The Effect of Jasmonic Acid on The Growth of Dendrobium Stratiotes in Vitro

. D. stratiotes is one of the endemic dendrobiums from Indonesia that grows in lowland rainforests. D. stratiotes is listed as a threatened orchid species due to the lack of available data, which results in a high uncertainty and insuﬃcient information. This study aims to provide preliminary information of jasmonic acid eﬀect on D. stratiotes growth in vitro in order to improve propagation techniques in agricultural practices. The exogenous application of jasmonic acid exerts a regulatory role on the plant's growth and development. This study was designed as a completed randomized design (CRD). VW medium was supplemented with jasmonic acid (0, 0.2, 0.8, 1, 1.5, 2 mg.L -1 ). There were 5 replications. The observed variable in this study includes leaves number, plant height, plant weight, roots number, root length, leaf anatomy, and root anatomy. Statistically, 1 mg.L -1 of jasmonic acid signiﬁcantly inﬂuenced the plant height, leaves number, plant weight, and roots number which resulted in 10.16 cm, 6.53, 1.18 g, and 9.53, respectively. .


Introduction
Dendrobium sp. is one of the largest and most popular groups of orchids, with more than 1500 species.Dendrobiums are extensively distributed worldwide, from Australia to tropical and subtropical Asia and several Pacific islands [1].Dendrobium stratiotes is an endemic species from Indonesia that grows in lowland rainforests.The World Checklist of Vascular Plants (WCVP) details the D. stratiotes distribution as Sulawesi, Maluku (Halmahera and Morotai) and the Lesser Sunda Islands [2].D. stratiotes is an orchid from the spatula section, commonly known as ceratobium, with distinctive morphological characteristics of spiral flower petals and labellum.D. stratiotes have white flowers with purple-striped labellum, green twisting petals, suitable fragrance, and a shelf life of up to 1-2 months, depending on the environment.D. stratiotes is unique and has high floricultural value.The natural habitat of D. stratiotes in the wild is raising the potential to be threatened by exploitation and over-collection for commercial purposes.Moreover, the high rate of deforestation also contributed to the degradation and loss of the orchid's natural habitat, which eventually leads to their extinction [3].In 2019, the International Union for the Conservation of Nature (IUCN) included D. stratiotes in the Red List of Threatened Species due to a lack of available data, resulting in a high level of uncertainty and insufficient information [4].Mass production of orchids, especially D. stratiotes is necessary to meet the market demand, innovation for the floriculture industry, and biodiversity.
Orchids have small seeds that do not have endosperm but have a very tough and thick protective shell.which makes the generative reproduction very difficult and lengthy.Tissue culture has been developed to facilitate the propagation of plants, particularly plants have generative issues.Plant tissue culture techniques can be used for rapid commercial-scale plant production.The growth of orchids in the culture glass jar, from the seed sowing to the acclimatization takes about one year and requires many sub-cultures.The application of many growth regulators in growing media with different concentrations and combinations is continuously explored and improved in order to produce high-quality plants in a short time.Various combinations and concentrations of plant growth regulators were added to growth media, resulting in the successful micro-propagation of numerous orchid species [5].
Research and development of agricultural technique and technologies is essential for modern agriculture.The study of jasmonic acid has not been as widely reported compared with other growth regulators such as auxins, cytokinin, ethylene and gliberiline.Jasmonates, such as jasmonic acid and its derivatives, belong to the group of plant hormones that are involved in the defense of plants from herbivory and pathogens, induce abiotic stress tolerance such as ozone, UV radiation, temperature and drought, and regulate plant development such as root growth, stamen formation, flowering, and leaf senescence [6].Some researchers have found that jasmonic acid regulates several physiologic induction mechanisms, such as root growth, reproductive system development, and plant senescence [7,8] Numerous factors affect the development of the orchids in vitro, including the source of explants, sterilization, type of medium, and plant growth regulator [9].The main role of exogenous phytohormones and plant growth regulator is to stimulate and increase the levels of hormones present in plants in order to promote the plant growth [10].This study aims to provide preliminary information on jasmonic acid effect on D. stratiotes growth in vitro in order to improve propagation techniques in agricultural practices for sustainable development.

Plant material
The second subculture of D. stratiotes (60 days after planting) was used as plant material in this study obtained from the Plant Physiology and Biotechnology Laboratory, Faculty of Agriculture, Sebelas Maret University, plantlets grown from primary seed and maintained on Vacin and Went (VW) media.

Experimental design, variables, data collection, and analysis
The treatments were organized in a completely randomized design (CRD).There were 5 replications and 30 experimental units in total.Variables observed included, roots number, leaves number, plantlet height, root length, plantlet weight, leaf anatomy and root anatomy.Data were analyzed by ANOVA (analysis of variance) and for significant differences between treatments, means were compared using DMRT (Duncan Multiple Range Test) at α=5%.Furthermore, a three-degree (cubic) regression analysis was carried out on the variables that showed a significant influence.The growth of D. stratiotes was observed four months (16 weeks) after the third subculture.The anatomical preparations used in this study were freshly prepared inspected under a microscope at 4x magnification for leaves and 10x magnification for roots and photographed using Optilab.

Results and discussion
Growth is the enlargement of plant by the increase in the number of plant cells.Many factors such as leaves, roots, plant height and weight are often observed to define the growth of a plant.The concentrations of jasmonic acid effect on D. stratiotes growth in vitro as measured by leaves number, roots number, plant height, root length and weight, is summarized in Table 1 and the anatomical structure in Figures 2 and 3.The application of jasmonic acid showed the presence of significant differences between the treatments in the leaves number, roots number, plant height, and plant weight (Table 1). 1 mg.L -1 of jasmonic acid was significantly influenced the leaves number and plant height compared to control.The highest roots number and plant height were 9.53 and 1.18, respectively, obtained by 1 mg.L -1 of jasmonic acid, but there are no significantly difference compared to the control, which produced 8,47 roots and 1.05 grams of weight.However, the applications of jasmonic acid in various concentrations do not significantly affect the root length.Other studies have shown that jasmonic acid is able to stimulate the growth of plants under normal or stress conditions.Moreover, jasmonic acid is linked to signaling during stressful conditions to maintain plant survival [11,12].Comparable findings were found in other orchids' in vitro propagation.The growth of Castetum fimbritum was significantly affected by the application of jasmonic acid in different concentrations, which resulted in a significant growth in the root number, leaf number, longest root length, largest leaf size, total seedling length and fresh and dry mass [13].The results obtained indicate that jasmonic acid acts as a plant growth regulator, exerting a favorable influence on plant growth.
The role of jasmonic acid depends on plant species and environmental circumstances [14].Jasmonic acid concentrations were associated with height growth in calendula plants [15].Jasmonic acid shortened the time of shoot initiation, increased shoot and root development and node number in Solanum tuberosum grown on MS media [16].However, the research on in vitro reproduction of Lavandula angustifolia Mill, found the opposite, jasmonic acid at low concentrations has no effect on plant height or mass as compared to the control, and jasmonic acid at higher concentrations significantly reduced shoot height compared to the control [17].The inhibition of primary root growth induced by jasmonic acid is a complex process involving a different set of molecular processes in different root tissues.Under some conditions, jasmonic acid can inhibit the elongation of roots through a reduction in cell number and cell volume [18,19].According to the regression analysis (Figure 2), the increment of the jasmonic acid concentrations affected both positive and negative to the growth variable observed.Leaves number, roots number and plant weight were decreased as the concentration of jasmonic acid increased.The level of endogenous hormones in each orchid plant is different, so the addition of exogenous growth regulators will induce different physiological responses.Phytohormones are organic compounds produced naturally by plants in small amounts.The chemical compounds, which have unique characteristics and compositions stimulate plant growth and development in many ways through various biochemical mechanisms [20].Jasmonic acid can promote or inhibit plant development depending on the concentration in the media [21].The balance between endogenous and exogenous hormones must match plant needs.The interaction between the different hormones is very important for the development of the root.Ethylene and jasmonic acid signalling are integrated with auxin in root development, mostly via transcription factors that act as important communication points [22].
Leaf anatomy serves a critical function in plants.It accommodates the biochemical and biophysical processes involved in photosynthesis.Orchid leaves consist of several anatomical features, including the epidermis, mesophyll, stomata, and vascular bundles (xylem and phloem).The leaf components facilitate the production and distribution of carbohydrates to the whole of the plant's body.The epidermis is a protective tissue consisting of upper and lower epidermis.Stomatal control of transpiration regulates leaf temperature and prevents overheating in the presence of intense radiation.As a result, it protects the internal mesophyll tissues and creates a stable environment for chemical reactions such as photosynthesis to take place effectively.The mesophyll cells contain small photosynthetically active organelles known as chloroplasts, which play a vital role in photosynthetic process [23].The plant vascular system consists of two conducting tissues, the xylem and the phloem.Like two vascular partners in a co-existing relationship, both rely on each other to accomplish their functions effectively.The phloem transports carbohydrates produced by the leaves throughout the plant, while xylem is responsible for transporting both water and nutrients to the leaves [24].Figure 3., shows that the application of jasmonic acid affected the mesophyll area of the D. stratiotes.The results show that the application of 0.4 and 1.5 mg.L -1 of jasmonic acid leads to the largest mesophyll area.The mesophyll's thickness is closely related to chlorophyll's efficiency in photosynthesis since chlorophyll exists within the mesophyll [25].A thicker mesophyll layer supports leaf freshness and has a greater ability to hold water, which is necessary for survival in high-radiation habitats.larger epidermal cells also can support species that are more adaptable to warmer environments [26].A study of several B. oleracea showed an increase in total chlorophyll content in B. oleracea L. italica following the use of jasmonic acid.The increase in chlorophyll pigment content may be an effect of the increased activity of several enzymes related to chlorophyll biosynthesis [27].
The root structure of D. stratiotes (Figure 4), comprises several layers arranged in a sequence from outermost to innermost.These layers include the epidermis, velamen, exodermis, cortex, endodermis, xylem, phloem, and pith.The epidermis mostly acts as protective tissue.The velamen refers to a root epidermis typically characterized by its spongy, multi-layered structure and consists of cells that have reached maturity and are no longer biologically active [28].The cortical tissues possess a wide range of functions.The endodermis and exodermis regulate the uptake and transport of water and nutrients within the root more specifically, act as barriers to the apoplastic pathway.The layers also have other functions, such as mechanical protection against pathogens and parasites [29].The plant's vascular system comprises two transport tissues.Xylem delivers nutrients and water   4., shows that the anatomy of D. stratiotes' roots exhibited a variation in root diameter across jasmonic acid treatments.The results showed that the treatment without jasmonic acid produced the largest root diameter.The application of 0.4 mg.L -1 of jasmonic acid leads to the largest root among jasmonic acid the treatments with the addition of jasmonic acid.Moreover, it results in anatomical structural arrangements that do not show a clear boundary between various components.The absence of velamen was observed in doses measuring 0.8 and 1.5, presumably due to their small size.Variations in cell size and number are considered to affect the plants' ability to survive.Differences in the number of cell layers indicate orchid adaptation to the environment.For example, orchid species from dry areas have multiple layers of velamen, while orchid species from humid areas have few velamen layers [31].

Conclusions
The application of jasmonic acid was shown to influence the leaves number, roots number, plant height, and plant weight.However, the increase of the concentration of jasmonic acid decreased the observed variables.1 mg.L -1 of jasmonic acid promoted the plant height, leaves number, plant weight, and roots number which resulted in 10.16 cm, 6.53, 1.18 g, and 9.53, respectively.The application of 1.5 mg.L -1 of jasmonic acid leads to the largest mesophyll area.Research and development of agricultural technology is essential for the advancement of modern agriculture.Jasmonic acid has the potential to be a growth regulator for orchid.Further research is still required to identify the specific use and ideal concentration of jasmonic acid on growth as well as development among different orchid species in vitro in order to improve propagation techniques in agricultural practices for sustainable development.
This research was supported by Sebelas Maret University, namely The Grant Research Group (HGR) 2022 with registration number 0001060073512022.

Fig. 2 .
Fig. 2. 3-degree regression analysis of jasmonic acid concentration with A: Leaves number, B: Plant height, C: Root number, D: Plant weight

Figure
Figure 4., shows that the anatomy of D. stratiotes' roots exhibited a variation in root diameter across jasmonic acid treatments.The results showed that the treatment without jasmonic acid produced the largest root diameter.The application of 0.4 mg.L -1 of jasmonic acid leads to the largest root among jasmonic acid the treatments with the addition of jasmonic acid.Moreover, it results in anatomical structural arrangements that do not show a clear boundary between various components.The absence of velamen was observed in doses measuring 0.8 and 1.5, presumably due to their small size.Variations in cell size and number are considered to affect the plants' ability to survive.Differences in the number of cell layers indicate orchid adaptation to the environment.For example, orchid species from dry areas have multiple layers of velamen, while orchid species from humid areas have few velamen layers[31].

Table 1 .
The growth of D. stratiotes plantlets in vitro under the influence of jasmonic acid in different concentrations Each value represent mean ± standard deviation.Means with joint letter in the same column are not different significantly at 5% DMRT.