Organic Compounds: Contents and Their Role in Improving Seed Germination and Protocorm Development in Orchids
In nature, orchid seed germination is obligatory following infection by mycorrhizal fungi, which supplies the developing embryo with water, carbohydrates, vitamins, and minerals, causing the seeds to germinate relatively slowly and at a low germination rate. The nonsymbiotic germination of orchid seeds found in 1922 is applicable to in vitro propagation. The success of seed germination in vitro is influenced by supplementation with organic compounds. Here, we review the scientific literature in terms of the contents and role of organic supplements in promoting seed germination, protocorm development, and seedling growth in orchids. We systematically collected information from scientific literature databases including Scopus, Google Scholar, and ProQuest, as well as published books and conference proceedings. Various organic compounds, i.e., coconut water (CW), peptone (P), banana homogenate (BH), potato homogenate (PH), chitosan (CHT), tomato juice (TJ), and yeast extract (YE), can promote seed germination and growth and development of various orchids. They also stimulate seedling development, formation of protocorm-like bodies (PLBs), plantlet growth, and multiple shoot formation. The addition of organic compounds to culture media, individually or in combination, accelerates seed germination and seedling development. Different types and concentrations of organic nutrients are needed for the success of in vitro cultures, depending on the species and genotype.
Orchids, member of the Orchidaceae, are one of the largest and most diverse families of flowering plants, consisting of 763 genera and more than 28,000 accepted species . Orchids are found in various habitats, primarily (70%) attached to tree trunks in forests as epiphytes, growing in the shade and comprising almost two-thirds of the world’s epiphytic flora; the remaining 25% are terrestrial, while 5% are found on various support systems [2, 3].
Orchid seeds have limited food storage tissue, which is needed for germination and protocorms development, making seed germination in natural conditions relatively low (<5%) [4, 5]. Under natural conditions, mature seeds depend on compatible mycorrhizal fungi for germination and early development [6, 7]. Therefore, in vitro orchid seed germination is a crucial aspect in propagation and conservation programs. Seeds cultured in vitro can develop into complete seedlings without the aid of fungi, which is a suitable approach for commercial orchid production [8–12].
Initially, in vitro seed germination used mycorrhizal fungi isolated from various natural environments for stimulation and was known as “symbiotic seed germination.” The addition of organic nutrients to in vitro culture was meant to stimulate seed germination. In 1922, Knudson  successfully developed a method to stimulate protocorm production in orchids by culturing the seeds in vitro and sprinkling them on sterile nutrient media plus sucrose. This technique was known as “asymbiotic seed germination” because it did not involve mycorrhizal fungi. Both approaches were effective.
2. The Orchid Seed
Orchid seeds are the smallest of those produced by flowering plants and are therefore called “dust seeds” . However, despite their microscopic structure, they exhibit a wide variety in shape, size, color, weight, testa (cell number, size, ornamentation), and embryo characteristics.
According to Molvray and Kores , orchid seeds can be crescent, broadly ellipsoid, filamentous, spindle-shaped, fusiform, oblong, irregular, or clavate. Verma et al.  established that ovoid, filiform, and spatulate-shaped seeds are present in Androcorys monophylla, Goodyera biflora, and Platanthera clavigera, respectively.
Seed color also varies, including orange yellow in Dendrobium formosum and Dendrobium densiflorum, brownish yellow in Dendrobium hookerianum, yellow in Cymbidium bicolor, white in Eria dalzellii, pale yellow in Liparis elliptica and Pholidota pallida, golden yellow in Bulbophyllum mysorense, and light yellow in Coelogyne breviscapa [16, 17]. Molvray and Kores  showed that seed size varied from 150 to 6,000 μm and weight ranged from 0.31 to 24 μg.
The cellular organization of seed is simple and consists of an undifferentiated mass of embryonal cells and a rudimentary endosperm, covered with a transparent testa . According to Arditti and Ghani , orchid embryos are relatively small and simple, generally oval or spherical in shape, and sometimes consist of only a few cells, mostly without an endosperm.
3. The Role of Organic Nutrient Supplements
The development and regeneration of in vitro cultured plant tissues can be improved by adding a variety of organic nutrients [19–21]. These may include coconut water (CW), peptone (P), potato homogenate (PH), banana homogenate (BH), chitosan (CHT), tomato juice (TJ), and yeast extract (YE). Organic nutrients are a source of vitamins, amino acids, fatty acids, carbohydrates, peptides, and growth factors, which all facilitate growth . Few organic nutrients have been studied for their contents and role in improving seed germination and development of the protocorm in orchids using in vitro models (Table 1, Figure 1). Some of these studies are discussed in the following sections.
4. Coconut Water
CW is a colorless liquid endosperm obtained from Cocos nucifera. It is often added to culture media containing auxin for the rapid induction of propagation and cell growth. The use of CW in tissue culture was first attempted by Van Overbeek et al. [64, 65], who reported that adding it to the culture medium was essential for the development of young Datura stramonium embryos.
CW contains soluble sugars as a natural source of carbon, as well as amino acids and vitamins, such as thiamin, pyridoxine, ascorbic acid, and minerals [66, 67]. It also consists of various organic ions such as phosphorus, magnesium, potassium, calcium, iron, and manganese [68, 69], all of which facilitate germination . Furthermore, CW contains indoleacetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA), and zeatin , which are generally used as growth supplements in plant tissue culture.
Orchid seed germination is usually increased by adding CW to the medium (Table 1). For instance, Thomas and Michael  reported 93% germination of Rhynchostylis retusa seeds after the addition of 150 mL·L−1 of CW to a half-strength Murashige and Skoog medium (½ MS; Murashige and Skoog ). Similar conclusions were reached by Piri et al. , who stated that the addition of CW 150 mL·L−1 to Mitra medium (M; Mitra et al. ) was most suitable for seed germination of Acampe papillosa.
According to Huh et al. , MS medium supplemented with CW enhances seed germination and protocorm formation of Cypripedium macranthos. In a study of different organic supplements, including CW, birch sap (BS), maple sap (MPS), BH, and P, at various concentrations, the use of 100 mL·L−1 CW resulted in the highest germination rate (70.8%) and protocorm formation rate (74.2%). Zeng et al.  also established that ½ MS medium containing 0.5 mg·L−1α-naphthaleneacetic acid (NAA) and 1.0 g·L−1 activated charcoal (AC) supplemented with CW, enhanced seed germination and seedling formation of Paphiopedilum wardii. Among the different organic supplements, including CW, PH, BH, tryptone (T), and P with various concentrations, 100 mL·L−1 CW increased seed germination (65.33%) and had the highest seedling formation (35.67%) compared to other treatments, while Wu et al.  concluded that ¼ MS medium containing 0.5 mg·L−1 NAA and 1 g·L−1 AC, supplemented with 200 mL·L−1 CW and 1 g·L−1 P, after 75 days in culture, was suitable for seed germination and protocorm development of Renanthera imschootiana.
CW not only affects germination and protocorm development, but also plays an important role in the formation of protocorm-like bodies (PLBs), plantlet growth, and multiple shoot induction. According to Baque et al. , the Hyponex medium, Kano , supplemented with CW effectively enhanced plantlet growth of ornamental orchid Calanthe hybrids. Of the various CW concentrations tested (0, 10, 30, 50, and 100 mL·L−1), a concentration of 50 mL·L−1 significantly increased plantlet dry weight. Maharjan et al.  assayed the effect of different concentrations of CW (0, 50, and 100 mL·L−1) on shoot formation of medicinal orchid Vanda pumila protocorms and found that the number and length of shoot increased when cultured on ½ MS medium supplemented with CW. Similar conclusions were made by Punjansing et al.  who determined that the addition of CW (50 mL·L−1) to ½ MS medium containing 50 g·L−1 PH was the best for promoting shoot multiplication of Phaius tankervilleae var. alba. This result was supported earlier by Jainol and Jualang  who observed the effects of different organic supplements (CW, TJ, BH, P, and YE) at various concentrations on multiple shoot formation in Dimorphorchis lowii and found that CW at 150 mL·L−1 resulted in the highest number of shoots. Kaur et al.  investigated the effects of the addition of various organic supplements (BH, CW, and control without organic additives) at various concentrations on the in vitro multiplication of protocorms in a medicinal orchid (Dendrobium nobile). In their study, M medium supplemented with 200 mL·L−1 CW was the most suitable for the enhancement of protocorms multiplication.
P is occasionally used as a medium supplement in orchid cultivation, facilitating explant growth and development. Numerous studies have shown that P supplements enhance germination (Table 1). For example, Utami et al.  evaluated the effect of different P concentrations (0, 1, 2, and 3 g·L−1) on seed germination and shoot formation of Dendrobium lasianthera, observed at 4, 8, and 12 weeks after culture. Among the different concentrations of P tested, seed germination rate (100%) was obtained after 12 weeks of incubation in all treatments. However, they also reported that shoot formation was the highest (84.0%) in the Vacin and Went medium (VW; Vacin and Went ) supplemented with 2 g·L−1 P. Seed germination rates of Epidendrum ibaguense Kunth and ornamental orchid Spathoglottis plicata Blume were higher on M or Phytamax medium (PM; Phytamax™, Sigma Chemical Co., USA) supplemented with 2 g·L−1 P than on treatments that did not contain P [34, 35]. Similarly, Buyun et al.  found a seed germination rate of 100% for Dendrobium parishii with Knudson medium (KC; Knudson ) supplemented with 2 g·L−1 P. According to Srivastava et al. , the seed germination rate of Aerides ringens increased significantly when cultured on KC medium containing 8.9 μM benzylamino purine (BAP) and supplemented with P. Among the different concentrations of P tested (0.25, 0.5, and 0.75 g·L−1), the highest germination rate (89.28%) was obtained in 0.5 g·L−1 P as an additive after 30 days of incubation.
P is also known to stimulate the multiple shoot and protocorms formation. Devi et al.  reported that P improved the in vitro multiplication of protocorms of medicinal epiphytic orchid Taprobanea spathulata. Of the different concentrations tested, 5 g·L−1 P was a suitable concentration for the induction of multiple protocorms. Kaur and Bhutani  evaluated the effects of different organic additives (BH and P) at various concentrations on the in vitro multiplication of shoots in Paphiopedilum venustum and discovered that modified terrestrial orchid medium (BM-1; Waes and Debergh ) supplemented with P (1 g·L−1) was the most effective in stimulating multiple shoots, with three shoots per explant. In addition to promoting the formation of multiple shoots and protocorms, peptone promotes seedling growth  because it contains high levels of amino acids  and vitamins, including thiamin, biotin, pyridoxine, and nitrogen .
6. Potato Homogenate
Orchid seed germination was significantly affected by the presence of PH in the medium (Table 1). Islam et al.  evaluated the effect of PH on in vitro germination of Vanda roxburgii and showed that Hyponex medium supplemented with PH increased the seed germination rate and promoted seedling development. Similarly, Bakar et al.  evaluated the effect of different organic supplements (PH, CW, and TJ) at various concentrations on seed germination of Dimorphorchis lowii and found that 10% (w/v) of PH resulted in the highest seed germination rates. Additionally, PH enhanced seedling growth of Dendrobium tosaense  and stimulated the multiplication and growth of Dendrobium officinale shoots . Besides, 75 g·L−1 PH in VW medium containing 100 mL·L−1 CW was most suitable for in vitro proliferation in Bulbophyllum nipondhii pseudobulbs .
PH contains important vitamins, such as C, B1, and B6, and mineral elements, such as potassium, iron, and magnesium, as well as carbohydrates and amino acids, which facilitate seed germination [80, 81]. Miransari and Smith  reported that seeds consume large amounts of nitrogen during germination, and therefore PH in the medium can influence germination rates.
7. Banana Homogenate
Seed germination and protocorm development were affected by BH in the media (Table 1). Pereira et al.  evaluated the effects of different organic additives (BH and TJ) on the germination of Chloraea gavilu and showed that Malmgren modified terrestrial orchid medium (MM; Malmgren ) supplemented with BH successfully improved seed germination.
Islam et al.  investigated the effects of different BH concentrations (0, 2.5, 5, 10, and 20%; w/v) on the growth and development of PLBs Dendrobium sp. and showed that ½ MS medium supplemented with 10% BH was the most suitable for PLBs regeneration. According to Zeng et al. , plantlet formation of ornamental lithophytic orchid Paphiopedilum hangianum increased significantly when PLBs were subcultured on HO26 medium (Zeng et al. ) containing 1 g·L−1 P and 1 mg·L−1 NAA and supplemented with BH. The same study concluded that Harvais medium (Harvais ) with various organic supplements effectively induced seedling growth of Cypripedium macranthos. Among the various organic supplements studied (CW, PH, and BH), BH at a concentration of 25 or 50 g·L−1 improved shoot and root number and shoot and root length . Gonçalves et al.  found that the number of Hadrolaelia purpurata seedlings produced was the highest on KC medium containing 90 g·L−1 of BH. Furthermore, Utami and Hariyanto  observed the effect of supplementation with 150 mL·L−1 CW with different BH concentrations (0, 5, 10, 15 g·L−1) on the plantlet development of Phalaenopsis amboinensis and found that the organic supplementation of CW together with BH had significantly increased the growth of plantlets. Banana has often been used as an organic additive in in vitro cultures because of its high levels of potassium, manganese, calcium, sodium, iron, zinc, thiamin, riboflavin, niacin, pyridoxine, pantothenic acid, ascorbic acid, folic acid , and natural growth regulators such as zeatin, gibberellin, and IAA [87, 88]. It is also rich in carbohydrates, supplying energy to heterotrophic plants during the early stages of in vitro cultivation .
Numerous studies have shown that orchid seed germination is affected by the addition of CHT to the media (Table 1). Kananont et al.  studied the effects of various types of CHT at different concentrations on the seed germination of various orchid species and found that none of the six CHT types tested (CHT polymers: P70, P80, P90; CHT oligomers: O70, O80, O90) at five concentrations (0, 10, 20, 40, and 80 mg·L−1) had a significant effect on seed germination in Dendrobium bigibbum var. compactum; however, the addition significantly increased germination in Dendrobium formosum. The highest germination rate (91.2%) was recorded using CHT type O80 at 10 mg·L−1. Restanto et al.  evaluated the effect of CHT on zygotic embryo development of Dendrobium sp. in vitro and observed that CHT significantly affected zygotic embryo growth and differentiation. Of the different CHT concentrations tested (0, 5, 10, 15, 20, and 25 mg·L−1), the PLB number, PLB fresh weight, and number of plantlets were the highest using 15 mg·L−1 of CHT. Nge et al.  also studied the effect of shrimp CHT at different concentrations (0, 5, 10, 15, 20, and 25 mg·L−1) on the meristematic bud growth of Dendrobium phalaenopsis and showed that VW medium supplemented with 15 mg·L−1 CHT resulted in the highest fresh weight of PLBs. Additionally, the same study concluded that ½ MS liquid medium supplemented with CHT effectively induced PLB development of Grammatophyllum speciosum. Of the different concentrations of CHT tested (0, 5, 10, 15, 20, 25, 50, and 100 mg·L−1), a concentration of 15 mg·L−1 resulted in the highest growth rate (4-fold increase) . Samarfard and Kadir  investigated the effects of CHT on PLB proliferation in Phalaenopsis gigantea, and among the six concentrations (0, 5, 10, 15, 20, and 25 mg·L−1) tested, 10 mg·L−1 CHT in VW medium resulted in the highest fresh weight after 20 weeks of cultivation.
Chitosan is also known to stimulate plantlet growth and development under greenhouse conditions. Pitoyo et al.  studied the effects of CHT on the growth of Grammatophyllum scriptum plantlets and suggested that CHT had significant effects on some parameters, including plantlet height and leaf length. Kumari et al.  investigated the effects of different CHT concentrations (2.5, 5, 7.5, and 10 mg·L−1) on the growth and development of Dendrobium cv. Sonia 17. Of the four concentrations tested, 7.5 mg·L−1 resulted in the highest number of spikes per plant, an increased flower diameter, an enhanced spike length, and a higher number of florets per spike. Similarly, Charoenwattana  studied the effects of CHT on the growth of plantlets of Dendrobium orchids and reported that an optimal leaf number was achieved at a concentration of 100 mg·L−1 within 10 weeks of transplanting. The increase in plantlet growth was allegedly under the influence of photosynthesis. According to Barka et al. , the addition of CHT derivative, i.e., Chitogel, improved CO2 fixation by 1.5-fold and O2 production by 2-fold, indicating that CHT derivatives have the potential to increase photosynthesis, thereby increasing plant growth. According to Uthairatanakij et al. , the beneficial effect of CHT may induce a signal to synthesize plant hormones, auxin and gibberellin.
9. Tomato Juice
TJ significantly influences the germination of orchid seeds and subsequent protocorm development (Table 1). David et al.  investigated the effect of different complex additives (TJ, CW, P, and YE) at various concentrations on seed germination in Vanda helvola in vitro. In their study, KC medium supplemented with 15% (v/v) TJ was the most suitable for promoting germination. Muthukrishnan et al.  also examined the effects of various organic supplements (TJ, CW, and PH) at different concentrations on seed germination in Geodorum densiflorum and showed that ½ MS medium supplemented with 5% (v/v) TJ resulted in the highest rate of seed germination and protocorm growth. Similarly, Gnasekaran et al.  reported that 20 and 30% (v/v) TJ improved PLB proliferation in Vanda Kasem’s Delight more than the addition of papaya extract (PE) or PH.
Tomato juice contains carbohydrates, vitamins and minerals , glucose, and fructose , which benefit cellular division and support systems. Tomatoes also contain lycopene, a vigorous antioxidant with the ability to eliminate the formation of free radicals, repair injured cells, and suppress DNA oxidation . Tomatoes also contain other antioxidants, such as α-carotene, β-carotene, and ascorbic acid [97–100]. According to Gnasekaran et al. , sugars and antioxidants play an important role in cell proliferation and the production of healthy PLBs.
10. Yeast Extract
YE is also an important source of amino acids and vitamins, especially inositol and thiamin, and has been effectively used to increase seed germination and regeneration in many orchid species . Numerous studies have shown that orchid seed germination and protocorm regeneration are affected by YE in the cultivation media (Table 1). Gansau et al.  investigated the effect of different YE concentrations (0, 0.1, 0.2, and 0.3%; w/v) on protocorm proliferation and growth of Dimorphorchis rossii. In their study, MS medium supplemented with 0.2% YE increased protocorm regeneration. Earlier, Jualang et al.  evaluated the effect of different organic supplements (YE, P, BH, CW, and TJ) at various concentrations on seed germination of Vanda dearei and found that 0.5% (w/v) YE enhancement seed germination.
Based on the literature survey, we conclude that the addition of organic supplements, including CW, P, BH, PH, CHT, TJ, and YE, to orchid seed germination media supports seed germination, precipitates seedling formation, and yields vigorous plantlets. They also effectively increase the number of PLBs, induce multiple shoot formation, and stimulate the growth and development of plantlets under greenhouse conditions. These organic supplements represent natural sources of amino acids, vitamins, minerals, organic acids, sugars, proteins, and natural growth regulators, assisting in orchid propagation by stimulating the development and morphogenesis in asymbiotic seed culture. Many orchid species are threatened by land conversion and habitat mismanagement. Asymbiotic seed germination with the addition of organic supplements is an excellent technique for the mass propagation and efficient acclimatization of orchids for reintroduction to natural habitats, which will facilitate the conservation of endangered orchid species.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare no conflicts of interest.
Both authors contributed to literature search, participated in manuscript writing, and read and approved the final manuscript.
The authors gratefully acknowledge the financial support provided by the article review program of Universitas Airlangga, no. 955/UN3.14/LT/2019.
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