Paphiopedilum is one of the Orchidaceae family that faces many threats to their natural habitat, including land degradation for plantation and housing, over-exploitation by society, and land fire. About 23 species of the genus included in The International Union for Conservation of Nature (IUCN) Redlist with status endangered (EN) to Critically Endangered (CR). The use of Paphiopedilum as a popular ornamental plant had threatened its existence in the natural habitat. Meanwhile, the propagation of this genus to support the orchid industry still faces problems and needs more attention, such as the continuity of the explants and the propagation methods.

Paphiopedilum propagation can be done in vitro through seeds (Yao et al. 2021), but it isn’t easy to obtain a viable seed. Paphiopedilum has unique and distinct flowers from other genera. Some species, including P. parishii and P. glaucophyllum have liquid pollen or anthers and unclear stigma cavity. The liquid anthers flow over the stigma to bring the pollination process (Madan et al. 2013) (Fig. 1B). These conditions make them challenging to pollinate and indirectly affect the difficulty in producing fruit.

Figure 1. (a) Flower of Paphiopedilum glaucophyllum. (b) Anther structural position (blue arrow), liquified anther reaching the stigma (black arrow).

Another problem is the small percentage of seed germi-nation of most Paphiopedilum species (Lin et al. 2000). The research that was carried out by Chen et al. (2004) and Ding et al. (2004) showed the germination percentage of P. armeniacum S.C. Chen and F.Y. Liu seeds from the fruit, harvested 120 days after pollination, were only 25.2% and 18.4% on Robert Ernst’s medium. The seeds also cannot be stored for a long period (more than one year). In addition, Handini et al. (2016) stated that the seed storage period of P. supardii Braem and Löb was only about one year, after that the seeds were not viable for germination. Furthermore, the results of a review by Zeng et al. (2015) showed that propagation of Paphiopedilum through tissue culture is generally difficult to execute, including vegetative pro-pagation with in vitro techniques using plant tissue parts.

The reports about propagation of Indonesian Paphio-pedilum species is very limited. Research by Azmi and Wiendi (2013) was studied on P. glaucophyllum J.J. Sm. through the proliferation of adventitious shoots in vitro by using plant growth regulators (PGR) 6-Benzylaminopurine (BAP) and 2,4-Dichlorophenoxyacetic acid (2,4-D). More-over, there are no tissue culture research results have led to the mass production of seedlings for their development. The experiments have only reached the stages of shoot multiplication, enlargement, and acclimatization, with no optimal results.

Mass propagation by callus culture to form adventitious shoots is still rare for Paphiopedilum. However, the use of PGR such as 2,4-D and Thidiazuron (TDZ) have been conducted in orchid species to form callus. The study on Papilionanthe hookeriana Rchb.f, or known as pencil orchid, gave the results that 2,4-D with doses of 0.25-1 mg/L can accelerate the growth and multiplication of explants and could maintain the green colour of the explants and produce transparent green friable callus (Romeida et al. 2016). Meanwhile, Da Silva (2012) stated that culture media with low macroelements with TDZ could stimulate callus formation in Cymbidium hybrid orchids. Other research on Dendrobium lineale showed that the addition of 2,4-D (1 and 5 mg/L) and TDZ (1 mg/L) was the best combination of callus initiation (Hoesen et al. 2008).

The limited number of explants is also one of the obstacles in Paphiopedilum in vitro culture propagation. The study by Chen et al. (2002) used stem internode explants for this purpose. Based on this, the needs of Paphiopedilum explants can be fulfilled by multiplying explants with the stem elongating technique. This technique has been used by Nhut et al. (2007) on P. delenatii Guillaumin by using Murashige and Skoog (MS) medium supplemented with 1.5 mg/L TDZ. Whereas the research of Luan et al. (2015) showed that the medium suitable for elongation treatment was Schenk & Hildebraant (SH) medium added with 5-1 mg/L BAP and 0.5-1 mg/L NAA by using the same species with red and blue LED lighting.

Other research states that alternating dark and bright light modification will stimulate shoots or stems elongation in P. callosum cultures (Huy et al. 2019). Elongation treatment stored the culture in a dark room for 14 dark days and one light day and repeated alternately for four months of observation. This condition will spur etiolation due to a lack of light and is advantageous for obtaining explant candidates in the form of long young stems. This elon-gation method was carried out to maintain the pigments and normal photosynthesis process.

This study aims to determine the effect of media and plant growth regulators on seed germination and callus induction with shoot tip as an explant, and also using the planlet from elongation treatment result. Through this research, an in vitro callus induction protocol is expected to be obtained on Paphiopedilum glaucophyllum using shoot tip as one of the suitable explants source.

Plant material and seed germination

The explants used in this research were seeds of P. glaucophyllum from mature fruit (five months after pollination). The capsule fruit was sterilized by cleaning the fruit with dish washing soap, then rinse in running water. The next sterilization process was located in Laminar Air Flow. The fruit was dipped in alcohol 96%, then briefly burned with a bunsen flame for about 30 seconds, and repeated thrice. Furthermore, the capsule fruit was cut in the vertical direction, and the seeds were sown on the ger-mination medium. The germination medium used Knudson C (KC) (Knudson 1946) modified medium with the addition of organic matter coconut water 150 ml/L, and bean sprout extract 150 g/L with three concentrations of NAA (0; 5; 10 mg/L). The media then were completed with sugar 20 g/L, 8 g/L agar, and active charcoal 1 g/L. The seeds of this orchid will germinate two months after planting (MAP). The germination percentage was counted and the protocorms then grown into plantlets. Those plan-lets were ready to be used for elongation treatment and callus formation.

Elongation treatment

The plantlets of P. glaucophyllum for this study were derived from the seed germination in July 2006, were given elongation treatment, both in the dark and with light. The plantlets used were 5.0 cm in height with 4-5 leaves. They were planted in 4 types of modification media with Murashige and Skoog (MS) medium as basic medium (Table 1). All media types were added with sugar 20 g/L and 7 g/L of agar. The culture bottles were then placed in a dark room for 13 days and one day in light alternately for four months for the plantlets elongation process. Obser-vations were made monthly with parameters of the increase in plant height and the number of leaves.

Table 1 . Type media used for elongation treatment.

Type of medium	Content
½P medium	Half-strength of MS medium with addition of banana extract 100 g/L
(½P5 medium	Half-strength of MS medium with banana extract 100 g/L and NAA 5 mg/L
MP medium	Full-strength of MS medium with banana extract 100 g/L
MP5 medium	Full-strength of MS medium with banana extract 100 g/L and NAA 5 mg/L

Callus induction

Callus induction study used two methods with two types of explants. The first was direct planting into callus media using plantlets from in vitro culture of P. glaucophyllum with 2.5 cm in height. It used the shoot tip (first node) with a length ± 0.5 cm and removed the leaves and the roots (Fig. 2). The explants were planted in the media for callus induction, Schenk and Hildebrant (SH) medium as the basic medium (Schenk and Hildebrant 1972) with the addition of 2,4-D 1 mg/L and TDZ with five concentrations (0; 0.5; 1; 1.5; and 2 mg/L). All media were solidified with the addition of gellan gum 3 g/L. The cultures were placed in the dark (without light) and light conditions (with LED lighting for 16 hours/day). The observation was conducted monthly to obtain data on living and the number of explants that produce callus.

Figure 2. The used part of Paphiopedilum glaucophyllum plantlet for direct callus induction (yellow circle).

The second method was using the plantlets resulting from the elongation technique. Similar to the first method, this study used the first node as an explant for callus induction. According to Udomdee et al. (2012), this part was excised horizontally and vertically. The explants then were planted in callus induction media, which consisted of half concentration of macro and microelements of MS (½ MS) medium, full concentration of MS vitamin, PVP 250 mg/L, peptone 2 g/L, gellan gum 3 g/L, sugar 20 g/L, and NaH₂PO₄ 170 mg/L (Wattanawikkit et al. 2011), with the addition of TDZ as the first factor with five concentrations (0; 0.5; 1; 1.5; and 2 mg/L) and 2,4-D as a second factor with four concentrations (0; 1; 5; and 10 mg/L) (Table 1). Cultures were placed in the dark at room temperature <25℃. Observations were carried out monthly for five months after planting by observing the number of living explants, the explants that produce callus, and grew shoots.

The calluses formed in the study were subcultured in the half-strength MS medium as the basic medium containing 2,4-D 1 mg/L and three concentrations of TDZ (0; 1; 1.5 mg/L). The development of those calluses was observed three MAP.

Data analysis

The research was designed in a completely randomized design with three repetitions for the germination study, fifteen repetitions for elongation study, and 19 repetitions for callus induction for each treatment. All data were analyzed by using IBM SPSS Statistics 26 software. If there were significant differences between treatments in the analysis of variance, a further test was performed using the Duncan test with a significance level of 0.05.

Seed germination

Based on the statistical result, there was no difference in germination percentage between the control and auxin (NAA) applications as PGR. Still, the media with 5 mg/L NAA gave the highest germination percentage (9.3%) (Table 2). The seed germination time for all media was similar (two months after planting), but there was a dif-ference in growth speed. The media with no auxin (control) gave slower growth, and the growth increased in line with the addition of NAA (Fig. 3). Germinated embryos are globular in shape and have started to form cotyledon. In this study, various protocorm phases formed in 60 DAP by using KC media supplemented with bean sprout extract or micronutrients modified medium (Fig. 3).

Table 2 . The effect of NAA on the germination percentage of the seeds of Paphiopedilum glaucophyllum.

Media type with NAA	Germination percentage (%)
KC (control)	7.39±2.86
KC with 5 mg/L NAA	9.3±1.53
KC with 10 mg/L NAA	8.22±2.45

Figure 3. The development of the germinated seed of Paphiopedilum glaucophyllum on KC (control) (a), KC with 5 mg/L NAA (b), and KC with 10 mg/L NAA (c) media 2 months after planting. Swollen seed (red arrow), globular shape of the germinated seed (black arrow), and embryo in the cotyledon phase (yellow arrow).

Elongation treatment

The results showed that the increase in plant height for all types of media was not significantly different sta-tistically, but 1/2P medium has the highest value. The increase in the number of leaves and internode ratio was statistically different (Fig. 4). Explants in the MP medium (full-strength of MS medium with banana extract 100 g/L) had the most leaves (1.24 ± 0.14). Specifically, the best media for the elongation process in this study is the media that can trigger the highest internode ratio, which is the ratio between explant height and the number of leaves. The results showed that the highest internode ratio was found in MP5 medium (modification of full strength of MS with banana extract 100 g/L and NAA 5 mg/L) (1.73 ± 0.26) and was significantly different from MP media (Fig. 4).

Figure 4. The elongation treatment result on planlets of Paphiopedilum glaucophyllum 5 MAP.

The in vitro elongation process of P. glaucophyllum plantlets was carried out by changing the lighting technique for two weeks without light and continued for one day with light, alternately between dark and light up to 7 times of changing lighting conditions. The elongation condition was signed by the lengthening of the internode part with whitish colour of the stem as the effect of etiolation process (Fig. 5, yellow arrow).

Figure 5. Elongation treatment results on planlets of Paphiopedilum glaucophyllum 5 MAP. The lengthening of internode in each medium (yellow arrow).

Callus induction

All methods for callus induction in this study used the first node from the upper part of the explant because of the meristem cells contained in that part. In the first method, the addition of TDZ and storing the culture bottles in dark and light affected explant viability. Table 3 shows that the number of shoot tip explants that survived for five months was more when exposed to the light. In this condition, even the media with no TDZ, the planlets were still survive and grow with 36.84% of living explants. Explants grown on media with TDZ concentrations of 0.5, 1, and 1.5 mg/L gave almost a similar effect in dark or light conditions. The percentage of viable explants ranged from 15.79% to 42.11%. But when the TDZ concentration was added up to 2 mg/L, the effect of light reduced the percentage of living explants to 26.32%, while in conditions without lighting (dark condition), the rate of living explants was 47.37% (Table 3).

Table 3 . Number of living explants and explants that were growing callus from the first node of Paphiopedilum glaucophyllum with addition of TDZ in light and dark conditions five MAP.

SH media with TDZ (mg/L)	Percentage of living explants (%)			Percentage of callus explants (%)
	Dark	Light		Dark	Light
0	0	36.84		0.00	0.00
0.5	42.11	42.11		15.79	15.79
1	15.79	21.05		15.79	0.00
1.5	36.84	36.84		15.79	0.00
2	47.37	26.32		10.53	10.53
Total				57.89	26.32

The basic SH medium used was a combination of single concentration of 2,4-D (1 mg/L) and TDZ with various concentrations (0; 0.5; 1; 1.5, and 2 mg/L), and in condition without lighting, those media seems to produce more callus.

The second method was using explants from the elon-gation treatment. The result showed that the most optimal medium for callus induction in 5 MAP on P. glauco-phyllum was T1.5D1 medium (TDZ 1.5 mg/L and 2,4-D 1 mg/L) with a percentage of explants with callus 33.33% from total number of used explants. Meanwhile, the medium of TDZ 1.5 and 2 mg/L without 2,4-D had no callus formation (Table 4; Fig. 6).

Table 4 . Callus induction result of Paphiopedilum glaucophyllum on ½MS media with the addition of TDZ dan 2,4-D in 5 MAP.

Media treatment	Callous explant (%)	Budding explant (%)	Living explant (%)	Number of callus/explant	Number of shoot/explant
T0D0	4.76	66.67	71.43	0.20	6.80
T1D0	13.33	66.67	100.00	0.20	1.87
T1,5D0	0.00	43.48	56.52	0.00	4.15
T2D0	0.00	50.00	57.69	0.00	6.47
T0D1	26.32	15.79	73.68	0.79	0.29
T1D1	0.00	8.33	41.67	0.00	0.20
T1,5D1	33.33	33.33	50.00	1.33	1.17
T2D1	0.00	0.00	33.33	0.00	0.00
T0D5	9.09	4.55	22.73	0.40	0.20
T1D5	8.33	0.00	16.67	2.00	0.00
T1,5D5	26.32	21.05	26.32	2.20	0.80
T2D5	13.33	6.67	33.33	1.20	1.00
T0D10	21.05	15.79	26.32	2.20	1.00
T1D10	27.27	0.00	36.36	1.00	0.00
T1,5D10	9.09	0.00	18.18	0.50	0.50
T2D10	6.25	6.25	25.00	0.25	0.25

Figure 6. Condition of Paphiopedilum glaucophyllum explants in ½MS medium with the addition of 2,4-D and TDZ in 4 MAP.

The medium without any PGR (T0D0) stimulated shoot (bud) growth with 66.67% of total explants used and the highest number (6.80 shoots) compared to other treatments (Table 4; Fig. 6a). This percentage rate is similar to T1D0 medium, using TDZ 1 mg/L without 2,4-D produced more shoots (66.67%) than callus (13.33%). Then, in T2D1 medium (2 mg/L TDZ and 1 mg/L 2,4-D) there were no callus and shoot bud emergenced and no growth response from the explants. It only showed green leaves (Fig. 6h).

The callus produced in this experiment was then sub-cultured in a callus medium containing 2.4-D 1 mg/L with few concentrations of TDZ. The results of callus develop-ment are shown in Fig. 6, where on media without TDZ (Fig. 6a), the callus is bigger in shape (±1.0 in diameter) with brown yellowish color and compact shape. The callus condition also gave rise to potential shoots. Meanwhile, the callus in medium with 2.4-D 1 mg/L and TDZ 1 mg/L is ±0.75 cm in diameter and small part of it turned to get browning. And in medium with TDZ 1.5 mg/L, the callus had more severe degree of browning. The addition of TDZ 1 and 1.5 mg/L inhibited the growth and then the callus started being brown in 3 MAP (Fig. 7b, c).

Figure 7. The callus growth in ½MS media with 2,4 D 1 mg/L and various TDZ concentrations (0; 1; dan 1,5 mg/L3 MAP. Magnifier 7,5×.

Seed germination

The success of Paphiopedilum seed germination is influenced by many factors, including the level of fruit maturity, medium composition, culture conditions, and culture methods (Yao et al. 2021; Khamchatra et al. 2016). In this study, the nutrient content of KC modified medium with or without NAA had no significant effect on growth response. Thus Knudson C nutrition alone is enough to encourage changes in the shape of seeds to germinate embryos.

Several studies of Paphiopedilum seed germination were carried out using a variety of media, including ½ MS, ¼ MS, 1/8 MS (Imsomboom et al. 2017; Yao et al. 2021), VW (Vacin and Went. 1949), RE: Robert Ernst (Arditti 1982), Orchimax media (Ragu et al. 2022). It took a minimum of 95 days after planting (DAP) for the seed to develop into protocorm in 1/8MS medium and 110 DAP on ¼ MS media (Zhang et al. 2015). The study conducted by Handini et al. (2016) showed that the KC modified medium with the addition of the same organic matters gave high germination rate in 90 DAP for P. supardii. Meanwhile, in this research, the germination time of P. glaucophyllum seeds was shorter (60 DAP) in the KC modified medium. This result showed that the KC medium is suitable for the germination of the Paphiopedilum species, specially P. glaucophyllum.

Elongation treatment

The most apparent effect of changing lighting is the growth of nodes and an increase in the number of leaves. Changing lighting then cause etiolation and de-etiolation (Armarego-Marriott et al. 2019). When there is light, the chloroplasts will convert water, carbon dioxide, and light into food for plants, but in conditions with no light, the chloroplasts will become etioplasts that do not contain chlorophyll (Johnson. 2016).

This etiolation and de-etiolation also related with internode ratio and in this study the highest internode ratio is in the MP5 medium (modification of full strength of MS with banana extract 100 g/L and NAA 5 mg/L). The addition of NAA as plant growth regulator in the medium would promote cell division and enlargement, thus effected on the growth of the explants. The work of auxin is also optimalized by the dark condition (I’anatshshoimah et al. 2020). In other side, light is also essential aspect for morphogenesis, then the combination between dark and light is needed to bring the ideal condition of explants growth for etiolation.

In the case of Arabidopsis plants, sunlight (660 nm) changes Pr (the inactive form of the phytochrome) into Pfr (the active form of the phytochrome). The Pfr phytochrome moves from the cytoplasm into the nucleus, stimulating the DELLA protein’s activity. This protein regulates transcrip-tion and suppresses almost all processes involving the hor-mone gibberellins, which affect plant growth and develop-ment. The protein binds to and inhibits a protein called PIF (phytochrome interaction factor), a transcription factor that binds to and turns on the promoter of a gene that stimulates cell elongation. It can be said that the DELLA protein pre-vents PIF from binding to the target gene promoter, thereby preventing cell elongation or etiolation (Kusnetsova et al. 2020).

Callus induction

The explant used in this study is the first node from the upper part because it contains meristem cells called shoot apical meristem (SAM), which are a group of proliferating embryonic cells. This SAM is localized at the ends of stems, branches, and axillary side (Traas and Vernoux 2002). Using the first bud node as an explant is expected to have a high cell division rate to trigger callus formation.

The first method of callus initiation by using the first node of the explants gave result to low number of explants that forming callus (<20%). Similar research was con-ducted by Huy et al. (2019) on P. callosum by using the first to fifth nodes. More explants produced the callus (31.25%) with the same basic media with the combination of 2,4-D 1 mg/L and TDZ 1 mg/L. (Huy et al. 2019). The research conducted by Chen (2018) with Paphiopedilum Alma Gavaert used stem, leaf and root explants, and only root explants that grow callus. In that study, the combination of TDZ 1 mg/L + 2,4-D 5 mg/L and TDZ 2 mg/L + 2,4-D 5 mg/L was able to produce 62% and 68% callus during 90 days of the incubation period. The differences of the result were caused by the age of the explants used and the callogenesis process’s efficiency, which may be specific for each type of orchid (Huy et al. 2019).

The explants in all treatment media, that stored in the dark, produced callus except control medium (Table 4). Meanwhile, in light condition, only 0.5 and 2 mg/L TDZ that emerge callus. Dark condition could reduce browning effect on the explants, then the callus could grow without any inhibition (I’anatshshoimah et al. 2020). Study con-ducted by Herawati et al. (2012) on Artemisia cina resulted in higher production of callus for explants placed in the dark. The inhibition in the light condition could be resulted from photochemical alteration which can change the medium component.

The second method of callus induction study used the explants produced from the elongation process by applying the best environmental condition (dark condition) and replacing the basic media. This media is expected to be more capable of inducing callus based on the research of Wattanawikkit et al. (2011) and using the first node of the plantlet. The combination of 1.5 mg/L TDZ and 1 mg/L 2,4-D gave maximum number of callous explants. The research conducted on Dendrobium lineale showed that maximum number of callous explants was reached with similar combination, which is 1 mg/L TDZ and 1 and 5 mg/L 2.4-D (Hoesen et al. 2008).

Thongpukdee et al. (2013) in their research on Paphiopedilum ‘Delrosi’ obtained a combination of TDZ 0.1 mg/L and 2,4-D 1 mg/L, which was able to increase shoot growth with the highest percentage of explants sprouting (90% of explants). While the results of the research by Wattanawikkit et al. (2011) with a combination of TDZ 0.1 mg/L and 2,4-D 1 mg/L produced 1.4 shoots per explant. This number was less than this study (1.6 shoots per explant) found on media containing TDZ 0.1 mg/L with no 2,4-D. This small number of shoots is probably due to the explants not being injured so that new shoots do not appear.

The elongation process is needed to increase the number of Paphiopedilum stem nodes, which can be used as material for callus induction and is intended to activate its meristematic cells. Injury by wounding explants vertically and horizontally increases the possibility of callus emergence. The dose 1 mg/L of 2,4-D is sufficient for callus induction of Paphiopedilum glaucophyllum. But, when combined with TDZ for callus induction, this dose of 2,4-D is fixed, while the appropriate concentration of TDZ is 1.5 mg/L.

This research was funded by Research Organization Life Sciences and Environment-The National Research and Innovation Agency.