Dendrobium sp. subcultures with instant Vacin Went media against many concentrations of fruit waste liquid organic fertilizer plus

Orchids are ornamental plants in great demand by collectors and the public due to the varieties colors of Orchid flowers and the price. This study aims to determine the effect and obtain the concentration of the application of fruit waste liquid organic fertilizer plus on the growth and development of a Dendrobium sp. sub-culture on VW media in vitro. The research had been carried out from May to August 2022 at the Biotechnology Laboratory of Kupang State Agricultural Polytechnic. The study design used a single-factor complete randomized design consisting of six treatments and four replications. The tested factors were six level of concentrations of fruit waste liquid organic fertilizer plus instant VW media in vitro, which are: instant VW media; instant VW media + 2.5 ml/L; instant VW media + 5 ml/L; instant VW media + 7.5 ml/L; instant VW media + 10 ml/L; and instant VW media + 12.5 ml/L. The results showed that the best concentration of Fruit Waste Plus LOF for the growth of Dendrobium orchid explants is the treatment instant VW media + 7.5 ml/L LOF, which is shown by the time of bud emergence (10.75 days after planting), number of shoots (2.38), shoot height (3.5 mm), number of roots (1.38), and root length (2.5 mm).


Introduction
Orchids are ornamental plants that are in great demand because their flowers are durable, they are not easy to wither and fall off, and their colors are diverse and have a high selling value [1].In 2021, orchid production in NTT as cut flowers will be 320, while the total orchid production in Indonesia will be 11,351,615 [2].So that the level of demand for orchids from collectors, ornamental plant businesses, and consumers is increasing.The increasing demand for orchids has not been met by orchid producers.Although orchid production in NTT increases every year, it is still in small numbers and has not been able to meet the demand for orchids in the country.The causes are the lack of availability of orchid plant seeds, inefficient cultivation, and poor post-harvest handling [3].
Propagation of orchid plant seeds using tissue culture propagation techniques is one solution to producing orchid plant seeds in large quantities in a relatively short time compared to conventional propagation.Propagation of orchids is conventionally carried out by cuttings (vegetative), that is, the separation of clumps and the separation of branches from the stem.
The main barrier to conventional orchid propagation is slow growth, which takes a long time to produce seeds [4].For this reason, a more effective alternative propagation method is needed, namely in vitro culture with seeds or buds.This method is effective in providing plant seeds in large quantities and uniformly, and it takes a relatively short amount of time.
Success in vitro techniques is influenced by several factors, which include culture media, explants, an aseptic culture environment, and nutrient addition (ZPT) to the media.Success in the use of tissue culture methods depends on the media used.
Media is a determining factor in the propagation of tissue culture.Growing media in tissue culture have a great influence on the growth and development of explants and the seeds they produce.Good culture media should provide nutrients both macro and micro, sources of vitamins and amino acids, sources of carbohydrates, and growth regulators [5].Vacin and Went (VW) media is an orchid culture medium made up of macro and micro nutrients, inorganic compounds, energy sources (sucrose or sugar), and vitamins [6].
The use of VW media with the addition of organic matter tends to give different results to the formation of Dendrobium orchid explant organogenesis, such as budding organs, leaves, and roots that grow more on fresh green and wider leaves, while the roots grow longer [7].With the use of VW media without organic matter, fewer shoots, leaves, and roots are visible.It can be proven that the addition of organic matter shows optimal results in the formation of Dendrobium orchid explant organogenesis compared to only using VW media without organic matter.One of the organic materials that can meet the nutrient needs of orchid culture media is liquid organic fertilizer.Liquid organic fertilizer is also included in complex organic matter because it has macro-and microelements and other nutrients needed by the plant.The medium with liquid organic fertilizer has the advantage of optimizing vegetative and generative plant growth, increasing plant resistance and health, and being simple to apply.Therefore, it is necessary to test VW Media's added liquid organic fertilizer.
The application of liquid organic fertilizer Fertile, Fish Emulsion, and Nasa shows that the use of liquid organic fertilizer Fertile 1.5 ml/L on VW media can affect the number of leaves and the highest number of orchid buds, with a value of 4.67 and 6.00 pieces, respectively [8].Stated that the application of Amino-Age liquid organic fertilizer with a concentration of 2 ml/L has a real influence on leaf growth and development in Vanda limbata and Vanda tricolor orchid plantlets [7].
Fruit Waste Plus liquid organic fertilizer is a biological liquid fertilizer made from fruit waste and selected natural materials that are processed through extraction and fermentation so that liquid organic fertilizer is produced that contain a variety of dissolved organic nutrients and is environmentally friendly to optimize soil and plant health, plant growth and development, and improve the quality and quantity of cultivated plant production.Waste Fruit Plus liquid organic fertilizer contains functional microorganisms that work as nitrogen-providing agents in soil and plant root zones by dissolving phosphates and other mineral elements in the soil and producing plant growth hormones.However, the use of liquid organic fertilizer, Fruit Waste Plus, in in vitro culture media is known.Therefore, this study was conducted.The purpose of this study is to determine the effect and best concentration of liquid organic fertilizer (LOF) fruit waste on the growth and development of a Dendrobium orchid subculture on VW media in vitro.

Materials and Method
From May to August 2022, this study was conducted at the Biotechnology Laboratory of the Department of Food Crops and Horticulture at Kupang State Agricultural Polytechnic.The materials used are multiplication phase bottle orchid, 90% alcohol, 70% alcohol, disinfectant solution, CD paper, and wipes.The equipment used is UV lamps, culture bottles, measuring cups, petri dishes, tweezers, pipettes, culture racks, laminar airflow (LAF), ovens, and bunsen.The culture laboratory rooms used are preparation rooms, planting media material rooms, transfer rooms, planting rooms, and growth rooms.
This study was designed using a randomized group design with a concentration treatment of liquid organic fertilizer plus fruit waste on Vacin and Went (VW) media consisting of 5 levels of treatment with 5 repeats, so that there were 25 experimental units.The treatment is: instant VW media; instant VW media + 2.5 ml/L; instant VW media + 5 ml/L; instant VW media + 7.5 ml/L; instant VW media + 10 ml/L; and instant VW media + 12.5 ml/L.
The implementation of the research begins with several stages, namely: sterilization of the work environment (laboratory), namely the room (preparation room, planting media material room, transfer room, planting room, and growth room) and laminar air flow (LAF); sterilization of equipment (metal and glass tools); preparation of explants; creation of VW culture media; sterilization of the substrate.Initiation (planting) of multiplication-phase orchid explants; observation (daily).
The experimental data obtained were then analyzed using fingerprints, and to determine the difference between treatments, further tests were carried out using the Honest Real Difference (BNJ) test at a level of 5% [9].Observation variables are supporting variables (type of contamination, percentage, and time of contaminated media) and main variables (bud emergence time (days), number of shoots, shoot height (mm), number of roots, root length (mm).

Types of contamination
Contamination is a disturbed cultural environmental condition due to the entry of good contaminants, such as small animals such as ants, fungi, and bacteria [10].Contamination requires daily observation during activities.Contaminant microorganisms that grow are fungi, where mold grows on the surface or edge of the media in direct contact with the wall of the bottle.If left unchecked, the fungus will cover the entire surface of the media in the form of fine white threads, then brownish and blackish, forming a circle covering the media or explant in the bottle.This results in the media not being used, and the explant cannot grow and die.The occurrence of contamination in plant propagation activities by tissue culture is caused by three sources of contaminants: the explants used, media factors, and the implementation process.
Contamination originating from the implementation process occurs in the manufacture of media and the use of tools that are less sterile so as to provide opportunities for fungi to grow and develop in the media [11].While contamination originates from explants, where the explants are carriers (carriers of microorganisms, either fungi or bacteria), and at the time of sterilization of the explant.If done less sterilely, fungi on the explant will easily grow and develop in the culture bottle, both in the media and on the explant.Contamination caused by mold can be seen in Figure 1.

Percentage and time of contaminated media
The occurrence of contamination in orchid tissue culture is caused by bacteria, fungi, and from plants, in the research process contamination occurs when plants have been in the incubation room, which is suspected by an incubation room that is less sterile resulting in several contaminated bottles.The percentage of contamination can be seen in Table 1.  1 shows variations in contamination time.Contamination occurs from day 9 to day 28.The day with the most contamination was day 17, then on days 9, 12, and 28, the amount of contamination was the same at 5%.The highest amount of contamination was found in the instant VW media + 5 ml/L treatment, as much as 10% from days 9 to 17. Then followed treatment instant VW media + 10 ml/l which is as much as 5% on day 12, treatment instant VW media +7.5 ml/L which is as much as 5% on day 17, and treatment instant VW media + 12.5 ml/L which is as much as 5% on day 28.
Stated that contamination of planted explants can occur due to external and internal infections.External contamination usually comes from non-sterile tools and materials, small animals that get into culture bottles, and the air.While internal contamination comes from within the plant tissue itself.Based on the time of contamination, it can be seen that the sterility of the room and equipment used in orchid culture activities is very important to pay attention to so as to reduce contamination and allow the explant to grow properly [3].

Bud growing time (days), number of shoots, shoot length
The time of emergence of shoots (Table 2) is observed daily, starting from the first day the explant is planted until the emergence of shoots after the number has reached 50%.Table 2 shows that the average time of emergence of the fastest shoots is 10.75 days after initiation in treatment instant VW media + 12.5 ml/L, which is significantly different from other treatments.The rapid time of budding is thought to be caused by the sufficient microbial content of Azotobacter sp.(12.5 x 107 CFU), Pseudomonas (12.5 x 108 CFU), and Saccharomyces (12.5 x 108 CFU), which play a role in producing nutrients and hormones that are sufficient for shoot growth, so that it can spur the process of faster shoot growth compared to other treatments.While the average time for the longest shoots to emerge is 23 days after initiation contained in the instant VW media treatment without liquid organic fertilizer.This is because of the lack of availability of nutrients needed by orchid plants to form buds, even though in each treatment there is the same concentration of coconut water, which has the same ZPT but is not enough for maximum growth of shoots.
Stated that Azotobacter can live in liquid organic fertilizer (LOF), which contains macronutrients C, N, P, and K as well as microelements Fe, Mn, Cu, Zn, Co, Mo, and B, which will support the growth of Azotobacter.The presence of N, even at low concentrations, at the beginning of growth can accelerate the growth of bacterial cells before fixing nitrogen and producing hormones.The hormones produced by Azotobacter are cytokinin.Cytokinin produced by Azotobacter can spur the growth of Dendrobium orchid shoots [12].
Stated that the best type of media composition in the treatment of media is 1/2 Nitsch and Nitsch + BAP 1 ppm + coconut water 150 ml with the average age of the fastest shoots being 10.80 days after initiation.The nutrient composition of the media meets appropriate standards for the growth of orchid explants from subcultures [13].Using the combination of benzyl amino purine (BAP) with organic matter extract on VW media, showed that the average time of emergence of the fastest shoots was 13.3 DAP (Days after planting) [14].
The highest average number of shoots (Table 2) was found in the treatment instant VW media + 12.5 ml/L, which was 2.38 buds, significantly different from other treatments.The highest number of shoots found in the treatment instant VW media + 12.5 ml/L liquid organic fertilizer is thought to be caused by the fact that it contains nutrients needed by orchid explants for bud growth.There is also ZPT cytokinin that plays a role in stimulating bud growth.While the average number of the lowest shoots found in the treatment instant VW media without liquid organic fertilizer is 1.13, it does not differ significantly in the treatment instant VW media + 2.5 ml/L, which is 1.13.It is suspected that the nutrients contained in instant VW media without liquid organic fertilizer and instant VW media +2,5 ml/L LOF are not enough to meet the needs of Dendrobium orchid explants during bud growth.
Adding LOF to orchid plant tissue culture will have a positive impact on the growth of orchid shoots in tissue culture.The content of cytokinin and gibberellin hormones in LOF can stimulate plant germination and growth, function as stimulators in tissue proliferation, facilitate metabolism, and facilitate respiration; therefore, coconut water has a great ability to facilitate the process of differentiation [8].
The highest average shoot length (Table 2) of 3.5 mm was found in the treatments instant VW media + 12.5 ml/L and 10 ml/L LOF, which was not significantly different from the treatment instant VW media + 7.5 ml/L LOF, which was 3.4 mm, but significantly different from other treatments.Stated that Azotobacter bacteria found in LOF can produce cytokinin.It is thought to be high in cytokinin, so that it spurs on shoot growth and suppresses root growth [12].While the average length of the lowest shoots of 2.1 mm resulting from instant VW media without liquid organic fertilizer treatment was significantly different from treatment instant VW media + 2.5 ml/L, which was 2.3 mm.This is thought to be caused by the fact that the instant VW media without liquid organic fertilizer and instant VW media + 2,5 ml/L LOF nutrients contained in it do not meet the needs of explants for the process of elongating shoots resulting in less than optimal growth of shoots in Dendrobium orchid plants.
Exogenous growth regulators are given to provide balance against endogenous hormones in order to be able to influence physiological responses as a driver of cell division and elongation during bud multiplication and bud morphogenesis [14].Found that the combination of benzyl amino purine (BAP) with organic matter extract on VW media in BAP treatment 3 ppm + tomato extract showed the highest average number of shoots of 14 pieces, and in BAP treatment 2 ppm + tomato extract showed the highest average shoot length of 2.06 cm.The more shoots that appear, the greater the chance to get more orchid seeds [14].

Number of roots, and root length
The highest mean number of roots (Table 3) was produced by treatment instant VW media + 7.5 ml/L, i.e., 1.38, which differed insignificantly from treatment instant VW media + 10 ml/L, i.e., 1.13.Plant roots number can indicate how widely the plant absorbs nutrients; the more roots there are, the more nutrients are absorbed.The lowest average number of roots produced by without LOF is 0.5, and instant VW media + 2.5 ml/L is 0.25.In general, shoot growth is influenced by ZPT and nutrients contained in the media used.Basically, bud induction is influenced by the use of ZPT, one of which is cytokinin.[14] Found that if the concentration of auxin is low, it will affect poor root growth due to high levels of cytokinin concentrations that can inhibit plant root growth [15].
The longest average root length (Table 2) was produced by treatment instant VW media + 7.5 ml/L, i.e., 2.5 mm, which differed insignificantly from treatment instant VW media + 10 ml/L, i.e., 2.1 mm, and instant VW media + 12.5 ml/L, i.e., 1.0 mm.The roots growing on the explant are very small.This is thought to occur because the presence of auxin acts as a root stimulant, but if the content is low, the roots that appear will be small.While the lowest average root length was produced by instant VW media + 2.5 ml/L, which is 0.4 mm, it was not significantly different from the treatment of instant VW media without liquid organic fertilizer, which is 0.5 mm, and instant VW media + 5 ml/L which is thought to have a low auxin content, resulting in poor root growth.
Stated that the addition of cytokinin affects cell division and morphogenesis, while auxins play a role in regulating cell growth and elongation [16].The highest average root length in treatment without the addition of ZPT is thought to be due to the endogenous auxin content that has been high enough to induce root growth; as a result, the addition of auxin will suppress root elongation.Anthurium side shoot explants planted on culture media produce endogenous auxin with a high enough concentration so that explant growth is more directed at cell elongation and root formation [17].

Conclusion
This research can be concluded that the concentration of Fruit Waste Plus Liquid Organic Fertilizer (LOF) added to instant VW media has a real effect on the growth of Dendrobium orchid shoots, namely at the time of bud emergence, number of shoots, height of shoots, number of roots, and root length.
The best concentration of Fruit Waste Plus LOF for the growth of Dendrobium orchid explants is the treatment instant VW media + 7.5 ml/L LOF, which is shown by the time of bud emergence (10.75 days after planting), number of shoots (2.38), shoot height (3.5 mm), number of roots (1.38), and root length (2.5 mm).

Figure 1 .
Figure 1.Contamination by mold in tissue culture media during research

Table 1 .
Percentage of contaminated bottles observed data

Table 2 .
Average bud emergence time, number of shoots, and shoot length of Dendrobium orchid plants due to giving various LOF concentrations of fruit plus waste on instant VW media treatment

Table 3 .
Number of roots, and root length of Dendrobium orchid plants due to giving various LOF concentrations of fruit plus waste on instant VW media treatment The numbers followed by the same letter in the same column show no real difference on the 5% BNT test