Members of the Cypripedium genus are commonly called lady’s slippers because of the slipper-like appearance of their flowers. Cypripedium macranthos Sw. is one of the most attractive species among the lady’s slipper orchids and widely distributed from Eastern Russia, Northern China, Japan, Korea and Taiwan (Cribb 1997). Unfortunately they are becoming extinct due to the destruction of native habitats and illegal collection (Cribb and Sandison 1998). The most commonly recommended propagation method for conservation and commercialization of Cypripedium species is micropropagation, however, many cultural conditions including physical and nutritional factors can affect the germination and regeneration of Cypripedium species critically. Numerous studies on efficient micropropagation of orchid plants through callus or protocorm-like bodies (PLBs) have suggested that their proliferation methods are species-specific and the major obstacles to successful micropropagation are involved in in vitro germination of seeds (Arditti 1977; Colli and Kerbauy 1993). So it is very important to find the efficient in vitro seed germination conditions for each Cypripedium species and establish their optimum artificial propagation system.

Orchids produce a number of minute seeds but they have low propagation rate in nature, because ovules are not present or poorly developed at the time of anthesis, and endosperms which contain sufficient nutrient reserves for germination are absent from mature seeds. Therefore, asymbiotic germination has been a beneficial and common technique for micropropagation of orchids (Arditti 1967). However, in vitro germination of terrestrial orchids, such as Cypripedium (Leroux et al. 1995; Rasmussen 1995), Spiranthes (Zelmer and Currah 1997), Platanthera (Zettler and MacInnis 1994), Ophrys (Kitsaki et al. 2004), Calanthe (Lee et al. 2007) and Epipactis (Rasmussen 1992) was found to be more complicated as compared to the other epiphytic orchids in tropical areas. Because they have some unfavorable physiological characteristics resulting in poor germination. The mature seeds of terrestrial orchids contain commonly the rigid and hard seed coats which have strong hydrophobicity, besides, inhibitory substances such as abscisic acid are accumulated in mature seeds (Lee et al. 2007; Van der Kinderen 1987; Van Waes and Debergh 1986). Miyoshi and Mii (1998) also mentioned the similar result that the germination rate of mature seeds was less than 5% in C. macranthos. Therefore various strategies have been applied, such as pretreating seeds with hypochlorite solution (Vujanovic et al. 2000), pre-chilling treatment (Masanori and Tomita 1997), immature seed culture (De Pauw and Remphery 1993; St-Arnaud et al. 1992) and development of optimum medium composition (Rasmussen 1995). Furthermore, medium types, plant growth regulators, carbohydrates, organic amendments, vitamins and other components have been studied for the improvement of Cypripedium seed germination (Deng et al. 2012; De Pauw et al. 1993, 1995; Harvais 1982; Piao et al. 2011; Rasmussen 1995; Yan et al. 2006).

Growth and morphogenesis of plant tissues can be promoted by the addition of various organic supplements and plant extracts (Fonnesbech 1972). Many organic additives including coconut water, banana powder, the bleeding sap of birch trees, peptone, tomato juice, honey, date palm syrup, corn extract, papaya extract and beef extract have been used effectively for enhancing the development of cultured cells and tissues, though they have undefined mixture of organic nutrients and growth factors (Islam et al. 2003; Murdad et al. 2010). Actually a large number of organic amendments were used successfully for orchid production (Arditti 1967; Arditti et al. 1990; Islam et al. 2003; Pyati et al. 2002). Therefore we tried to find the effect of various organic additives on in vitro growth of seedling of C. macranthos Sw. during asymbiotic seed culture and identify the most suitable organic additives to enhance its proliferation significantly in this study.

Plant materials

C. macranthos Sw. has been cultivated in field and greenhouse at Chungcheongbuk-do Agricultural Research and Extension Services. During blooming season (from April to June), the flowers were cross-pollinated manually by transferring pollinia onto the stigma of the other flower. Seed capsules were harvested at 75 days after cross-pollination. These premature seeds were brought to the laboratory and prepared for inoculation onto media on the same day. In a laminar flow hood, whole seed capsules which were not injured by insects such as leaf-miners were surface-sterilized in 3% (v/v) sodium hypochlorite solution for 15 min and rinsed three times in sterile distilled water. Capsules were cut and their seeds were scooped out with forceps onto the culture medium. The seeds were incubated in darkness at 23 ± 2°C during 5 months.

Asymbiotic seed culture with media containing various organic additives

Seeds from surface-sterilized capsules were sown on each basal medium with different organic additives including coconut water (0, 50, 100, 200 ml·L^-1, Sigma-Aldrich, USA), birch sap (0, 50, 100, 200 ml·L^-1), maple sap (0, 50, 100, 200 ml·L^-1), banana powder (0, 15, 30, 60 g·L^-1, MBcell, USA) and peptone (0, 1, 2, 4 g·L^-1, MBcell, USA). Saps were obtained from the birch (Betula pendula) and maple (Acer platanoides) trees growing in the field of Chungcheongbuk-do Agricultural Research and Extension Services. They were collected in the early spring (from the end of March to the beginning of April) during the intense period of their production. For the sap collection, the trunk was tapped and the plastic spout which had a tube was connected into the taphole. From this tube, sap water ran into the sterile container, and these collected saps were immediately stored at -20°C. The basal medium was made with quarter-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) which was supplemented with 10 g·L^-1 sucrose, 7 g·L^-1 agar and each organic additive. Its pH was adjusted to 5.8 before autoclaving (at 121°C and 1.2 kgf·cm^-2 pressure for 15 min). 100 ml of medium was poured onto each plant culture dish (Φ100 x h40 mm). This experiment was designed randomly. Each treatment had ten replicates and was conducted three times. Seeds from each capsule were equally distributed into each replicate. After sowing, the seeds were incubated in darkness at 23±2°C during 5 months. After 2 months of culture, the germination rate (the percentage of the number of seeds germinated among the total countered number of seeds) was calculated. Germination indicated the emergence of the full embryo from the testa. After 3 months of culture, the protocorm formation rate was also calculated as the percentage of the number of young protocorms with promeristem among the total germinated seeds. After 5 months of culture, the growth characteristics of young seedlings developed from protocorms were measured.

Determination of constituents in coconut water, birch sap and maple sap

Sugar contents were measured by high performance liquid chromatography (HPLC) with a refractive index detector (Agilent 1100 series, Agilent, USA) using ZORBAX carbohydrate analysis column (4.6×150 mm, Agilent, USA). A mobile phase consisting of 75% acetronitrile was used at a flow rate of 1.0 ml·min^-1. Among inorganic ion contents, the cations such as K⁺, Mg²⁺ and Ca²⁺ were analyzed by HPLC with a conductivity detector (Breeze HPLC, Waters, USA) on IC-Par CM/D column (3.9×50 mm, Waters, USA, eluent: 0.5 mM EDTA / 2 mM HNO₃), and the anions such as NO₃^- and SO₄^- were analyzed using IC-Par anion HR column (4.6×7 5mm, Waters, USA, eluent: 1.6 mM NaHCO₃ / 1.4 mM Na₂CO₃). According to the published methods (Amin and Reusch 1987), vitamin B and C group were measured by HPLC with diode array detector (Agilent 1100 series, Agilent, USA) using ZORBAX Eclipse Plus C-18 column (4.6×150 mm, Agilent), its mobile phase channel A was 25 mM NaH₂PO₄ (pH 2.5), and mobile phase channel B was methanol at a flow rate of 1.0 ml·min^-1. Organic acids were also analyzed by HPLC with refractive index detector (Agilent 1100 series, Agilent, USA) using Hi-Plex H column (7.7×300 mm, Agilent), and its mobile phase was 0.004 M H₂SO₄ at a flow rate of 0.4 mL·min^-1. Endogenous plant hormones such as indole acetic acid (IAA), trans-zeatin riboside (trans-ZR) and abscisic acid (ABA) were measured according to the methods described by Nakurte et al (2012). They were analyzed with a modular HPLC system with UV and fluorescence detectors (Agilent 1100 series, Agilent, USA) using a reverse-phase Zorbax Eclipse XDB-C8 column (4.6×150 mm, Agilent, USA), its mobile phase was methanol containing 1% acetic acid (60 : 40 v/v) in isocratic mode at a flow rate of 1 ml·min^-1.

Statistical analysis

Data from each experiment were subjected to Two-way ANOVA and Duncan`s multiple range test using SAS program (Version 6.21, SAS Institute Inc., Cary, NC, USA).

Table 1 showed the nutrient components of coconut water, birch sap and maple sap. All of them contained around 2.5% sugars which were almost composed of sucrose, glucose and fructose. Sucrose was the main sugar type in coconut water and maple sap, on the other hand, birch sap was mostly composed of fructose and glucose. They possessed variable inorganic ions such as potassium (K), phosphorus (P), calcium (Ca), magnesium (Mg), iron (Fe) and manganese (Mn). Water-soluble vitamins such as thiamin (V_B1), riboflavin (V_B2), niacin (V_B3), pantothenic acid (V_B5), pyridoxine (V_B6), myo-inositol and ascorbic acid (V_C), which were recommended for orchid growth, were also detected in coconut water and maple sap. But they were not detected in birch sap, except for ascorbic acid. Organic acids including citric, malic and succinic acid were contained in three organic additives. Phytohormones such as IAA, trans-ZR and ABA were detected only in coconut water.

Table 1 . Comparison of nutrient constituents in coconut water, birch and maple sap added to the culture medium.

Nutrient constituents		Coconut water (100g)	Birch sap (100g)	Maple sap (100g)
Sugars (g)	Total	2.5	2.3	2.8
	Sucrose	1.2	0.2	2.3
	Glucose	0.8	0.9	0.3
	Fructose	0.5	1.2	0.2
Inorganic ions (mg)	Ca2+	24.2	58.6	63.4
	Fe3+	0.3	0.1	0.6
	Mg2+	25.3	11.3	14.5
	PO43-	20.4	6.4	2.7
	K+	242.1	120.4	204.2
	Mn2+	0.1	1.1	3.3
	Cu2+	0.04	0.03	0.7
	Na+	36.5	5.3	10.4
	SO42-	21.4	31.2	38.8
	NO3-	2.8	3.1	3.5
Vitamins (mg)	Thiamin (VB1)	0.03	-	0.01
	Riboflavin (VB2)	0.05	-	0.01
	Niacin (VB3)	0.08	-	0.03
	Pantothenic acid (VB5)	0.04	-	0.03
	Pyridoxine (VB6)	0.03	-	0.002
	Myo-inositol	0.01	-	-
	Ascorbic acid (VC)	2.4	0.3	0.9
Organic acids (mg)	Malic	289.3	359.1	141.7
	Citric	23.7	6.8	15.0
	Succinic	8.4	11.5	12.2
Phytohormones (nM)	IAA	25.6	-	-
	trans-ZR	10.2	-	-
	ABA	8.5	-	-

Table 2 explained the different effects of each organic additives on in vitro germination and protocorm formation of C. macranthos Sw. With 100 ml·L^-1 coconut water, the highest germination rate (70.8%) and protocorm formation rate (74.2%) were obtained, they increased by 6 times as compared with non-treatment. Both birch sap and maple sap also induced the facilitating effect to improve the germination and protocorm development. When 100 ml·L^-1 birch sap and maple sap were added, the germination rate and protocorm formation rate showed more than 65%. But the ddition of banana powder and peptone could not make the favorable culture condition significantly. Table 3 and Table 4 also indicated the beneficial effect of organic amendments including not only coconut water but also phloem sap such as birch sap or maple sap. When these additives were included at the level of 100 ml·L^-1, the fresh and dry biomass of seedling increased significantly by 4 times as compared with non-treatment. Roots and buds of seedlings grew vigorously in the medium containing 100 ml·L^-1 coconut water or phloem sap. In

Table 2 . Effects of organic additives on in vitro seed germination and protocorm formation in C. macranthos Sw. after 2 and 3 months of culture.

Organic additives		Germination (%)	Protocorm formation (%)
Type (A)	Conc. (B)
Coconut water	0 ml·L-1	12.6 gz	14.4 e
	50 ml·L-1	60.9 b	62.0 b
	100 ml·L-1	70.8 a	74.2 a
	200 ml·L-1	64.4 ab	60.8 b
Birch sap	0 ml·L-1	12.8 g	13.1 e
	50 ml·L-1	55.8 c	62.1 b
	100 ml·L-1	65.2 ab	68.2 ab
	200 ml·L-1	56.9 c	53.4 bc
Maple sap	0 ml·L-1	10.9 g	12.7 e
	50 ml·L-1	59.1 b	60.5 b
	100 ml·L-1	66.4 ab	66.9 ab
	200 ml·L-1	61.1 b	61.4 b
Banana powder	0 g·L-1	11.1 g	12.2 e
	15 g·L-1	25.5 f	21.3 de
	30 g·L-1	30.6 ef	35.4 d
	60 g·L-1	36.8 e	41.9 c
Peptone	0 g·L-1	9.6 g	10.3 e
	1 g·L-1	35.2 e	38.2 cd
	2 g·L-1	43.3 d	43.7 c
	4 g·L-1	40.9 d	35.0 d
Significancey
A		**	**
B		**	**
A×B		**	**

^zMean separation by Duncan’s multiple range test at P ≤ 0.05.

^yNS, *, ** not significant or significant at P ≤ 0.05 and 0.01, respectively.

Table 3 . Effects of organic additives on the fresh and dry weight of seedlings developed from C. macranthos Sw. after 5 months of culture.

Organic additives		Fresh weight (mg)	Dry weight (mg)
Type (A)	Conc. (B)
Coconut water	0 ml·L-1	76.8 ez	7.8 e
	50 ml·L-1	228.1 b	24.2 bc
	100 ml·L-1	296.4 a	34.1 a
	200 ml·L-1	221.9 b	23.7 bc
Birch sap	0 ml·L-1	65.3 e	6.4 e
	50 ml·L-1	210.3 c	21.9 c
	100 ml·L-1	260.5 ab	28.7 ab
	200 ml·L-1	205.6 c	22.0 c
Maple sap	0 ml·L-1	71.4 e	6.9 e
	50 ml·L-1	207.6 c	22.4 c
	100 ml·L-1	273.2 ab	30.9 ab
	200 ml·L-1	231.7 b	25.5 b
Banana powder	0 g·L-1	72.9 e	7.2 e
	15 g·L-1	126.7 d	13.3 d
	30 g·L-1	178.4 cd	18.6 cd
	60 g·L-1	168.6 cd	16.9 cd
Peptone	0 g·L-1	68.4 e	6.8 e
	1 g·L-1	135.1 d	14.3 d
	2 g·L-1	169.9 cd	18.0 cd
	4 g·L-1	173.3 cd	18.2 cd
Significancey
A		**	**
B		**	**
A×B		**	**

^zMean separation by Duncan’s multiple range test at P ≤ 0.05.

^yNS, *, ** not significant or significant at P ≤ 0.05 and 0.01, respectively.

Table 4 . Effects of organic additives on the growth of seedlings developed from protocorm of C. macranthos Sw. after 5 months of culture.

Organic additives		Root			Bud
Type (A)	Conc. (B)	No.	Length (cm)	Diameter (mm)	Formation (%)	Length (cm)	Diameter (mm)
Coconut water	0 ml·L-1	3.1 d	1.0 d	1.0 a	20.2 gz	3.7 e	1.4 b
	50 ml·L-1	6.7 ab	2.7 b	0.9 a	62.5 c	6.8 b	2.0 a
	100 ml·L-1	7.1 a	2.9 ab	0.9 a	74.6 a	8.0 a	2.1 a
	200 ml·L-1	7.0 a	2.8 ab	0.9 a	69.5 abc	8.1 a	2.1 a
Birch sap	0 ml·L-1	3.2 d	0.9 d	0.8 a	23.9 g	3.9 e	1.4 b
	50 ml·L-1	5.9 b	2.6 b	0.9 a	57.1 cd	6.4 bc	1.7 ab
	100 ml·L-1	6.6 ab	3.0 ab	1.0 a	70.4 ab	7.8 a	2.2 a
	200 ml·L-1	6.1 b	2.7 b	0.9 a	67.3 b	7.4 ab	2.0 a
Maple sap	0 ml·L-1	3.2 d	1.1 d	1.0 a	23.0 g	3.5 e	1.4 b
	50 ml·L-1	5.4 c	2.5 b	0.8 a	61.1 c	7.0 b	1.6 ab
	100 ml·L-1	6.7 ab	3.3 a	0.9 a	73.5 a	7.9 a	2.1 a
	200 ml·L-1	6.6 ab	2.9 ab	0.9 a	68.0 b	7.8 a	2.1 a
Banana powder	0 g·L-1	3.0 d	1.4 cd	0.8 a	21.3 g	3.4 e	1.3 b
	15 g·L-1	4.0 cd	1.8 c	1.0 a	31.5 f	4.2 de	1.5 ab
	30 g·L-1	5.2 c	2.1 bc	1.0 a	36.9 ef	4.4 de	1.6 ab
	60 g·L-1	4.6 cd	1.7 c	0.9 a	29.0 f	5.3 d	1.5 ab
Peptone	0 g·L-1	2.8 d	1.2 d	1.0 a	21.0 g	3.7 e	1.3 b
	1 g·L-1	4.4 cd	1.9 c	0.9 a	44.2 e	5.2 d	1.6 ab
	2 g·L-1	5.1 c	2.3 bc	0.8 a	56.8 cd	6.0 c	2.0 a
	4 g·L-1	4.5 cd	1.9 c	0.8 a	52.6 d	5.3 d	1.9 a
Significancey
A		**	**	NS	**	**	*
B		*	**	NS	**	**	*
A×B		*	*	NS	**	**	NS

^zMean separation by Duncan’s multiple range test at P ≤ 0.05.

^yNS, *, ** not significant or significant at P ≤ 0.05 and 0.01, respectively.

particular, the bud formation rates were raised up to 70%, and their length and diameter also increased by 2 and 1.5 times respectively. Similarly, the number and length of roots increased by 2 and 3 times individually. It was found that 100 ml·L^-1 of coconut water or phloem sap showed the best response to the germination and protocorm development in the quarter-strength MS basal medium supplemented with 10 g·L^-1 sucrose. The optimal osmotic potential should be maintained in culture medium for the successful in vitro germination and seedling growth of Cypirpedium species (Piao et al. 2011; Van Waes and Debergh 1986). So when coconut water or phloem sap which contain not only high sugars but also various rich minerals are used as organic supplements, they have been recommended to be applied at the proper concentration. In this study, when these organic additives were adjusted at the level of 100 ml·L^-1, the most balanced osmolality might be kept during the long culture period, in comparison with their lower or higher concentrations. From Figure 1, it was also confirmed that the roots and buds of seedling grew most vigorously in the culture medium supplemented with 100 ml·L^-1 coconut water or phloem sap.

Figure 1.

Morphology of seedlings developed from protocorms of C. macranthos Sw. depending on organic additives after 5 months of culture

It is one of the advisable methods to use organic additives in orchid culture medium, because they have been reported to be an easy way to improvise the current plant tissue culture media towards commercial production (Ichihashi and Islam, 1999). As organic additives, coconut water, apple homogenate, banana homogenate, potato homogenate, date palm syrup, corn extract, papaya extract, beef extract, casein hydrolysate, pineapple extract, yeast extract, tryptone, peptone or pure amino acids such as glutamine were used successfully in orchid production (Islam et al. 2003; Murdad et al. 2010; Pyati et al. 2002; Rasmussen 1995). They possess a wide spectrum of growth factors, resulting in the beneficial effects producing more PLBs, shoots and leaves (Akter et al. 2007), increasing the size of somatic embryos (Al-Khateeb 2008) and promoting the development of asymbiotic seeds as well as regeneration of plantlets (Tawaro et al. 2008). Cypripedium seed germination and protocorm development are known to be stimulated or inhibited by organic amendments (Chu and Mudge 1994; Deng et al. 2012; DeMarie et al. 1991; Steele 1995; Yan et al. 2006).

Coconut water was reported to promote the development of orchid tubers and roots (Mclntyre et al. 1974; Rasmussen 1995). 100 ml·L^-1 coconut water extracted from a fully ripe coconut could induce the germination of C. reginae, in particular, quarter-strength MS medium supplemented with 100 ml·L^-1 coconut water led to synchronous germination, but casein hydrolysate and yeast extract had a deleterious effect (Chu and Mudge 1994). Huang and Hu (2001) reported that 5% coconut water, banana homogenate or potato homogenate in Harvais medium could accelerate the seed germination of C. flavum. Similar results have been reported in different plant species. For instance, the fresh and dry biomass of Anoectochilus formosanus increased significantly when 50 ml·L^-1 coconut water and 0.5 g·L^-1 activated charcoal were added during bioreactor culture (Yoon et al. 2007). Hyponex medium supplemented with 50 ml·L^-1 coconut water proved for the enhancement of fresh and dry biomass, number of roots, leaf area as well as development of healthy plantlets of Calanthe hybrids (Baque et al. 2011). In the proliferation of PLBs of Dendrobium Alya Pink, 10% coconut water was selected as the best organic additive (Nambiar et al. 2012). Due to the difficulty of obtaining coconut endosperm and its expensiveness in Europe, some alternatives were tested. The bleeding sap of birch tress could enhance the growth of Brassia, Cattleya, Phaleanopsis and Cymbidium explants instead of coconut water (Pieper and Zimmer 1974; Zimmer and Pieper 1975, 1976, 1978). Natural organic compounds such as bleeding sap of birch trees as well as coconut water have been used as important constituents of culture medium for micropropagation of Phalaenopsis (Homa and Asahira 1985).

Coconut water is the colorless liquid endosperm of green coconuts (Cocos nucifera) which contain soluble sugars as a natural source of carbon, amino acids, phenols, fiber and vitamins, moreover, it also contains diphenyl urea which functions as cytokinin that can enhance the explant growth and regeneration by inducing cell division (Gnasekaran et al 2010; Texeira da Silva et al. 2006). The faster regeneration of plantlets was observed in PLBs of Dracaena purplecompacta L. when coconut water containing cytokinins and auxins was supplied, in fact, IAA extracted from coconut water showed the beneficial effect to facilitate the adventitious root development (Agampodi and Jayawardena 2009). Similarly, Chung et al. (1995) and Kūka et al. (2013) reported that birch and maple saps were rich in bioactive compounds and mineral substances including carbohydrates, proteins, organic acids, B complex vitamin, vitamin C and phenolic compounds, which have been related to the bioactive activities.

In conclusion, our results demonstrated that the organic additives including the phloem sap such as birch and maple sap as well as coconut water could promote the seed germination, increase the protocorm formation and led the vigorous seedling growth of C. macranthos Sw. during the culture period. Organic amendments, as a natural source supplying carbohydrates, inorganic ions, vitamins and phytohormones might be one of the most important components in the culture medium and helpful for in vitro orchid propagation.