Effectiveness of media B5 (Gamborg) for multiplication of Sengon (Paraserianthes falcataria L. Nielsen) in vitro

Paraserianthes falcataria (L.) Nielsen, also known as sengon, is one of the most important and adaptable pioneer species. The Sengon plant is one of the quickest-growing tropical plants. The objective of this study was to identify the optimal medium for sengon multiplication with the addition of ZPT, cytokinin, BAP, and kinetin, either singly or in combination. The research design employed was an entirely random design (CRD). Media M2 (B5 + BAP 0.1), which was the medium with the highest number of shoots, was determined to be the optimal medium for sengon plantlets in the study. At 1 MST, the fastest medium for leaf emergence was (B5 + BAP 0.2) media. The poisson test analysis shows that, in comparison to other media, the M2 medium (B5 + BAP 0.1) has a very significant impact on the quantity of leaves. The medium with the highest average height of sengon plantlets was M1 (B5 Control), with 1.73 cm. The highest percentage of dead plantlets on M4 medium was 33%, while on M9 medium it was 17%, due to browning leading to cell death. Only media M4 (BAP 0.2), M5 (Kinetin 0.2), and M9 (BAP (0.2) + Kin (0.2) contained plantlets contaminated by bacteria at a concentration of up to 17%, which inhibited plantlet growth.


Introduction
Paraserianthes falcataria (L.) Nielsen, also known as sengon, is one of Indonesia's most significant versatile pioneers.The sengon plant is one of the fastest-growing tropical plants.Java has extensively developed Sengon plantation forests to meet the community's needs, particularly for building materials and industry [1].Sengon began to be widely cultivated as a folk forest because it is adaptable to a wide range of climatic conditions and does not require a high growing altitude.Due to the fact that the annual demand for sengon wood exceeds 500,000 m 3 , the prospects for planting sengon are quite favorable.In tandem with the development of this type of planting area for industrial plantation forests and community forests in the present day, the demand for this high-quality wood and sengon seeds is rising.Most sengon industrial plantation forests are still established using seeds of unknown origin, resulting in low wood productivity [2,3].
Currently cultivated Sengon plants have a very diverse growth pattern and low productivity.Therefore, sengon plants with relatively uniform growth and high yield are required.Solomon's sengon is recommended as one of the sengons.Sengon seeds are typically propagated using conventional methods, making it more time-consuming to produce enough seedlings to meet the needs of the woodworking industry [1].
Utilizing in vitro propagation is the solution for overcoming this issue.This method of propagation permits the production of numerous seedlings in a relatively short amount of time.In tissue culture, the growing medium has a significant impact on the growth and development of explants and the seedling they produce.The substrate's composition depends on the type of plant being propagated.Typical media components include gelatin, mineral salts, vitamins, and growth regulators [4].Auxins and cytokinins are two Growth Regulators (ZPT) commonly used in tissue culture media.Auxin is a ZPT that plays a role in inducing rooting during in vitro propagation, whereas cytokinins play a role in inducing explant bud formation.Benzyl Amino Purine (BAP) is a commonly used cytokinin because it is the most effective at stimulating bud formation, is more stable, and is oxidation-resistant.Kinetin is a type of cytokinin that promotes cell or tissue division in explants and stimulates the growth of bud shoots [4].
Sengon might be propagated vegetatively through tissue culture.Seed propagation can produce plant saplings with a wide range of properties, necessitating lengthy field tests to achieve uniformity in superior properties.The system of tissue culture propagation through organogenesis of shoot buds is anticipated to meet the demand for uniform sengon seedlings in a short amount of time and at a relatively low cost.When meristem tissue is utilized, the success rate of tissue culture will increase.Buds contain a portion of the meristem tissue found in plants [5].
Explants in the form of shoot buds have the highest percentage of producing planlets, particularly when grown on auxin-free media [6].The type of media used is B5 media, which has been used in a number of previous studies, including research conducted by Karjadi et al. [7], which demonstrated that the best results for each change were obtained on B5 media with treatments such as, for the number of leaves at the NAA level of 0 mg/l with a BAP of 2.5 and 7.5 mg/l, and for plantlet height at NAA level 0 mg/l and BAP 2.5 mg/l, as well as for In light of this, it is crucial to conduct this research in order to obtain the optimal media by adding ZPT cytokinin in the form of BAP and kinetin to the zincon multiplication phase.

Materials and methods
This research was carried out at the Laboratory of Tissue Culture, 2 nd Regional of Forest Seed/Seedling Center (BPTH), Sulawesi from December 2021 to February 2022.The materials used in this study included of sengon seeds from Papua.

Tools and materials
B5 (Gamborg) media containing macro stock solutions, micro stock solutions, Fe stock solutions, vitamin stock solutions, Myo-Inositol, glucose obtained by compacting gelatin, 70% alcohol.Additional ingredients include the growth regulators Benzyl Amino Purine (BAP) and Kinetin.

Implementation of activities
Beginning with the sterilization of tools and materials, making media, preparation and sterilization prior to planting, planting explants on culture bottles containing media, growing explants, and observing and collecting data are the steps involved in the conduct of this study.

Research design
The research design employed was a Complete Randomized Design (RAL) with one factor, namely the ZPT concentration, consisting of nine treatments and six tests, so that the total number of observation units was 54, with two explants planted in each unit.The used ZPT concentration is shown in Table 1.

Observation parameters
Observations are carried out 1 time a week for 2 months.Parameters observed include, the number of sprouted shoots, the time they appear (Sunday After Planting / MST), the number of leaves (strands), plant height (cm), and percentage of explants living and dying (%), calculated at the end of the observation.

Data analysis
The research design used was a complete Randomized Design (RAL) with 1 factor.The RAL mathematical models used are as follows: Information: yi= number of events to ij, i= 1.2,.., ; μi = means the number of occurrences in the period it ; ɛi= error error or residual.

Number of shoots
After planting, the response at the stage of multiplication for sengon planlets is the emergence of new shoots.Observation begins following the planting date.Figure 1 depicts the result of the calculation of the average number of shoots.The addition of ZPT to the medium is one of the factors that affect the growth of explants, particularly in inducing the development of shoots into planlets [8].The results (Figure 1) indicate that M2 media (B5 + BAP 0.1) is the medium with the highest number of shoots, while M4 media (B5 + BAP 0.2) has the lowest number of buds.The explants that form buds are visible as greenish protrusions on the undersides of the leaves, which correspond to the characteristics of the buds.Observations indicate that all BAP treatments are capable of inducing shoots in vitro.According to Larekeng et al. [9], BAP is one of the growth regulators that has a significant impact on the development of explants, including bud formation, bud multiplication, and promoting cell division to form the required organs.
According to the research of Hariyanti et al. [10], the addition of cytokinins with a high concentration has a positive effect on bud formation and the production of the most buds.Due to the proliferation of buds, some explants are capable of producing new shoots from the base of the axillary buds.Similar to Yuniastuti et al. [11] findings, BAP at 2 ppm, 4 ppm, and 6 ppm can cause Teak plants to produce between 1.8 and 2.4 buds/explants (Tectona grandis).In addition, according to the research of Ibrahim et al. [12], the use of BAP at a concentration of 2 ppm is able to induce 4.6 buds per plant on coffee plants (Coffea arabica L.).Since the addition of BAP to the propagation medium in vitro plays a natural role in organogenesis, the correct concentration of BAP can effectively stimulate the doubling of buds.
BAP growth regulators are one of the cytokinin groups that can stimulate and induce flowering, according to Hossain et al. [13].However, the type and concentration of BAP growth regulators depend on the type of plant.In accordance with [14] assertion, the cell will divide when cytokinins are added in small amounts and the proportion is increased.Nitrogen also plays a role in the formation of plant tissues during organogenesis, and the addition of BAP to the media can increase the production of endogenous hormones, such as zeatin in leaf tissue, so that endogenous and exogenous hormones (BAP) cooperate to form bud growth.
This indicates that ZPT BAP is superior to ZPT Kinetin in its ability to induce flowering.The addition of BAP, a type of cytokinin that induces budding more effectively, is utilized.As a result of the number of shoots data, there are varying quantities in each treatment and repeat.This is consistent with the findings of [15], who state that this variation in bud growth is not only due to differences in ZPT concentrations, but also to other factors such as the clone type and the medium used.It is believed that the use of basic media explants, growing environments, and suitable regeneration systems will increase the multiplication capacity of shoots.

When leaves appear
The timing of the emergence of leaves after planting characterizes the response at the multiplication stage of sengon explants.Observation begins following the planting date.Figure 2 displays the results of the calculation of the average time of leaf emergence.M4 (B5 + BAP 0.2) (Figure 2) is the media with the quickest leaf emergence time at 1 MST.According to [17], the effect of exogenous ZPT in in vitro media is determined by the presence of identical or distinct endogenous growth regulators (in plant tissues).In other words, the effect of exogenous BAP and endogenous growth regulators in the growing medium influences the growth of planlets and the number of leaves.According to Mashud [16], a single factor of BAP concentration in in vitro media has a significant effect on the rate of leaf emergence.In in vitro cultures, a greater ratio of cytokinins to auxins will stimulate the growth of shoots and leaves, whereas an excessive concentration of BAP will inhibit leaf growth.According to Yuniastuti et al. [11], treatments with high BAP concentrations are incapable of producing leaves.This is due to the explants' genetic ability to respond to various growth regulators.The metabolic capacity of plants is largely determined by the plant's genetic makeup (endogenous factors).Some plants will not respond to a particular growth regulator (exogenous factor).In addition, growth is also affected by the equilibrium between endogenous and exogenous factors [17].

Number of leaves
The leaves are where photosynthesis occurs because there is a process of carbohydrate formation occurring on the leaves.A large number of leaves will generate numerous photosystems, resulting in enhanced plant growth [18].Conducting a Poisson Test Analysis on the effect of media treatment on the number of leaves will reveal the difference between each level of treatment in the parameter of the number of leaves.The results of the observations in Table 2. indicate that the Poisson Test Analysis on M2 media (B5 + BAP 0.1) reveals a significantly greater effect on leaf number than other media.The addition of a high concentration of BAP is believed to inhibit leaf formation by reducing the number of leaves; the higher the concentration of BAP, the lower the number of leaves.The number of leaves is proportional to the number of tumas; the greater the number of shoots, the greater the number of leaves on the planlet.Similarly, cytokinin hormones affect the development of leaves in tissue cultures.According to Purwanto [19], the addition of cytokinins of the BAP group with a higher ratio of cytokinins to auxin can stimulate an increase in leaf count.4 of the Tukey Advanced Test Results reveals that M1 media (B5 Control) has the highest average value of sengon planlet height at 1.73 cm, while M4 media (B5 + BAP0.2) has the lowest average value of planlet height at 0.88 cm.This is possible due to the fact that the concentration of cytokinins used does not synergize with endogenous hormones found in sengon plants.Based on this research, it has been determined that the use of B5 basic media without ZPT is superior to the use of MS basic media because B5 basic media contains sufficient nutrients for plant growth, such as magnesium elements [14].Several previous studies, such as that of [20], have used B5 media without ZPT.According to their findings, stem lengthening is caused by the division, elongation, and enlargement of new cells in the stem end meristem, which results in the plant growing taller.In vitro plant growth and morphogenesis are governed by the interaction and balance of growth regulators in the explant, which determines the direction of culture development.The results of [21] it is stated that the growth of plant shoots, particularly high growth, is the result of the use of photosynthesis in plants, then in cells metabolic processes occur so that plant cells continue to develop and increase in buds, and that these activities can be stimulated by giving growth regulators to plants.Description: The numbers followed by the letters are not the same on that line the same shows a real difference

Percentage of live planlets and dead planlets
The seed coat of sengon explants began to crack during the first week of observation, which marked the beginning of the growth response.On fresh green to yellowish-green-colored media M1, M2, M3, M5, M6, M7, and M8, the percentage of living planlets reaches 100 percent.M4 media has the highest percentage of dead planlets, at 33%, while M9 media has the lowest, at 17%, due to browning, which causes cell death.Figure 6 depicts the percentage of living and nonliving planlets.According to Hartati [22], the death of the explant is caused by contamination, less-than-careful cutting of tissue that damages a portion of the explant, and the influence of heat from the tools used to cut or plant the explant, which can kill the cells.According to Khaniyah et al. [23], the possibility of cessation of the explant response is dependent on the concentration of ZPT used; if the concentration of ZPT is too low, it is incapable of inducing callus or budding, whereas if it is too high, it will be toxic to the explant, causing death in the explant.Planlets that perish as a result of the use of a hot instrument for cutting and relocating explants during multiplication are likely the result of this practice.

Percentage of contaminated planlets
Contamination is one of the limiting factors in plant propagation through tissue culture.Explants (both internal and external), small organisms that enter the medium (such as ants), less sterile culture bottles or tools, and less sterile work environments and culture rooms can cause contamination (spores in the air).This is due to the fact that colonies of bacteria frequently do not appear when they are cultured for the first time, but instead appear a few weeks later.The two types of contamination that frequently occur in plant tissue culture are contamination by bacteria and contamination by fungi.Physical characteristics present in explants and culture media can be used to distinguish contamination.Due to the fact that bacteria directly attack the plant's tissues, bacterial contamination can cause plants to become wet or secrete mucus.During contamination by fungi, the plant will become drier, and white-to-gray stripes (thread-like) will appear on the affected plant, indicating the presence of fungal hyphae.
Only on M4 (BAP 0.2), M5 (Kinetin 0.2), and M9 (BAP (0.2) + Kin (0.2) were planlets contaminated by bacteria to the extent of 17%, resulting in stunted planlet growth.At 8 MST, such bacterial contamination appears.Figure 5 depicts the percentage of sengon planlet contamination data.Nisa et al. state that fungi and bacteria cause contamination [4].The media and explants are encased in white cotton-shaped spores, while the explants contain yellow mucus, some of which are attached to the media in the form of wet clots, indicating bacterial contamination.According to Hartati [22], contamination is a limiting factor in the success of tissue culture.Contamination can be caused by (1) external and internal plant material, (2) small organisms that enter the media, (3) less sterile culture bottles and equipment, (4) the work environment and culture room, and (5) carelessness in implementation.Contamination of media and explants is caused by the presence of fungi or bacteria that survive media sterilization or enter the media during planting or maintenance.

Conclusion
According to the findings of this study, M2 media is the most effective medium against the zincon multiplication stage when supplemented with ZPT, cytokinin, BAP, and kinetin.M2 media has the greatest impact on all variables when compared to other media.Except for M4 and M9 media, which are characterized by browning and cell death, planlets are able to survive on treatment media.
Information: Yij= Response or observation value of the I-th treatment and the j-th replay; μ= Common middle value; αi= Effect of i-th treatment; ԑij= Effect of trial error from I-th treatment and j-th replay; i= Treatment (M1, M2, M3, M4, M5, M6, M7, M8 and M9); j= Deuteronomy (1, 2, 3, 4, 5 and 6).R Statistics software (R Development Core team, 2013) and Microsoft Excel were used to analyze the data (Microsoft Corporation).Using Poisson regression, the number of branches was analyzed as data.The Poisson Regression Model is a Generalized Linear Model (GLM) whose response data is assumed to be Poisson distributed using the following formula: ỳi = µi+ɛi = ti exp (xiβ) + ɛi

Figure 4 .
Figure 4. Percentage Diagram of Life and Death Planlets on Each Treatment of Sengon Growing Media (Paraserianthes falcataria (L.) Nielsen).

Figure 5 .
Figure 5. Diagram of the percentage of contaminated planlets on each treatment of sengon growing medium (Paraserianthes falcataria (L.) Nielsen).
Figure 6 depicts planlets subjected to bacterial contamination.

Table 2
below displays the outcomes of the Poisson test analysis.

Table 2 .
Results of poisson test analysis of the effect of media treatment on the number of leaves on sengon planlets (Paraserianthes falcataria (L.) Nielsen).