ANALYZING SUITABLE COMBINATIONS OF POTTING MIXTURES AND HORMONE LEVELS FOR VEGETATIVE PROPAGATION OF Excoecaria agallocha (MILKY MANGROVE) FROM MADU GANGA RAMSAR WETLAND

Mangroves are the foundation species that control ecosystem dynamics and identifying their vegetative propagation methods is important to conserve them. This study was conducted to identify suitable combinations of potting media and indole-3butyric acid (IBA) concentrations for the propagation of Excoecaria agallocha (milky mangrove) stem cuttings sourced from Madu Ganga Wetland in south west rainforest region of Sri Lanka. Three IBA concentrations (2500, 3000 and 3500ppm) and three potting media (P1. lagoon silt, P2. clay and sand (1:1), and P3. topsoil, coir-dust, compost and sand (4: 4: 4: 1) were factorially combined in a Completely Randomized Design with five replicates. The control treatment was 2500 ppm IBA with P2 potting media. There was no interaction effect for survival percentage, root dry weight and shoot number between potting media and IBA concentration. For survival percentage, and root dry weight, significantly highest values were observed in 3500ppm IBA. Shoot number was significantly higher in P3 while the survival percentages were significantly greater in P1 and P3 potting media than control. The average number of roots, root length, and root volume, were significantly greater in 35000ppm IBA with P1 media. According to the vigor scales, the highest root vigor was depicted by lagoon silt treated with 3500 ppm IBA while the highest shoot vigor was observed from P3 media treated with 3500 ppm IBA. Results revealed that treating stem cuttings with 3500ppm IBA and using P1 or P3 potting media are the most effective method to propagate E. agallacha through stem cuttings out of the treatments tested in this study.


INTRODUCTION
Mangroves are fulfilling several essential ecological functions that are vital to maintaining sustainability within natural resources (Nagelkerken et al. 2000). They act as a carbon sink and a type of carbon lock in the world. Hence, it is a potential plant species to store carbon in different saline conditions (Banerjee et al. 2016). Mangrove ecosystems are acting as nurseries for aquatic life while giving enormous benefits for mankind (Karunathilake 2003). Excoecaria agallocha (milky mangrove)species belongs to the family Euphorbiaceae is prominent among other mangrove species (Arulnayagam 2020). It is small to medium size, briefly deciduous, and growing up to 10 -30 meters (Mondal et al.

ANALYZING SUITABLE COMBINATIONS OF POTTING MIXTURES AND HORMONE LEVELS FOR VEGETATIVE PROPAGATION OF Excoecaria agallocha (MILKY MANGROVE) FROM MADU GANGA RAMSAR WETLAND
2016). There are numerous advantages taken from E. agallocha, but it considers as a poisonous tree due to skin irritation, blistering and blindness that occurred when contaminate. It is called blinding tree as this latex makes blindness when contaminating (Kumarasinghe and Seneviratne 1998). Its leaf and stem sap are used to treat leprosy, epilepsy, conjunctivitis, dermatitis and so many other diseases (Bandaranayake 1998). It consists of many pharmacological potentials and many other bioactivities including antioxidant and antiviral properties (Chan et al. 2018). Further, there is a high impact of Exoecaria agallocha on diabetes mellitus, pathogenic microbial strains, pandemic diseases, anti-oxidant, and free radical scavenging efficiency, as well as have potential in nanoparticles biosynthesis and *Corresponding author: emuindeewari@gmail.com  (Manueke et al. 2018). It is widely used as a larviciding agent as well as a mosquito-repelling agent (Pradeepa et al. 2015), (Thangam and Kathiresan 1993). E. agallocha was tested for its allelopathic effect against seed germination and seedling growth of some grain crops such as green gram, black gram, groundnut, pearl millet, and finger millet (Desai and Gaikwad 2015). Similarly, E. agallocha root exudates have an inhibitory effect on the hyphal growth and colonization of fungi such as Glomus mosseae (Kumar et al. 2007).

SHORT COMMUNICATION
Due to several anthropogenic and natural pressures, these mangroves are being destroyed every year globally and need immediate conservation strategy for the restoration of degraded mangrove ecosystems (Pillai and Harilal 2018). Intensive afforestation and conservation of mangrove habitats are essential to developing fast methods to raise superior seedlings. Restoration of mangrove ecosystems, mangrove re-plantation, and propagation, declare protected zones, take public support for the conservation are effective remedies to conserve mangroves including E. agallocha (Mathanraj and Kaleel 2015). Rather, the nutritional limitation is also an impact for successful reforestation of E. agallocha. Hence, applying nutrients rationally is important to promote the survival of E. agallocha (Chen and Ye 2014). These species are dioecious and male and female flowers are separately born. Normally male to female trees ratio is 2.2:1, and the male to female flowers ratio at the inflorescence level is 16:1 (Karamsetty and Aluri 2018). These flowering habits and seed predation is another barrier to the natural regeneration of milky mangroves (Maharana and Basak 2020). Calliphara nobilis insect mainly feeds on the seeds of this tree and they are often seen in large numbers during the fruiting stage. E. agallocha is the preferred local food plant for Iscadia pulchra, Selepa celtis (hairy caterpillar), Achaea janatas (castor semi-looper or croton caterpillar) and of the genus Archips, Sauris and Phyllocnistis (Karamsetty and Aluri 2018). Less seed germination further endangers this species. Therefore, a protocol for the micropropagation of E. agallocha by invitro propagation is a timely need (Manickam et al. 2012). In Sri Lanka, most of the fish-aggregating devices such as brush parks are constructing using branches of E. agallocha species (Costa and Wijeyaratne 1994). Although E. agallocha is one of the 21 widespread mangrove species in Sri Lanka, there is a threat of disappearing overall composition of mangrove forests (Jayathissa 2012) and poisonous nature leads to cut down and destroy E. agallocha habitats.
Madu River wetland is the second largest wetland in Sri Lanka and E. agallocha is a readily abundant true mangrove species in the wetland (Ratnayake et al. 2017). It is a hotspot of biodiversity while 303 species of flora and 248 species of vertebrates are nest in it (Amarathunga et al. 2010). Conserving this wetland including valuable mangrove species is a need for sustaining ecological, cultural, and scenic value. It needs to create a framework on ethnobotanical traditions, uses taken by small-scale fishery communities and local people in order to conserve mangrove species. There is an essential need to develop a new growing scheme of E. agallocha for biomass production (Hossain et al. 2015). Developing sustainable mangrove management policies is vital to conserve biodiversity (Dahdouh-Guebas et al. 2006). Socio-economic and socio-ecological studies on mangroves are very essential to understand the importance of natural vegetation. Mangroves have a very low potential to reestablish in the cleared areas which are stressed environments (Lugo et al. 2014). It also leads to the loss of mangrove species from the gene pool and favorable means of vegetative propagation should identify for the re-establishment of mangroves (Mathiventhan and Jayasingham 2012). Thus the present study was aimed to find out the best composition of potting media and IBA concentration for the effective rooting of stem cuttings.

MATERIALS AND METHODS
The present study was conducted in a plant house at the Department of Crop Science, Faculty of Agriculture, University of Ruhuna from December 2020 to February 2021. A Completely Randomized Design was applied to the experiment with five replicates having five single propagators each. Nine different treatments were used, where three levels of IBA concentrations (2500, 300, and 3500 ppm) were used with three potting mixtures (original on-site lagoon silt clay: sand 1:1(w/ w), sand: topsoil: coir dust: compost 4:4:4:1). The treatment with IBA hormone 2500 ppm with clay: sand 1:1(w/w) potting mixture was used as the control treatment (Eganathan et al., 2000).
A well-grown female E. agallocha plant was selected from the Madu River Ramsar site. Stem cuttings were taken from a matured, healthy female plant, and 5-10 cm lengthy semi-hardwood cuttings were selected for propagation ( Fig. 1). Excess leaves were removed from the cuttings to avoid touching them inside the polythene layer of the single propagator and burning the leaves. A wax layer was applied on the top end of the cutting to avoid dehydration. The cuttings were dipped in Captan fungicide solution (PENTOGEN CAPTAN captan 50% (w/w) WP )for 5 minutes prior to the hormone treatment. Three IBA (Indole Butyric Acid Water Soluble IBA-K >98% Pure) concentrations 2500ppm, 3000ppm, 3500ppm were prepared by dissolving 2.5g, 3 g, and 3.5 g in 1 liter of water respectively. Semi-hardwood cuttings were dipped in respective hormone concentrations for 2 minutes. White color polythene with 300 gauge was used to prepare single propagators (16"×8"). Electrical Conductivity (EC) and pH were checked in all three potting media prior to filling propagators.

Figure 1: (A) Stem cutting used for vegetative propagation, (B) Single propagators with stem cuttings (C) Male plant of E. agallocha, (C) Female plant of E. agallocha
All single propagators were placed in a protected house (80-82 0 F at night and 96-98 0 F in the daytime). Survival percentage, root (the average number of roots, root length, root volume, root dry weight), and shoot parameters (number of shoots, chlorophyll content) were collected 45 days after the establishment by carefully removing cuttings from potting media. Data were analyzed using SAS Package version 9.1. The vigor scales were developed for analyzing qualitative characteristics for roots and shoots as follows.

RESULTS AND DISCUSSION
All three potting media were checked for their EC and pH before the establishment of the cuttings (Table 1). The highest EC was observed in potting media with lagoon silt while the lowest was from the potting media with clay: sand 1:1. pH of all potting media was within the range of 5.4-7.5.As reported by Zhang (2019) climate, topography, parent materials, and the horizon that variedly influence the pH of the soil samples.
The survival percentage of cuttings was influenced by the potting medium and IBA concentration but, not due to their synergistic effect (Fig 2). The survival percentages were significantly greater in P1 and P3 potting media and P2 which has the lowest EC showed the lowest survival percentage. Manickam et al. 2012,Meena (2000  higher than that of male cuttings, and IBA treated male cuttings failed to produce roots. Eganathan et al. 2000 also showed the maximum rooting was observed when E. agallocha cuttings were treated with IBA alone up to 2500 ppm and higher concentrations of IBA showed a better response for rooting. The average number of roots, root length, roots volume, and average chlorophyll content were significantly affected by the interaction effect of potting media and IBA concentration. Potting media 1 consist of lagoon silt with 3500ppm showed the highest number of roots, root length, and roots volume. The highest chlorophyll content was recorded from potting media 3 consist of sand, topsoil, coir-dust, compost 4:4:4:1 with 3500 ppm IBA (Table 2). Root dry weight was significantly affected by the IBA concentrations. But potting media was not significantly affected for root dry weights. The significantly highest root dry weight was observed in IBA 3500 ppm while the least was recorded in the 2500 ppm IBA (Fig. 3) and this observation is collaborated by previous researches. Govindan and Kathiresan (2014) have found that higher rooting occurred in 0.5 g IBA treated Avicennia    et al.(2000) also concluded that the highest number of adventitious roots was emerged from 2500 ppm IBA and 5000 ppm NAA treated E. agallocha stem cuttings. The average number of new shoots was significantly different among the potting media (P <0.0001). The highest number of new shoots was obtained from the P3 while the least by P1which consists higher EC (Fig.  3).According to Rahman (2020), dominant mangrove species in Bangladesh are adversely affected by the increased salinity and top dying disease leads to a drastic reduction in mangrove density .  T1  T2  T3  T4  T5  T6  T7 T8 T9 Figure 4: The appearance of uprooted stem cuttings from different treatments, 45 days after the establishment. (T1-2500 ppm IBA with lagoon silt, T2-3000 ppm IBA with lagoon silt, T3-3500 ppm IBA with lagoon silt, T4-2500 ppm IBA with clay: sand 1:1(w/w), T5-3000 ppm IBA with clay: sand 1:1(w/w), T6-3500 ppm IBA with clay: sand 1:1(w/w), T7-2500 ppm IBA with sand, topsoil, coir-dust, compost 4:4:4:1, T8-3000 ppm IBA with sand, topsoil, coir-dust, compost 4:4:4:1, T9-3500 ppm IBA with sand, topsoil, coir-dust, compost 4:4:4:1) Propagation through stem cuttings is an easy and quick method that does not lead to genetic variation among the new propagules. Nevertheless, the root and shoot vigor of the new plantlets can be changed through the provision of plant growth promoters and an environment with optimum temperature, water, humidity, light, and appropriate potting media (Abbasi et al.2014).
According to the vigor scale, the highest mean value for root growth was obtained by T3 and the highest mean value of shoot growth was obtained by T9 (