhibitor hormone. The ABA accumulation showed the same trend as ethylene biosynthesis and endogenous concentration was highest in short-lived hibiscus flowers. This may act by increasing ethylene sensitivity as observed by exogenous applications of ABA in hibiscus flowers²⁴. Physiological studies during hibiscus flower senescence showed that ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) content increased in petals during flower development and senescence. Exposure to ethylene accelerated the onset of senescence as indicated by petal in-rolling and stimulated ethylene production. Senescence was also hastened by basal application of ACC²⁵.

Light and phytohormones both influence leaf senescence. The effect of light on leaf senescence has been proposed to act on photosynthesis²⁵ and a photomorphogenic effect involving phytohormones²⁶. Phytohormones like cytokinins and in some system gibberellins delay the loss of chlorophyll whereas ethylene and ABA enhance the rate of chlorophyll loss²⁷. In this study, leaves treated with GA₃ contained the highest content of chlorophyll. This result is also similar to those reported by Dai and Paull¹¹ who found that loss of chlorophyll in leaves of alstroemeria cut flowers is strongly delayed by GA₃.

Chlorophyll pigments are accompanied by carotenoid ones, usually, from yellow to orange and is frequently masked by green chlorophylls²⁸. Carotenoids are important nutritive and biological constituents common in the plant world. In this study, chlorophyll a, b and total carotenoid contents in Hibiscus rosa-sinensis flowers were determined spectrophotometrically. Generally, pigments contained in flowers are much lower compared to that in the leaves.

CONCLUSION

From this study, it can be concluded that the application of GA₃ to H. rosa-sinensis was the best treatment in delaying the flowers senescence compared to other different treatments as it exhibited the least petal discoloration and wilting. Furthermore, it induced the highest number of buds and leaves and also biggest size of flowers and leaves. In addition, GA₃ treated flowers had higher full bloom duration which is 2-3 days compared to only one day for control. Thus, GA₃ application was the most effective method in prolonging the longevity of Hibiscus rosa-sinensis flowers.

SIGNIFICANCE STATEMENT

From this study, it can be recommended that the administration of GA₃ to H. rosa-sinensis was the best treatment (dipping method) in delaying the flower senescence compared to other different method of treatments like spray and sprinkle etc. Thus, GA₃ application was the most effective scientific method to prolong the longevity of Hibiscus rosa-sinensis flowers. This study will help researchers to uncover the potential of GA₃ and possible use in longevity of H. rosa-sinensis.

ACKNOWLEDGMENTS

Authors are thankful to the Ph.D., MS and UG students for assistance in this project and also thankful to the Department of Biology, Faculty of Science, University of Malaya for financial support and also thankful to the Undergraduate and MS students, Department of Biology, Faculty of Science, University of Hail, KSA.