Pitcher formation of Nepenthes ampullaria and Nepenthes rafflesiana on modified in vitro media

Nepenthes, known as pitcher plants, is one of the unique ornamental plants that are highly sought after for their unique shape and color of the pitchers, a modification of the leaves. The pitcher was reportedly formed under nutrient-poor conditions at its growth site. This study aims to investigate the effects of modification of the culture media on the formation of in vitro pitchers of Nepenthes ampullaria and N. rafflesiana. The experimental design in this study used a completely randomized design with two factors (the species of Nepenthes and the media). Nepenthes ampullaria and N. rafflesiana plantlets with a stem height of about 1-2 cm were planted in medium containing half strength of Murashige and Skoog (1/2 MS), in medium containing sugar, agar, and distilled water (SAW), in medium containing agar and distilled water (AW) and medium containing sterile distilled water (W). All media were adjusted to a pH of about 5.7. The filter paper was used as a buffer in a liquid medium to support the plantlets. Each treatment was replicate three times, with each replicate containing ten bottles of culture, each containing one plantlet. Research results were observed for 12 weeks after planting and showed that all cultures could grow well in each medium. The highest average number of pitchers was recorded for the N. ampullaria culture planted in AW medium, with 22 pitchers/plant, followed by the culture in W medium, with 20 pitchers/plant. For N. rafflesiana, on the other hand, the highest number of pitchers was found in W medium with 18 pitchers/plant.


Introduction
Nepenthes are commonly known as tropical pitcher plants, Asian pitcher plants, or monkey cups in English or kantong semar in Indonesian [1].Nepenthes comprises nearly 160-180 species worldwide and is probably the most-studied pitcher plant [2][3][4].Nepenthes is characterized by one of the morphological features of plants that produce modified foliage that forms hollow, water-containing vessels suitable for capturing and digesting animal prey [5].In most species, the traps, modified leaf tips called pitchers, adapt to the scarcity of nutrient resources [5].The pitcher actively attracts prey through lures such as nectar, scent, and conspicuous coloration.Animals, such as small vertebrates or insects that fall into the plants' hollow, water-filled traps cannot escape and eventually drown or die of exhaustion or starvation.Slowly, their remains are digested by the enzymes of the pitcher and microbial processes release nutrients that are absorbed directly into the plant tissue via special glands [6].
The variety of shapes, sizes, and color patterns of their pitchers has attracted interest for centuries.However, 121 species of Nepenthes are listed on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species [7].Among them, five species are classified as endangered, and 19 are critically endangered.Most are in the lower-risk category, including N. ampullaria and N. rafflesiana.The most common threats responsible for the disappearance of carnivorous species progressively are wild collection, pollution, and habitat loss due to agricultural expansion [8].Recently, negative impacts of climate change on the genus Nepenthaceae have also been reported, particularly for upland species, which comprise twothirds of Nepenthes species [9][10].Many efforts have been made by institutes, botanical gardens, foundations, and even national parks to develop strategies for their protection and conservation [11].
The Research Center for Plant Conservation, Botanic Gardens, and Forestry in the National Research and Innovation Agency (BRIN) or Bogor Botanic Gardens (BBG), initiated a program for the conservation of Nepenthes species by introducing propagation using tissue culture techniques and proposed a series of in-house research actions.These actions emphasize the importance of ex-situ conservation through in vitro propagation to maintain genetic diversity [12][13][14][15][16].However, to raise awareness of Nepenthes conservation and embed social inspiration for environmental protection, the BBG is developing educational methods that integrate research findings on Nepenthes and make them accessible to all.
One of the Nepenthes objects of interest to the public is the pitcher in the in vitro culture vessel.The pitcher is formed as an adaptation to nutrient-poor habitats and is considered an additional way to obtain nutrients such as nitrogen and phosphorus [17].In vitro propagation of Nepenthes has been developed by some researchers [13,18,19,20,21].Their studies revealed that the highest shoot multiplication was recorded in half-strength Murashige and Skoog (MS) media or Woody Plant Media (WPM) supplemented with growth hormone-like kinetin and BAP.
Different nutrient applications affected growth and root fertilizer treatment resulted in more biomass in leaves than in pitchers [22].A half-strength MS media induced the highest number of leaves and pitchers of N. rafflesiana.Nevertheless, the pitchers were more significant on a media with more nutrient deficiency (1/4 MS and 1/8 MS) [23].Another research reported that the number of pitchers formed was lower in N. mirabilis than in N. ampullaria and that the best media for forming pitchers was the 1/16 MS media [24].This study aims to determine the effects of modified media on in vitro pitcher formation of N. ampullaria and N. rafflesiana.

Materials and methods
The studies were conducted at the Research Center for Plant Conservation, Botanic Gardens, and Forestry or Bogor Botanic Gardens (BBG), Bogor, West Java, Indonesia.The experimental design in this study used a completely randomized design with two factors.The first factor is the species of Nepenthes, i.e.N. ampullaria and N. rafflesiana.The second factor is the type of media, i.e. halfstrength Murashige and Skoog (1/2 MS), sugar + agar + distilled water (SAW), agar + distilled water (AW), and sterile distilled water (W).
The plantlets of Nepenthes ampullaria and N. rafflesiana were obtained from the tissue culture laboratory collection of the BBG.Plantlets with a stem height of about 1-2 cm were planted in half-strength Murashige and Skoog medium (1/2 MS), sugar + agar + distilled water (SAW) medium, agar + distilled water (AW) medium and sterile distilled water (W) medium.All media were adjusted to a pH of about 5.7.The filter paper was used as a buffer to support the plantlets planted in a liquid medium.Each treatment was replicate three times, each replicate containing ten bottles of culture and one plantlet per bottle.All cultures were maintained at 25 ± 20°C under a 16-hour photoperiod with an 800-1000 lux TL lamp.
The percentage of explants producing pitchers and the number of leaves, pitchers, and roots per explant were recorded after 12 weeks of culture.The color of the leaves and pitchers was recorded and compared with the Royal Horticultural Society Color Charts.The phase of pitcher formation was observed according to the Schwallier classification [5] from 0-12 weeks after treatment (Figure 1).Quantitative data were analyzed using Microsoft Excel and the SAS program.Analysis of Variance (ANOVA) results showing differences were further analyzed using the Duncan Multiple Range Test (DMRT) at the α = 5% level.

Results and discussion
The observations carried out 12 weeks after treatment showed that all N. ampullaria and N. rafflesiana cultures could grow well and formed pitchers in each media used.The media sugar + agar + distilled water (SAW), agar + distilled water (AW), and sterile distilled water (W) have a good effect on the number of leaves, roots, pitchers, and pitcher phases.The results of the statistical test show that all experimental parameters do not differ in the media used but the Nepenthes species show a significant difference in the number of pitchers and leaves (Tables 1  and 2).Several studies were conducted to induce pitchers in Nepenthes.The study results showed that N. gracilis could form pitchers on ½ MS and ¼ MS media [25].Meanwhile, a preliminary study showed that the media concentration of 1/8 MS had the best effect on the number of pitchers, number of roots, percentage of live plantlets, percentage of plantlet's pitchers, and percentage of rooted plantlets in N. rafflesiana [26].Another report on research results on the formation of pitchers in N. mirabilis and N. ampullaria, which are better on ¼ MS and ½ MS media [24].Another study showed that the highest number of pitchers was found when N. ampullaria was cultured on an MS media without nitrogen [27].
Based on the ANOVA result, the Nepenthes species showed a significant difference in the number of pitchers and leaves.This result suggests that there is a correlation between leaf and pitcher formation.In well-studied research, protein expression of N. gracilis was reported at three stages of pitcher development: young leaves, leaf with a developing pitcher, and developed pitcher.It was found that the composition of the protein profile was similar in the three pitcher development stages.However, the protein content in the developed pitchers was much lower than in the other stages.This study showed that pitcher formation might be correlated with the gene expressed in the leaf.Thus, this study suggests that pitcher formation is part of leaf development [28].
The research result showed that the differences in the number of pitchers and leaves of the two Nepenthes species (N.ampullaria and N. rafflesiana) planted in the same nutrient media were probably due to the different characteristics of the pitchers and the different nutrient requirements.The two species require different conditions for pitcher formation and a favorable growing environment.The predominant color of N. ampullaria was green, and there were no visible nectar secretions.In contrast, N. rafflesiana produces nectar secretions and a sweet smell in red-colored pitchers [29].Therefore, it is assumed that the differences in pitcher characteristics, nutrient requirements, and optimal growth environment in the two species lead to significant differences in the same media treatment.
The pitchers inflated on agar+distilled water medium (AW) and sterile distilled water medium (W) showed the highest number of pitcher formations (Table 1).Both species have the highest number of pitchers on media containing only sterile distilled water (W) or a combination of agar and distilled water (AW) compared to ½ MS, which is an exciting result.Nepenthes are found in extremely nutrient-poor habitats such as heath forests (kerangas), peat swamp forests, and montane forests.In order to survive, the species forms a jug-shaped pitcher that has the function of attracting, trapping, holding, and digesting nutrients from animal prey.Thus, the result of this study shows that it corresponds to what occurs in the natural habitat and can be compared to what is cultured under in vitro conditions [30].
In some compositions of sugar + agar + distilled water (SAW), there is a brown coloration of the media (Figure 2) due to a phenolic compound.The leaves of N. ampullaria are primarily light green and belong to green group 143 A. Some leaves of N. ampullaria are yellowish-green or belong to the yellow-green group N144C.In contrast, the average of the dark green leaves of N. rafflesiana corresponds to the green group 141 A on the color chart.
The phenolic compound in plant tissue can inhibit plant growth because it controls the transport of phytohormones such as auxin.The phenolic compound controls the auxin concentration gradient by preventing polar transport and local auxin transport by inhibiting IAA oxidase.Due to this regulation, the phenolic compound can retard growth and reduce the growth rate of plants [31].The development of the pitchers of N. ampullaria starts with curvation (A), elongation (B), inflation (C) and ends with maturity (D) only on sterile distilled water (W), while the pitchers of N. rafflesiana grew well on all media except ½ MS media (Table 3).The result showed that all the plants had uniform pitcher color in each treatment, namely light green in the newly formed pitcher until the elongation stage and green in the maturation stage (Figure 3).Some pitchers could no longer develop in the two Nepenthes species, so they wilted and were grey-orange.There are four primary phases in the growth and development process of Nepenthes pitchers, namely A (curvation phase), B (elongation phase), C (inflation phase), and D (maturation phase) [5].The more deficient the nutrient content of the media, the more and larger the pitchers are likely to form [6]. Large pitchers formed on nutrient-poor media (1/16 MS) compared to the other nutrient-rich media [6,24].
The upper pitcher of Nepenthes rafflesiana was classified as type 2 (narrow-funnel), while N. ampullaria fell into the type 11 (pitfall) category [31].The fundamental differences between N. rafflesiana and N. ampullaria are the pitcher's target.The upper pitcher of N. rafflesiana catches more flying insects than the lower pitcher.This is due to the presence of a viscous liquid.The upper pitcher has a viscous fluid, but the lower one does not.In addition, the upper pitcher of N. rafflesiana catches more arthropods and has a broader range of prey than the lower pitcher [29,32,33].
In contrast, the target of food fulfillment for N. ampullaria is not an insect but leaf litter from the tree canopy.This is because the pitcher does not have an attractive color.In addition, scent and nectar production reduce efficiency in catching prey [32,34,35].
Generalist carnivorous species have a large pitcher opening, an acidic pitcher fluid, and a sweet odor.Ant capture increases with the liquid's acidity, extrafloral nectar, and slippery waxy walls.The number of termites caught increases with the narrowness of the pitchers, the symbiotic association with ants, and the presence of a rim with edible trichomes.The number of flying insects correlates mainly with the diameter of the pitcher opening and the presence of odors [29].

Conclusions
The Nepenthes species differed markedly in the number of leaves and pitchers.The highest number of pitchers in N. ampullaria was produced on a media containing agar and distilled water (AW).In contrast, on a media containing sterile distilled water (W), the highest number of pitchers was produced in N. rafflesiana.

Table 1 .
Analysis of Variance (ANOVA) result per treatments on observed parameters Treatments

Number of pitchers Number of leaves Number of roots
Note: ns = no significant differences; *= significantly different at the 5% level; the numbers following the same letter in each column are not significantly different at the 5% level using the DMRT test; ½ MS: half-strength Murashige and Skoog, SAW: Sugar + Agar + distilled Water, AW: Agar + distilled Water, and W: sterile distilled Water; CV= coefficient of variation.

Table 2 .
The effect of modified in vitro media on the Nepenthes characters Note: The numbers following the same letter in each column are not significantly different at the 5% level using the DMRT test.

Table 3 .
Development phase of the pitchers in each treatment media