In vitro culture of wild Vanda tricolor Lindley seeds from Kintamani, Bali, as initial step for conservation

Vanda tricolor is an endemic orchid from Indonesia that can be found in Bali. As part of Orchidaceae, Vanda tricolor is facing the same problem as other orchid species, their seeds have various requirements for germination which causes a low germination rate. Preventing this endangered orchid from extinction with tissue culture not only maintains the genetic diversity but also understanding the growth factors of this very species. The aim of this research was to analyze how sterilization, medium, and light affect the germination of V. tricolor. Wild V. tricolor pod from Buahan Village, Kintamani, Bangli Regency, Bali, was sterilized with 2g/L fungicide, 1,05% and 0,79% NaClO; while seeds were sterilized with 1,05% NaClO. The seeds from these two different sterilization methods were cultured on Murashige and Skoog (MS) medium, Vacin and Went (VW) medium, Organic 1 (O1) medium, and Organic 2 (O2) medium, then half of them were placed under LED light and the rest were kept on dark. Each one of the sixteen treatment combinations was replicated six times. Seed sterilization with 1,05% NaClO showed better results than pod sterilization. Seeds started to germinate 3 weeks after sown with the highest germination rate occurred on seeds cultured on MS medium under LED light. This study revealed tissue culture with different treatments will aid to understand the preferred germination condition for wild Vanda tricolor and support its conservation.


Introduction
Orchid, family Orchidaceae is a flowering plant with high popularity in Indonesia, with appealing colors and shapes.One of the most popular orchids in Indonesia is Vanda tricolor, which is an endemic species in Java and Bali [1].This commercially high-demand plant has been decreased in number in its natural habitat [2].Vanda tricolor, like other orchid species, have small-sized seeds without endosperm [3] and need mycorrhizae as a symbiont to supply nutrient for growth [4].Those features barrier the seeds' capability to germinate and produce more plants in nature.Conservation is needed for this very species to prevent it from extinction.Reproducing orchid through vegetative propagation is possible but it can only be resulting in clones (keiki) with the same exact genotype [5,6].Plants that grow from seeds, on the other hand, are genetically more diverse than keiki [7].
In vitro propagation with seed as propagules is an effective method for orchid conservation since it increases the orchid's germination rate, provides massive amounts of healthy plants with diverse genotypes, and can be done in all seasons throughout the year [8].This particular method requires external factors such as light and medium composition because orchid germination is a complicated process [9].Several orchid species need light for their germination, they are categorized as photoblastic plants while other species are negative photoblastic plants because they do not require light.Some orchid species that can germinate with or without light belong to neutral photoblastic plants [10].Alghamdi [11] revealed that light inhibits the germination of Orchi mascula, Encyclia phoenicea, Dactylorhiza fuchsii, and Goodyera repens.Different outcome showed on Dendrobium nobile and D. phalaenopsis where their germination are not affected by light [12].
Medium composition is considered as the most important factor that affect plant growth.The most common tissue culture media that have been used for orchid is Murashige and Skoog (MS) and Vacin and Went (VW).Highest germination rate of Vanda tricolor Lindley var.Suavis 'Queen Maxima' was showed in VW medium [13].Both MS and VW media are expensive therefore modified medium from 1255 (2023) 012026 IOP Publishing doi:10.1088/1755-1315/1255/1/012026 2 natural organic extract and foliar fertilizer can be an alternative option.Frequently-used organic extract are banana, tomato, and coconut water [9].
Explant sterilization is crucial step in tissue culture as it prevents the growth of contaminant such as bacteria and fungi.The presence of contaminant on medium or explant can damage the plant tissues that cause death [14].Surface sterilization of whole orchid pod with natrium hypochlorite (NaClO) as sterilant is the common method of sterilization before sowing the seeds.Seeds disinfection of Epidendrum nocturnum, Prosthechea garciana, Maxillaria rufescens and capsule disinfection of Zygopetalum hybrid showed to be 100% efficient [15].
There is lack of study regarding the effects and interactions of light, media, and sterilization.This study was conducted to analyze how sterilization, nutrition from medium, and light affect the germination of Vanda tricolor.

Materials and methods
Green-yellowish wild Vanda tricolor Lindley pod from Kintamani, Bali was stored in refrigerator for 7 days before used in this study to prevent pod dehiscence.The pod was sterilized with 2.000 ppm fungicide (Dithane ® M-45 80 WP) with Tween 20 (Sigma), 1,05% NaClO, and 0,79% NaClO for 15 minutes for each sterilant.The seeds (in needleless syringe) were soaked in 1,05% NaClO for 15 minutes and rinsed with distilled water before sowed.
Seeds were sowed on MS, VW, Organic 1 (O1), and Organic 2 (O2) medium in 16×150 mm test tube.Every 1 L of O1 was made with 30 g sugar, 2 g 'Gandasil D' leaf fertilizer, distilled water, 100 mL coconut water, 100 g banana homogenate, and 100 g tomato homogenate; while O2 was made with same ingredients but with 150 mL coconut water, 150 g banana homogenate, and 150 g tomato homogenate.Banana homogenate was made with 300 g banana flesh that smashed with blender then filtered once and weighed to gain 100 g and 150 g banana homogenates.Tomato homogenate was made with similar process.
Half of the cultures were exposed to LED light for 16 hours per day and the rest were placed in a box that covered with dark fabric.Factorial completely randomized design was used in this study with 4 × 2 × 2 factorial scheme (four different media, two different sterilization method, and two different light intensity) (Table 1).Each treatment combination was replicated six times.The relative humidity of culture room which was used to store the cultures was 70% with a temperature of 21℃.Note: I1 = light, I2 = dark; M1 = MS, M2 = VW, M3 = O1, M4 = O2; S1 = pod sterilization, S2 = seed The seeds were observed weekly and captured biweekly, started at three weeks after sown, using light microscope.Contaminated cultures were counted and the contamination percentage were calculated using Microsoft Excel.All seeds with embryo (non-germinated seeds/NS) and protocorms (chlorophyll propagules/CP) in each test tube were counted at 7 weeks after sowing (WAS) to compute the germination rate.The percentage of the germination rates (%G) were computed with this equation [12]: Three-way analysis of variance (ANOVA) was conducted after the %G data were transformed according to Templeton [16], proceed to Duncan's Multiple Range Test (DMRT).

Results and discussion
The observations of the sterilization method showed that all experiment units were not contaminated except for I1M2S2 and I2M3S1 (Table 2).One unit of I1M2S2 was contaminated by fungi and two unit of I2M3S1 were contaminated by fungi and bacteria.Contamination by fungi was indicated by the presence of thread-like structures (hyphae) and white to grey fungi colonies.Translucent to white mucoid structures that appeared on the media is bacteria colonies [17].Contaminations could happen due to the inability of sterilant to eliminated the contaminants from the surface of the explant.Seeds sterilization with 1,05% NaClO was proved to be the better than pod sterilization despite there is no significant difference from both methods.= bacterial contaminants The protocorms have formed on I1M1S1, I1M2S1, I1M1S2, and I1M2S2 at 3 WAS while protocorms on I2M1S1 formed at 5 WAS and protocorms on the rest of the treatment combinations have formed at 7 WAS.Seeds which were directly sterilized with NaClO showed better germination compared to seeds from sterilized pod.This happened because of NaClO as a sterilant which scarified the seed coat, helps to breaks seed dormancy [18].
Seeds which were sowed in MS showed significantly best germination rates among all media (Table 3).MS medium contains 19 nutrients, more complex than other media.Macronutrients such as nitrogen, potassium, and phosphor in MS medium are the substitution of nutrients that provided by mycorrhizae for orchid in their natural habitat.MS provides highest amount of nitrogen and potassium among all media.MS contains nitrogen in form of ammonium nitrate and potassium nitrate.Plants normally absorb ammonium and nitrate, which are inorganic form of nitrogen, to supply their need of nitrogen [19].Nitrogen in form of nitrate takes role as signaling molecule which helps germination process through breaking seed dormancy [20].Potassium in MS provided in form potassium iodide, potassium nitrate, and potassium phosphate.Potassium assists water transport in germination to break seed dormancy and accelerate germination process [21].
Germination rates of seeds that were irradiated with LED light proved to be significantly higher from seed that not exposed to light (Table 3).Thus, V. tricolor is a light-dependent species (photoblastic).Light helps plant's nutrients utilization, supports plant growth, and affect chlorophyll content [22].Protocorms that grow under light visually tended to be greener than protocorms that not irradiated (Figure 1).Protocorms with I1M1S1 treatment were already developed cotyledon and rhizoid (Figure 1a).Combination of MS medium and LED light irradiance was significantly different among all interaction of independent variables.For that reason, regardless of the sterilization method, MS and LED light irradiance are the recommended treatment for wild V. tricolor seeds germination.

Conclusion
Light intensity, sterilization method, and particularly medium affect the germination rate of Vanda tricolor seeds.Highest germination rate was found on seeds that directly sterilized by 1,05% NaClO and sowed on MS medium with 16 hours exposure of LED light, thus these treatments are recommended for in vitro germination of V. tricolor.Different kind of sterilant, media, or light spectrum should be examined further for better understanding of V. tricolor germination.

Table 1 .
Light intensity, sterilization, and medium treatment combinations.

Table 2 .
Percentage of contamination.