Ex vitro shoot induction of rodent tuber (Typhonium flagelliforme) with benzyl amino purine and naphthalene acetic acid

A medicinal plant called rodent tuber has the potential to both prevent and treat various diseases. There is no Standard Operating Procedure (SOP) governing the growth method of rodent tuber because it still grows wild and is rarely cultivated. This research aimed to study the optimal growth of rodent tuber for mass seed propagation based on the type of explants and the concentration of auxin and cytokinin combinations for inducing shoots. While generative parameters (harvesting of tubers/shoots) were observed up to 13 WAP (Week After Planting), vegetative parameters were observed up to 10 WAP. The results showed that the best type of explants for shoot induction and growth of rodent tuber were whole plants with a survival rate of 100%; the number of shoots was 6.65; the plant height was 21.27 cm with 6.34 leaves; 26.56 roots with a length of 16.05 cm; and 40.80% flowering plants. PGR (Plant Growth Regulator) combination of 50 ppm BAP (Benzyl Amino Purine) and 100 ppm NAA (Naphthalene Acetic Acid) became the best explant soaking solution by forming eight shoots and the height of plants was 24.13 cm with seven leaves. The explant produced the highest root length in a combination of 100 ppm BAP and 50 ppm NAA, which was 17.70 cm. The interaction between whole plant explants immersed in 0 ppm BAP and 100 ppm NAA solution resulted in the most significant number of roots (41). Those goals can support the SOP of propagated methods and ensure the consistency, quality, and availability of raw materials for traditional medicine.


Introduction
Rodent tuber (Typhonium flagelliforme) is an antiviral and antibacterial Indonesian medicinal plant.This plant has the ability to destroy or reduce the growth of cancer cells and decrease the side effects of contemporary medication, including the chemotherapy process in cancer patients, including hair loss, loss of appetite, nausea, and physical discomfort.Additionally, it can be used to treat conditions like scabs, ulcers, yaws, and drug poisons [1].Rodent tuber contains anti-cancer [2] and anti-leukemia [3] content.This plant contains biologically active compounds such as alkaloids, saponins, steroids, and glycosides [4].
In line with the government's policy to encourage innovation in the production of traditional medicinal raw materials, rodent tuber has a variety of potential to be cultivated as herbal medicinal plants.[5] The development of BBOT has the objective of increasing domestic production of traditional medicinal raw materials and decrease imports of high-quality, guaranteed imports.The traditional medicine sector and herbal medicines industry are still dealing with several significant challenges.One of the major issues is the quality, quantity, and consistency of raw materials for traditional medicines, both in the form of simplicial raw materials and extracted raw materials.The limited supply of plant materials and the mainly still-traditional processing methods are issues with simplicial raw resources.As a result, the Standard Operational Procedure (SOP) for cultivation is crucial.However, because rodent tubers are rarely cultivated, the SOP for their cultivation has yet to be discovered.
IOP Publishing doi:10.1088/1755-1315/1255/1/012018 2 Numerous factors that will impact plant productivity, in general, are listed in the SOP for the cultivation of medicinal plants.These variables include site selection and determination, seed supply, including seed selection and seeding, planting, fertilizing, treatment, pest and disease management, harvesting, and post-harvest.To guarantee stability and predictability of cultivation outcomes and to achieve uniform growth, the provision of seeds is a crucial aspect that strives to supply high-quality seeds [6].The success rate of growing medicinal plants is influenced by seed quality by about 40% [7].
Ex vitro technology is one of the ways to propagate plants.This method of vegetative propagation replicates all stages of in vitro culture in the lab, but it is simpler and cheaper to carry out outside the tube.The ex vitro technique starts with the selection of shoots, sterilization of the damaged parts of the shoots, roots induction through growth regulators immersion, planting in media containing organic matter, incubating in a controlled environment, and treatment through daily watering [8].
The most crucial ex vitro plant propagation phase is shoot induction or multiplication.According to several studies, in vitro shoot multiplication is affected by basic medium [9], type of Plant Growth Regulator (PGR) [10], combination and concentration of PGR cytokinin and auxin[11], explant size [12], type of explant [13], and interactions between PGR (cytokinin and auxin ratio) [14].
PGR plants are crucial for regulating biological functions in plant tissue.PGR controls how quickly each tissue grows and combines these components to form complete plants.PGR activity in growth is affected by the plant's type, chemical structure, concentration, genotype, and physiological stage [15].Auxin and cytokinin are two types of growth regulators.Auxin groups such as 2,4-Dichlorophenoxyacetic acid (2.4-D) are commonly used for callus production, whereas Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA), and Naphthalene Acetic Acid (NAA) are widely used for root formation.For the development of shoots, cytokinin groups such as Benzyl Adenine (BA) and kinetin are utilized [16].The cytokinin family also includes Benzyl Amino Purine (BAP), which promotes cell differentiation in meristems and the development of side shoots, dominance, and apical expansion of leaves [17].Explants are the first planting material utilized in micropropagation [18].Explants from plant organs can include roots, tubers, rhizomes, internodes, nodes, shoots, buds, seeds, and others.
The explants used in this study were whole plants and tubers.The explants were immersed in a solution containing PGR cytokinin Benzyl Amino Purine (BAP) and auxin Naphthalene Acetic Acid (NAA).This type of PGR was chosen since it is widely available and reasonably inexpensive compared to other types of auxin and cytokinin.The goal of this study was to obtain information about the types of explants, auxin and cytokinin concentration combinations, or their interactions, that led to the best shoot induction response and growth in rodent tubers so that it could be used to create SOP for their cultivation, particularly the provision of seeds that can support quality, quantity, and continuity of traditional medicine's raw resources.

Date and location of the study
This study was conducted for 13 weeks, from June to October 2020, in South Tangerang City, Banten Province, at the Screen House of the BRIN Biotechnology Laboratory Building 630 BJ.Habibie Serpong Science and Technology Area.

Materials and tools
Explants of rodent tubers Matesih accession in the form of tubers and whole plants collection of the Biotechnology Laboratory Deputy for Research and Innovation Infrastructure, PGR BAP (Benzyl Amino Purine) and NAA (Naphthalene Acetic Acid), fungicides and bactericidal, B1 freshener solutions, fertilizers liquid, mixed soil media: compost: husk charcoal (2:1:1), polybags 12 x 15 cm and 20 x 20 cm, soaking containers, and measuring cups were used as materials and tools.Facilities like a shade house with a plastic incubator are being utilized.

Experimental design
A completely randomized design (CRD) with two factors was used in this study.The first factor is the explant type (E), which has two levels: tubers (E1) and whole plants (E2).The second factor was a combination of BAP and NAA solution (P) with 25 levels (P1-P25) (Table 1), resulting in 50 treatment combinations (Table 2).Each treatment had five replications to obtain 250 experimental units.

Treatment method
x Explant preparation and immersion in PGR Two types of explants, namely tubers and whole plants, were used in this study.Tuber explants are obtained by cutting the main circular tuber.Whole plant explants are obtained by selecting healthy plants with complete parts such as roots, tubers, stems, and leaves.Additionally, 2 gL -1 of fungicide and bactericide were applied to tuber explants and whole plants for 10 minutes, followed by immersion in B1 refreshing solution and draining.Each explant was then immersed for 10 minutes in the prepared treatment solution.
x Planting, incubation, and acclimatization Explants were planted in 12 x 15 cm polybags made up of a 2:1:1 ratio of soil, compost, and husk charcoal.Labeled polybags are arranged according to the treatment plan and then incubated in a plastic incubator until the plants grow perfectly.During incubation, the 4 plastic is gradually opened.Acclimatization was accomplished at 10 WAP by relocating the plants into a single shading Screen House.

Observation and measurements
At the ages of 1, 3, 5, 7, 10, and 13 Weeks After Planting (WAP), observations and measurements were carried out six times during 13 weeks.Vegetative parameters were measured up to 10 WAP, while generative parameters (harvesting tubers/shoots) were measured up to 13 WAP.The following parameters were measured: live percentage (%) (13 WAP); the number of shoots by counting the number of shoots that either appeared as shoots that had leaves or prospective shoots in the form of small globes with plumula (13 WAP); shoot height (cm) by calculating the average of shoot height that appears for each treatment (10 WAP); the number of shoot leaves by calculating the average number of shoot leaves that occur in each treatment (10 WAP); the plant height (cm) by calculating the average increase in mother plant height (10 WAP); the number of leaves by calculating the average increase in mother plant leaf (10 WAP); the number of roots by calculating the average number of roots of each treatment without counting the root branches (13 WAP), root length (cm) by calculating the average of the longest roots in each treatment (13 WAP); and the percentage of flowering (%) by calculating the average of explants that had flowering (13 WAP).

Analysis of data
Observational data for each parameter were evaluated using Analysis of Variance (ANOVA).If the findings obtained were significantly different, the analysis will be followed by the Duncan Multiple Range Test (DMRT) at the test level α = 0.05.The IBM SPSS Statistics 26 application was used to perform the statistical analysis.

3.1.The effect of explant types, BAP and NAA combinations, and their interactions on the measured parameters of rodent tuber shoot induction
Table 3 summarizes the findings of the analysis of the several methods for immersing two types of explants in BAP and NAA combination solutions and their interactions on the growth of rodent tuber.The factor of explant types significantly affected the survival and flowering %, the number of shoots 13 WAP, the height and number of leaves of the mother plant, and the number and length of roots.The PGR treatment significantly changed the parameters of the plant height, number of leaves, root length, and number of shoots.The interaction between the two treatments greatly affected the number of roots parameter.The parameters of shoot number, height, and number of leaves during the vegetative phase did not affect the treatment of explant types, PGR combinations, and their interactions.
The explant type and the combination of BAP and NAA did not affect shoot induction during the vegetative phase (1-10 WAP).However, at 13 WAP, both treatments had a very substantial effect.This case might be because the treatment impacted specific plant ages.The other study about Phalaenopsis sp. was given BAP cytokinins, with the number of shoots having a substantial impact starting at 4 WAP [19].Another element that may affect it is that at the age of 10 WAP, a large number of potential shoots are there but cannot be counted since their plumula have not yet pierced the soil surface.Notes : ns = no significant; *= significant effect on α =5%, **= very significant effect on α =1% The explants used were tubers and whole plants (Figure 1).Rodent tuber shoots started sprouting at the age of three WAP.In tuber explants, the first shoot that grows as the mother plant is typically followed by the induction of other shoots from the tuber.In whole plant explants, shoots sprout from the mother plant's side from small globes, which are potential shoots (Figure 2).During shoot induction, whole plant explants generated a range of responses.Figure 3 shows the varied responses to shoot induction.The high response produced by whole plant explants could be caused by the fact that the source of whole plant explants was suspected to be stress-free due to cutting/injury, whereas tuber explants were cut/injured during planting, needing a longer growth time than whole plants.That case is shown by the tiny percentage of flowering plants at 13 WAP (15.2%), which shows that the percentage of plants entering the generative phase was much lower than whole plants (40.80%).One of the reasons that might lead to stress in plant tissue culture is cutting explants, along with other factors such as mother plant treatment, medium composition, atmosphere, and growth environment [20].

The effect of explant type on rodent tuber shoot induction
The size of the explants is related to the number of energy reserves contained in these explants; it is suspected that tuber explants have fewer energy reserves because the size of the explants is smaller; this is consistent with the findings of the other study about mangosteen that the initiation of shoots is influenced by the explant size; the smaller the explant, the longer it will take for shoots to initiate [21] and study about cassava that the length of stem cuttings that is too short indicates smaller energy reserves and a smaller number of shoots, so the time for shoots to sprout will be longer [22].Cutting materials with a high carbohydrate content and enough nitrogen will produce roots and shoots.Additionally, the available energy reserves will be adequate to carry out the process of cell division necessary to create new shoots [23].

The effect of PGR BAP and NAA combination on rodent tuber shoot induction
The results of Duncan's further test on the immersion treatment of PGR BAP and NAA combinations are displayed in Table 5.The best PGR combination treatment is P17 (50 ppm BAP and 100 ppm NAA), resulting in a growth response significantly different from other PGR combinations found in almost all test parameters.The treatment successfully produced eight shoots; the plant's height was 24.13 cm with seven leaves.In the treatment of 100 ppm NAA concentration followed by an increase of 25-100 ppm BAP, there was a tendency to increase growth in these three parameters along with an in BAP concentration to 50 ppm and then a tendency to decrease to 100 ppm BAP concentration (Figure 4).In the root length parameter, the PGR P23 combination treatment (100 ppm BAP and 50 ppm NAA) produced the best response (17.70 cm). Figure 5 illustrates how the plants performed.
The combination treatment PGR P17 (50 ppm BAP and 100 ppm NAA) resulted in a significantly higher plant response of 8.0 shoots.Still, it was not significantly different from P8 (0 ppm BAP and 75 ppm NAA) and P14 (50 ppm BAP x 25 ppm NAA) regarding the number of shoots.In addition, PGR P17 generated the tallest plant (24.13 cm) and the most leaves (7 leaves) but was not significantly different from P15 (50 ppm BAP and 50 ppm NAA) in the number of leaves.Treatments that showed a significantly lower growth response were P1 (0 ppm BAP and 0 ppm NAA), which resulted in 6.26 cm in the height of plant 10.00 cm in root length, and P10 (25 ppm BAP and 25 ppm NAA), which resulted in 2.2 shoots on the number of shoots and 2.9 leaves.
The study's findings showed that the interaction of cytokinin and auxin at specific concentrations significantly affected the rodent tuber shoot induction process.The interaction between cytokinins and auxins given to the media affects the number of shoots produced.Proliferation and shoot elongation might be increased by controlling the BAP and IBA concentration ratio to produce the optimal quantity and quality of shoots [24].The interaction of cytokinins and auxin may stimulate morphogenesis in the development of shoots [25].BAP also functions in cell division in meristematic tissues [17].The case is suspected of producing the best root length parameters by treatment with a higher cytokinin ratio than auxin (100 ppm BAP and 50 ppm NAA ) because the cells are more actively dividing roots.Notes: Numbers followed by the same letters in the same treatment and variables are not significantly different in the DMRT test on α = 5%, "-" means "up to", for example, a-d means abcd .
PGR are frequently used to control biological functions in plant tissues.During the shoot induction process, auxins and cytokinins are crucial.Study on the impact of PGR on rodent tuber mass multiplication, rodent tuber cultured on MS medium with 5 mgL -1 BAP and 1 mgL -1 NAA treatment produced the highest number of shoots (29.17 shoots) at 12 weeks after culture.The same treatment obtained the highest average wet weight of shoots [26].The results of another study indicated that MS medium with 0.5 mgL -1 BAP and 0.25 mgL -1 IBA was the optimum medium for in vitro shoot multiplication of rodent tuber (16 shoots in 6 weeks) [27].In the micropropagation of the medicinal plant Valeriana jatamansi Jones utilizing rhizome explants, it was discovered that adding BAP up to 1 -2 mgL -1 increased shoot length, number of leaves, and leaf growth when used in combination with NAA at 0-1 mgL -1 [28].
This is consistent with research on the P14 treatment (50 ppm BAP and 25 ppm NAA), which produced 8.10 shoots.However, they were not significantly different from the treatments P17 (50 ppm BAP and 100 ppm NAA), which had 8.0 shoots, and P8 (0 ppm BAP and 75 ppm NAA), which produced 8.4 shoots.The auxin ratio was higher than the cytokinin.This is probably because the explants already contain relatively high endogenous cytokinins.Therefore, the induction of shoots only requires the addition of 0-50 ppm of exogenous cytokinins.There is an interaction between exogenous growth regulators supplied to the medium and endogenous growth regulators produced by plant tissues during the formation of organs like shoots or roots [29].In a different experiment, rodent tuber grown in MS liquid medium with 3 mgL -1 BAP and 0.5 mgL -1 IBA produced the best number of shoots (4.4 shoots in 6 weeks) [30], and the same ratio between cytokinin and auxin was achieved with the maximum number of shoots obtained at the initiation stage of rodent tuber performed on MS medium with 0.5 mgL -1 BAP and 0.5 mgL -1 NAA (14 shoots) [31].

The interaction between explant type and PGR BAP and NAA combination on rodent tuber shoot induction
According to Table 6, the interaction between the types of whole plant explants immersed in a PGR 0 ppm BAP and 100 ppm NAA combination solution differed significantly from the other treatments, producing a higher number of roots (41 roots) than the other treatments (Figure 6).This is consistent with auxin's function in root development.Depending on the chemical composition, concentration, and plant tissue being treated, auxin can play various roles.By promoting cell elongation and division in the cambium tissue, auxin is often used to stimulate the production of callus, suspension cultures, and roots [32].The best root production of the rodent tuber was induced by MS0 medium containing 1.5 mgL -1 NAA, which resulted in 43.20 roots [33].

Figure 4 .
Figure 4. Responses to the number of shoots, plant height (cm), and number of leaves of rodent tuber (T.flagelliforme) in 100 ppm NAA treatment with an increase in the concentration of 25-100 ppm BAP

Figure 6 .
Performance of rodent tuber's roots with the treatment of (a) whole plants with PGR (0 ppm BAP and 0 ppm NAA), (b) whole plants with PGR (0 ppm BAP x 100 ppm NAA)4.ConclusionsThe best type of explants for rodent tuber's shoot induction and growth is whole plants with a 100% survival rate, a number of shoots of 6.65, a plant height of 21.27 cm with 6.34 leaves, 26.56 roots with a length of 16.05 cm, and 40.80% flowering plants.The best PGR combination treatment is 50 ppm BAP and 100 ppm NAA by forming 8.00 shoots, a plant height of 24.13 cm with seven leaves.The highest root length was produced by the PGR combination treatment of 100 ppm BAP and 50 ppm NAA which was 17.70 cm.The interaction between whole plant explants immersed in PGR combination treatment of 0 ppm BAP and 100 ppm NAA resulting the highest number of roots (41 roots).Further research suggestions can use the other prospective explants such as the long secondary tubers, adjusting auxin and cytokinin concentration ranges from 50 ppm to 150 ppm, and increasing treatment explant immersion time in PGR solution (hours).5.References[1] Katrin E, Novagusda F N, Susanto, Winarno H 2012 Characteristics and efficacy of irradiated rodent tuber (Thyponium flagelliforme (L.) Decne) leaves J. Sci.Isotop.Rad.App 8(1) 31-42 [2] Sianipar N F, Purnamaningsih R 2018 Enhancement of the contents of anticancer bioactive compounds in mutant clones of rodent tuber (Typhonium flagelliforme Lodd.) based on GC-MS analysis Pertanika J. Trop.Agric.Sci.41 (1) 305-320 [3] Mohan S, Abdul A B, Abdelwahab S I, AL-Zubairi A S, Sukari M A, Abdullah R, Taha M M E, Beng N K, Isa N M 2010 Typhonium flagelliforme inhibits the proliferation of murine leukeumia WEHI-3 cells in vitro and induces apoptosis in vivo J. Leukeumia research 34(11) 1483-1492 [4] Syahid S F 2007 Rodent tuber (Typhonium flagelliforme Lodd.) in vitro propagation J. Warta Puslitbangbun 13(3) 19-20 [5] Regulation of the Minister of Health of the Republic of Indonesia number 88 of 2013 concerning the master plan for the development of traditional medicine raw materials [6] Ministry of Agriculture Directorate General of Horticulture Directorate of Vegetables and Medicinal Plants 2019 Standard operational procedures for curcuma cultivation (Curcuma xanthorriza) in Sukabumi 4th Printing Ed Siregar I, Hartono B (Jakarta: Ministry of Agriculture) [7] Rahardjo M 2010 Application of cultivation SOP to support temulawak as a potential medicine raw material J Perspektif 9 (2) 78-93 [8] Karyanti, Sigit Y, Tajuddin T, Erwinda, Minaldi, Haska N 2016 Handling method of young suckers in ex vitro culture to produce vigor planlets of sago palm (Metroxylon sagu Rottb.)JBBI 3(1): 13-19 [9] Page S R G, Monthony A S, Jones A M P 2020 Basal media optimization for the micropropagation and callogenesis of Cannabis sativa L (USA : bioRxiv preprint) [10] Singh C K, Raj S R, Jaiswal P S, Patil V R, Punwar B S, Chavda J C, Subhash N 2016 Effect of plant growth regulators on in vitro plant regeneration of sandalwood (Santalum album L.) via organogenesis Agroforest Syst 90 281-288 [11] Kartiman R, Sukma D, Aisyah S I, Purwito A 2018 In vitro multiplication of black orchid

Table 1 .
The composition of the BAP and NAA combination solution used as an immersion solution for rodent tubers explants (T.flagelliforme)

Table 2 .
The combination treatment of rodent tuber shoots induction (T.flagelliforme) by immersing the explants in a combination of BAP and NAA

Table 3 .
Recapitulation of the effects of explant types, combinations of PGR BAP and NAA, and their interactions on the measurable parameters of rodent tuber shoot induction (T.flagelliforme)

Table 4 .
Table 4 displays the findings of Duncan's test on explant type parameters.The best response came from whole plant explants, which were significantly different from tuber explants for all observational parameters, including survival percentage of 100%, number of shoots of 6.65, plant height of 21.27 cm with 6.34 leaves, number of roots of 26.56 with a length of 16.05 cm, and 40% of the plants getting flowered.The results of the Duncan Multiple Range Test about the impact of explant types on rodent tuber shoot induction (T.flagelliforme) Notes: Numbers followed by the same letters in the same treatment and variables are not significantly different in the DMRT test on α = 5%

Table 5 .
The results of the Duncan Multiple Range Test about the impact of PGR BAP and NAA combination treatment on rodent tuber shoot induction (T.flagelliforme)

Table 6 .
The results of the Duncan Multiple Range Test about the interaction of explant type and PGR BAP and NAA combination on the parameter rodent tuber's number of roots (T.flagelliforme)