Abstract: Background and Objective: Natural Plant Growth Regulator (PGR) not only affects the growth and yield of binahong, but also affects the physiological characteristics of binahong. The objective of the research was to determine the impact of the foliar application of natural PGR (shallot extract, banana hump extract and bamboo shoot extract) on physiological characteristics of binahong. Materials and Methods: The research use a non-factorial randomized block design with 7 treatment and 4 replication. The natural PGR treatment in the research was without natural PGR ; shallot extract 40 mL nL–1; bamboo shoots extract 40 mL L–1; banana hump extract 40 mL L–1; shallot extract (20 mL L–1)+bamboo shoot extract (20 mL L–1); shallot extract (20 mL L–1)+banana hump extract (20 mL L–1); bamboo shoots extract (20 mL L–1)+banana hump extract (20 mL L–1). The variables observed were chlorophyll a, chlorophyll b, total chlorophyll, density of stomata and cuticle thickness. Data were analyzed by using analysis of variance at p = 0.05. Results: The result showed that the application of natural PGR decreased stomatal density compared to control (without natural PGR application), but increased chlorophyll a, chlorophyll b and total chlorophyll. Natural PGR of shallot extract 40 mL L–1 application increased total chlorophyll. Banana hump extract 40 mL L–1 application increased the cuticle thickness compared to other natural PGR. Conclusion: It was concluded that application of natural PGR decreased the stomatal density, but increased the total chlorophyll. Banana hump extract 40 mL L–1 application increased the cuticle thickness compared to other natural PGR.
INTRODUCTION
Binahong is one of the species from the family Basellaceae which has health benefits. The Basellaceae family has several species such as Anredera baselloides (Kunth) Baill, Anredera cordifolia (Ten.) Steenis and Anredera diffusa, Anredera leptostachys (Moq.) Steenis, Anredera vesicaria, Anredera spicata and others1. The species that is widely known in Indonesia is Anredera cordifolia with the local name "binahong". This plant was known to have extraordinary benefits and has been consumed more than thousands of years ago in China, Korea and Taiwan. Almost all parts of binahong plants such as; tubers, stems and leaves can be used as herbal therapy2,3.
Binahong is one of the medicinal plant used to be a raw material for phytopharmaca. Previous studies reported that binahong contains several flavonoids, alkaloids, terpenoids, saponins, anthocyanins, glucans, carotenes, organic acids, mucopolysaccharides such as; L-arabinose, D-galactose, L-rhamnosa, acid aldonat, also contains saponins, vitamins4-6 A, B and C. It was reported that binahong leaf paste can be used to heal wounds7. Binahong leaf extract has analgesic activity8. Binahong leaves are also proven to be able to improve kidney function that has been damaged9. In addition, binahong is effective for accelerating wound healing, burn wound healing, smoothing and normalizing circulation and blood, healing internal and external wounds after surgery, swelling and blood clots, restoring weak conditions after illness and also preventing strokes10,11.
Nowadays, binahong has been used as a raw material for the phytopharmaca industry. According to Balai Penelitian Tanaman Rempah dan Obat (Research Institution of Medicinal Plant and Spice)10, only about 20% of the raw material of binahong for industry is obtained from cultivation, while the rest is obtained from the forest. Therefore, in order to fulfil the needs of binahong as a medicinal ingredient, it is necessary to conduct cultivation in a directed manner, so that a quality of binahong can be obtained.
In fulfilling the needs of large-scale binahong plant material, it is necessary to apply the treatment to improve the physiological characteristics of binahong. The application of natural PGR is one treatment to improve the physiological characteristics of binahong such as; chlorophyll content, density of stomata and cuticle thickness. Natural PGR is the right choice because it is environmentally friendly, often the natural PGR that is naturally present in plants is below optimal, so external sources are needed to produce a maximum response12. Chlorophyll is the most important pigment for green plants because it can generate solar radiation and energy into chemical energy. Chlorophyll a plays an important role in the process of photosynthesis. The status of chlorophyll pigments in leaf tissue is very influential in the overall photosynthesis that occurs directly on plant growth, development and yield13-20.
Until now there is still very little information about the role of natural PGR on the physiological characteristics of binahong (chlorophyll content, density of stomata and cuticle thickness). Based on the background, the study was aimed to identify the physiological response of binahong as a potential medicinal plant through the application of natural PGR.
MATERIALS AND METHODS
Research area and materials: A pot experiment was conducted at the Experimental Field, Faculty of Agriculture Universitas Sumatera Utara, Medan on April-July, 2018. The materials used in this research were seedlings of binahong, shallot, bamboo shoots, banana hump, 2 kg polybag size, top soil, chicken manure, compost, acetone, kutex. The equipment used namely scale, sieve, bucket, hand sprayer, broach, microscope, spectrophotometer.
Procedures: This research was a pot experiment using a non- factorial randomized design with 7 types of treatment and 4 replications. There were 28 experimental units. The natural PGR treatments in the research was:
| P0 | : | Without natural PGR |
| P1 | : | Shallot extract 40 mL L–1 |
| P2 | : | Bamboo shoots extract 40 mL L–1 |
| P3 | : | Banana hump extract 40 mL L–1 |
| P4 | : | Shallot extract (20 mL L–1)+bamboo shoot extract (20 mL L–1) |
| P5 | : | Shallot extract (20 mL L–1)+banana hump extract (20 mL L–1) |
| P6 | : | Bamboo shoots extract (20 mL L–1)+banana hump extract (20 mL L–1) |
The variables observed were chlorophyll a, chlorophyll b, total chlorophyll, density of stomata and cuticle thickness. Determination of chlorophyll content is carried out by taking sample of 0.1 g of fully expanded leaves for each treatment and then macerated in mortar with 10 mL of aqueous acetone (v/v). Determination of chlorophyll a, chlorophyll b and total chlorophyll was done using the formula to the specific absorption coefficient as reported by Henry and Grime21. The content of chlorophyll were expressed as mg g–1 of fresh leaf:
Chlorophyll a = {(12.7×A663}-(2.69×A645)}/10
Chlorophyll b = {(22.9×A645}-(4.68×A663)}/10
Total of chlorophyll = {(8.02×A663)+(20.2×A645)}/10
Determination of stomatal density used leaf impression method. This method used clear nail polish to make an impression or cast of the leaf surface. The cast is removed with sticky tape and placed on a microscope slide. The stomatal density expressed as Unit mm–2.
Statistical analysis: Data were analyzed by two way analysis of variance (ANOVA) procedures the SAS version 12 computer program, if there were significant differences then proceed with Duncan's Multiple Range test at the level of α = 5%.
RESULTS AND DISCUSSION
Analysis of natural PGR: Based on Table 1, it can be seen that bamboo shoot contained the highest indole acetic acid (0.0084%), GA3 (0.0058%) and cytokinins (0.0045%) than shallot and banana hump. While, banana humps contained the lowest indole acetic acid (0.0022%), GA3 (0.0016%) and cytokinins (0.0021%).
Stomatal density: Based on Table 2, it can be seen that without natural PGR (P0) treatment produced the highest stomata content of 112 Unit mm–2. Natural PGR treatment decreased stomata density, where the lowest stomata density was found in treatment P6 (bamboo shoots extract (20 mL L–1)+banana hump extract (20 mL L–1). This showed that bamboo shoot extract and extract banana humps containing GA3 and cytokinin suppress stomata formation. This is in line with previous study that the application of GA3 resulted in a decrease in stomata in the epidermis12.
It was contrary to the previous reports that the stomata density was stimulated by cytokinin, the effect of specifics is correlated with the concentration of cytokinin and the method of application22,23.
Chlorophyll a, chlorophyll b and total chlorophyll: Based on Table 3, it can be seen that each natural PGR treatment has a different influence on the chlorophyll a, chlorophyll b and total chlorophyll. The highest of total chlorophyll content is found in the treatment P1 (shallot extract 40 mL L–1 water). This showed that GA3, cytokinin and IAA contained in shallot play a major role in the formation of chlorophyll. Natural PGR increased the changes in physiology and biochemistry and improved the plant productivity. Previous research also found the similar result that GA3 increases chlorophyll content in apple plants and Mentha piperita24,25. It was also reported that application of mepiquat chloride and GA3 combined or alone, improved the chlorophyll content and leaf area in grapes26.
| Table 1: | Content of auxin, gibberellin and cytokinin in bamboo shoots, shallots and banana hump |
Source: Research Institution of Medicinal Plants and Spice (Bogor) | |
| Table 2: | Stomatal density of binahong with application of natural PGR |
Means followed by the same letter indicated not significantly different based on Duncan’s Multiple Range test (p = 0.05) | |
| Table 3: | Chlorophyll a, Chlorophyll b and total chlorophyll content of binahong with application of natural PGR |
| Fig. 1: | Cuticle thickness of binahong on application of natural PGR |
Previous research also stated that the application of foliar GA3 increased chlorophyll levels in corn plants under saline condition27. Another researcher reported that application of foliar application of PGR can increase growth, photosynthesis and flowering in cashew28.
Cuticle thickness: Based on Fig. 1, it can be seen that the treatment of P3 (banana hump extract 40 mL L–1 water) produced the highest cuticle thickness, while treatment P2 (bamboo shoots extract 40 mL L–1) and P6 bamboo shoots extract (20 mL L–1)+banana hump extract (20 mL L–1) produced the lowest cuticle thickness.
Bamboo shoots contained GA3, Indole Acetic Acid and cytokinin were the highest among other natural PGR (Table 1), resulted in lower cuticle thickness. This indicated the influence of higher levels of natural PGR on the decrease in cuticle thickness. The banana hump contained the lowest IAA, GA3 and cytokinin produced the highest cuticle thickness. The results of this research were in line with previous study stated that an increase in GA3 application on radish seedlings increased the thickness of the cuticle29. Although, the results was not appropriate with previous literature that stated the foliar application of GA3 enhanced thickness of the cuticle as compared to the control and 2,4-D30. The thickness of the cuticle can affect the transpiration process in plants. Modification of cuticle thickness is a response to reduce the transpiration and reaction of plants to the entry of pollutants. It was studied that the cuticle is located in the outer layer of the epidermis consisting of two layers, the outermost layer which only consists of the layer of cutin (true cuticle) and the inner layer (the cuticular layer) which contains the cutin and other cell wall material. The outermost layer of leaves is used to maintain leaf moisture, because the cuticle layer can control transpiration or evaporation, so as to minimize water loss31. In addition to maintaining moisture, the cuticle also served as an initial defense against the entry of foreign objects including pollutants from the air into the leaves.
CONCLUSION
The use of natural PGR decreased stomatal density compared to control (without natural PGR application), but increased the chlorophyll a, chlorophyll b and total chlorophyll contents. Natural PGR shallot extract 40 mL L–1 application increased the total chlorophyll. Banana hump extract 40 mL L–1 application increases the cuticle thickness compared to other natural PGR.
SIGNIFICANCE STATEMENT
This research has discovered findings that there are differences in the response of stomatal density, chlorophyll content and cuticle thickness of binahong on application of natural plant growth regulator. Based on this research a new theory has been obtained that, the use of natural PGR decreases stomatal density compared to control (without natural PGR application), but increases chlorophyll a, chlorophyll b and total chlorophyll. Natural PGR shallot extract 40 mL L–1 application increases total chlorophyll. Banana hump extract 40 mL L–1 application increases the cuticle thickness compared to other natural PGR. It is concluded that application natural PGR decreased the stomatal density, but increase the total chlorophyll. Banana hump extract 40 mL L–1 application increased the cuticle thickness compared to other natural PGR. This research will help the researchers to cope the best natural PGR to increase the chlorophyll content that play an important role for photosynthetic process.
ACKNOWLEDGMENT
The author would like to thank the Research Institution, Universitas Sumatera Utara for funding this research through a Research Contract TALENTA Universitas Sumatera Utara, Fiscal Year 2018 Number: 2590/UN5.I.R/PPM/2018 dated March 16, 2018. The author also thank Panca Ramadhan for his assistance in this research.