Seed biology and in vitro seed germination of Cypripedium.

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–14 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2013.841117

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Seed biology and in vitro seed germination of Cypripedium Songjun Zeng1, Yu Zhang2, Jaime A. Teixeira da Silva3, Kunlin Wu1, Jianxia Zhang1, and Jun Duan1,4 1

Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Gardens, Chinese Academy of Sciences, Guangzhou, China, 2Beijing Botanical Garden, Beijing, China, 3P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken, 761-0799, Japan, and 4 Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China Abstract

Keywords

Cypripedium orchids have high horticultural value. The populations of most species are very geographically restricted and they are becoming increasingly rare due to the destruction of native habitats and illegal collection. Reduction of the commercial value through large-scale propagation in vitro is a preferable option to reduce pressure from illegal collection. Cypripedium species are commercially propagated via seed germination in vitro. This review focuses on in vitro seed germination and provides an in-depth analysis of the seed biology of this genus.

Acclimatization, Cypripedium, research advances, seed biology, seed germination

Introduction Members of the Cypripedium genus are commonly called lady’s slippers because of the slipper-like appearance of their flowers. This genus comprises 56 species and four varieties (Cribb 1997; Wu et al., 2009; World Checklist of Selected Plant Families, 2012). Cypripedium are becoming increasingly rare owing to the destruction of their native habitats and illegal collection (Cribb, 1998). Terrestrial orchid species from northern temperate climates are generally much more difficult to germinate in vitro than tropical epiphytic species (Hossain et al., 2013; Oliva & Arditti, 1984). Four genera (Cypripedium, Paphiopedilum, Phragmipedium and Selenipedium) of the subfamily Cypridedioideae are considered to be difficult to culture in vitro, while Cypripedium is the most difficult (Arditti, 1984, 2008). The most commonly recorded method for the propagation of Cypripedium species is in vitro seed germination (Supplementary 1). However, the capacity of Cypripedium seeds to germinate depends on capsule-related parameters such as age, location, season and growth stage (Arditti, 1967, 2008; Ballard, 1987; Harvais, 1982). Many cultural factors, physical and nutritional, affect the germination of Cypripedium. In this review, the research progress of in vitro seed germination of Cypripedium, including nutrient requirements, culture conditions, the effect of plant growth

Address for correspondence: Songjun Zeng, Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. E-mail: [email protected] Duan Jun, Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. E-mail: [email protected]

History Received 20 January 2013 Revised 24 June 2013 Accepted 2 September 2013 Published online 5 November 2013

regulators (PGRs), and pre-treatment methods to improve seed germination in vitro is summarized.

Cypripedium species propagated in vitro There is a large literature on the culture of Cypripedium species in vitro. In total, 17 species (including their varieties) and two hybrids have been successfully cultured in vitro (Supplementary 1). Understandably, each species has a different germination capacity. Curtis (1936) reported low levels of germination for C. acaule, while seedling development for this species was very poor. De Pauw & Remphrey (1993) noted differences in the germination of seeds of four Cypripedium species when the medium was changed, although the effects were inconsistent and depended on the year of experimentation. Seed germination percentage (GP) of C. reginae was higher than that of C. candidum and C. calceolus var. parviflorum. In another study, C. reginae also appeared to be more amenable to culture than other species including C. acaule, C. calceolus, C. californicum, C. candidum, C. montanum and C. pubescens var. parviflorum, and higher germination was observed with C. reginae and the protocorms were more vigorous (Oliva & Arditti, 1984). However, Harvais (1982) reported, based on over 10 years of experience, that the germination of C. reginae seed varied considerably from year to year and even within the same year, if collected from different habitats.

Pollination, fertilization and embryo development in Cypripedium In nature, Cypripedium species are reportedly pollinated by self- and cross-pollination, both forms of pollination requiring insects as the pollination vector. Moreover, self-pollination


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does not have severe reproductive limitations (Ballard, 1987). Cypripedium calceolus is one of Britain’s rarest orchids and was only found on a single natural site after the 1950s. Since this orchid was not able to complete pollination naturally, hand pollination was carried out to ensure seed set (Ramsay & Stewart, 1998). Tremblay (1994) reported that pollen tube competition of C. calceolus var. pubescens could result in increased seedling vigor, and multipaternal pollination usually performed better than uniparental crosses or self-pollination. The number of viable seeds also depends on effective pollination, which in turn is influenced by the age of the flower at the time of pollination (Ballard, 1987). Curtis (1954) and Ballard (1987) found that C. reginae flowers could be successfully pollinated at any time up to 10 days after they had opened. Light and MacConaill (1998) reported 30–70% pollen germination in C. calceolus var. pubescens while pollen contaminated with fungus from older (12 days old) flowers did not germinate. Further, using flowers 12 and 14 days old at pollination, the proportion of seeds with full embryos was lower than that with flowers pollinated at an earlier stage. A better understanding of zygotic embryogenesis of endangered orchids could provide insight into subsequent germination events and aid in their in vitro propagation. According to the literature, only Cypripedium seeds have a two-layer testa, unlike the single-layered testa for other genera within the Orchidaceae (Lee et al., 2005; Liu et al., 2012; Ren & Wang, 1987; Ye & Guo, 1995; Wu et al., 2004; Zhang et al., 2010a). The inner integument of C. formosanum (Lee

Crit Rev Biotechnol, Early Online: 1–14

et al., 2005), C. macranthos (Zhang et al., 2010a; Figure 1) and C. japonicum (Liu et al., 2012) was retained throughout the process of seed development and an impermeable endothelial layer was formed after fertilization, while the outer testa had two cell layers (Table 1). Lee et al. (2005) and Liu et al. (2012) observed that C. formosanum and C. japonicum growing in subtropical high-altitude areas took 210 or 205 days, respectively, from pollination to seed maturation. At 60 or 55 DAP, respectively, most of the ovules had been fertilized and embryo development had commenced. At 75 or 70 DAP, two- to three-celled embryos were commonly observed in C. formosanum seeds collected at 75 DAP; these germinated well (approximately 55%) with a noticeable increase in germination observed at 90 DAP (approximately 75%) (Lee et al., 2005). At this stage, part of the placenta of C. formosanum had attached to the seeds, which were yellowish white and moist, and early globular to globular embryos with a single-celled suspensor could be observed, while the suspensor was proposed as a channel for the conduit of nutrients and as a food storage site for the developing embryo. By 105 DAP, a globular embryo had formed, at 120 DAP, mitotic activity had ceased within the embryo, and after 135 DAP, the seeds germinated poorly (approximately 7%). During this period, the seeds began to turn light brown. The GP of seeds collected at 75 or 105 DAP in different years were significantly different (Table 2; Lee et al., 2005). During the maturity of C. formosanum seeds (135 DAP), a cuticular substance was deposited in the wall of the inner integument, enclosing the embryo, followed by

Figure 1. The embryo and integument development process of C. macranthos. (A) A just fertilized zygote and megaspore (days after pollination, DAP ¼ 28); (B) A two-celled embryo (DAP ¼ 35); (C) A T-shaped, four-celled embryo (DAP ¼ 35); (D) An early globular embryo with a two-celled elongated suspensor (DAP ¼ 42); (E) A globular embryo covered by outer and inner integuments (DAP ¼ 49); (F) A globular embryo at the stage of suspensor degeneration (DAP ¼ 56); (G) A globular embryo covered by endopleura in a near mature seed (DAP ¼ 70); (H) Globular embryo tightly covered by endopleura in a mature seed (DAP ¼ 90); (I) Air cavity and endopleura and episperm in a transverse section of mature seed (DAP ¼ 90); (J) Air cavity and endopleura and episperm in a longitudinal section in a mature seed (DAP ¼ 90) (adapted from Zhang et al., 2010a).

Seed biology of Cypripedium

DOI: 10.3109/07388551.2013.841117

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Table 1. Comparison of embryo development of five Cypripedium species. Days after pollination


Developmental process Zygote T-shaped proembryo Early globular embryo Globular embryo Late globular embryo Seed desiccation Mature seed Fruit ripens and splits

C. calceolus (Wagner & Hansel, 1994)

C. formosanum (Lee & Lee, 2005)

C. macranthos (Zhang et al., 2010)

C. japonicum (Liu et al., 2012)

C. debile (Hsu & Lee, 2012)

30

60 75 90 105 120 180 210 240

28 35 42 49 56 70 90 100

55 70 90 120 150 – 205 –

60

40 50 60

90 120 150

Table 2. Effect of seed maturity on seed germination of Cypripedium species.

Species

Media

C. acaule

Modified MS þ 1.0 mg/L Kn þ 0.1 mg/L NAA þ 1.2 g/L PE

C. formosanum

Thomale GD þ 10% CW þ 20 g/L sucrose

C. macranthos

VWD þ 1.2 mg/L BA þ 10% CW

C. macranthos

VWD þ 10% CW þ 0.5 g/L AC

Seed maturity (DAP)

Germination (%)

Germination period (days)

References

30 60 90 60 75 90 105 120 135 200 28 56 84 96 28 42 56 70 84

0 72.8 3.4 0 5–65 65–70 20–65 5–40 5 0 0 31.4 0 0 0 37.19 68.08 6.96 0

180

St. Arnaud et al. (1992)

120

Lee et al. (2005)

63

Zhang et al. (2010b)

56

Deng et al. (2012)

AC, activated charcoal; BA, N6-benzyladenine; CW, coconut water; DAP, days after pollination; Kn, kinetin; MS, Murashige and Skoog medium (1962); NAA, a-naphthaleneacetic acid; PE, potato extract; Thomale GD, Thomale (1957) medium; VWD, Van Waes and Debergh medium (1986b).

shrinking of the inner integument to form a tight layer or carapace, which may play a role in the seeds’ impermeability and may be a protective adaptation of this species to harsh natural environments. Mature Cypripedium seeds are generally brown and have an impermeable carapace enclosing the globular embryo while the suspensor channel is closed. Harvais (1980) suggested that the accumulation of suberin in the testa might contribute to the hydrophobic nature of the mature seed. At maturity (after 210 DAP), the embryo was only eight cells long and six cells wide. Most likely, had there not been a long dormant period, made possible by the presence of a double testa, and had embryos germinated in harsh natural conditions, the germinating seedlings would most likely have become injured and died. The hydrophobic nature of the integument tissues possibly prevented water and nutrition from entering the embryo, in turn preventing mature seeds from germinating in vitro and becoming an obstacle to artificial propagation (Lee et al., 2005; Zhang et al., 2010a). However, C. macranthos growing in sub-tropical highaltitude mountainous and temperate low-altitude areas, took only about 90 days from pollination to seed maturation, and 56 DAP was the most suitable period for aseptic seed germination

(Zhang et al., 2010a, b; Figure 2). Triphenyl tetrazolium chloride (TTC) was used to test the seed maturity of this species. The seeds of C. macranthos at 28 DAP were not viable because the embryo was too young to be stained, while approximately 50% of seeds at 56, 84 and 96 DAP were equally viable (Zhang et al., 2010b). Huang & Hu (2001) reported that 68% of hand-pollinated C. flavum seeds from the greenhouse had a full embryo, as observed under a dissecting microscope.

Morphogenesis of Cypripedium protocorms and seedlings When the embryo of almost all members of the Orchidaceae ruptures, the testa widens and becomes obovoid as a small starchy parenchymatous tuber termed the protocorm. Lipolysis is regarded to be a crucial step in the germination of orchid seeds. Later, starch accumulates in the embryo cells, particularly in asymbiotic cultures (Rasmussen, 1995). The nature of orchid protocorms has fascinated morphologists for more than a century (Yam & Arditti, 2009). Harvais (1973) and Leroux et al. (1997) reported, in detail, the development of C. reginae and C. acaute protocorms using in vitro seed germination. Harvais (1973) reported that the mature seed


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Figure 2. Propagation in vitro of C. macranthos. (A) Tetrazolium (TZ) viability test of 64 DAP seeds; (B) Asymbiotic seed germination on van Waes and Debergh (VWD) medium supplemented with 1.2 mg/L kinetin (Kn), 10% coconut water (CW; v/v) and 1.0 g/L activated charcoal (AC). (C) Protocorm-like bodies (PLBs) derived from protocorms on VWD medium supplemented with 1.0 mg/L N6-benzyladenine (BA), 10% (v/v) CW and 1.0 g/L AC; (D) Development of seedlings on VWD medium supplemented with 1.0 mg/L a-naphthaleneacetic acid (NAA), 100 g/L potato homogenate (PH), and 1.5 g/L AC; (E) Seedling growth on VWD medium supplemented with 1.0 mg/L NAA, 100 g/L PH and 1.5 g/L AC; (F) Transplanted seedlings after growing in soil from natural habitat of C. macranthos in the greenhouse for 2 months (unpublished photos).

(without indicating the DAP) of C. reginae was oval and consisted of about 80 cells, with more enlarged cells at the suspensor end, and a dormant meristem at the opposite end. The seed abounded in lipids but was completely devoid of starch; the converse was true after germination in vitro. Inside C. acaute seeds, the embryos consisted of about 100 cells and these were not exalbuminous. In the embryo, cells were arranged along a longitudinal axis according to size; these cells contained protein and lipid reserve material. In the first stages of seedling development in vitro, the embryo was transformed into a protocorm and meristematic tissue became organized into a meristematic dome (‘‘promeristem’’) at the anterior pole. This meristematic dome gives rise to a scale and the apex of the seedling. At first, the apex and the scale leaf develop synchronously. The development of the root always follows that of the apex and the protocorm may be interpreted as an extension of the proembryonic stage (Leroux et al., 1997). St. Arnaud et al. (1992) also studied the morphology and anatomy of C. acaute protocorms and seedlings. The testa first split and the embryo developed into a round protocorm 1–2 mm in diameter. After development of cylindrically shaped protocorms, the first organs to be formed were always shoots, which always appeared first as a narrow appendage on which the apical dome and the first squamous leaf developed. On most occasions, a root formed at another distinct point on the protocorm. This root grew faster than the first shoot. Adventitious roots also formed on the shoot. The protocorms developed in several ways and usually many roots and shoots were produced on each protocorm. Eight months after germination, some protocorms had as many as 10 shoots and 30 roots. Growth of the protocorm root was sometimes nearly simultaneous to shoot appearance, but always occurred after the appearance of the shoot apex.

Champagnat (1966) reported that all the leaves from Cypripedium protocorms associated with the apex had the same phyllotactic pattern. However, St. Arnaud et al. (1992), using an anatomical section of C. acaule at the base of the stem, showed that the first leaf does not display the same phyllotactic system as the ensuing leaves: the first leaf formed 90 relative to the subsequent leaf. It is possible that the protocormial root, the protocorm and the first leaf constitute a morphological and physiological system that differs from the rest of the seedling and would constitute the real (true) embryological stage in the plant. Oliva & Arditti (1984) found that roots and shoots generally appeared together in most Cypripedium seedlings, including seven species and two hybrids. However, in C. acaute, shoots formed after the roots, while the reverse was true for C. californicum. Protocorms and rhizomes formed simultaneously on C. californicum seedlings. In addition, the rhizome formed after the appearance of protocorms in C. reginae, but no rhizomes formed in any other Cypripidium species.

Effect of degree of seed maturity on germination in vitro and methods for breaking dormancy Immature terrestrial orchid seeds usually germinate better in vitro than ripe seeds (Kauth et al., 2008), which can be caused by high endogenous levels of abscisic acid (van der Kinderen, 1987), or due to physical and chemical properties of the testa and by illumination photodormancy. Dormancy could be broken by certain temperature regimes, lengthy imbibition, chemical breakdown of the testa, signals from fungi or germination in darkness (Rasmussen, 1995). In the in vitro germination of Cypripedium, refrigeration, softening the testa with NaClO and Ca(ClO)2), or mechanical damage, including treatment with ultrasound and vacuum infiltration,

DOI: 10.3109/07388551.2013.841117

are effective methods to improve seed germination, as discussed in detail below.


Effect of degree of seed maturity on germination in vitro Numerous studies have shown that immature seeds of Cypripedium species germinate better than mature seeds (Table 2). Lee et al. (2005) reported that the timing of seed collection outweighed the medium composition and seed pretreatment in terms of seed GP. Withner (1953) reported that immature seeds from green capsules of C. acaule germinated better than mature capsules, whereas St. Arnaud et al. (1992) reported no differences. Ballard (1987) reported that the degree of seed maturity had no effect on GP in C. reginae, but that GP was unpredictable (10% to 80%) as the seed matured, with approximately 50 DAP exhibiting the best GP (80%). However, Hsu & Lee (2012) reported optimum GP (75%) of C. debile when mature seeds (150 DAP) were collected rather than immature seeds (30–90 DAP for 0% GP, or 120 DAP for 72.7%) on 1/4 MS medium (Murashige & Skoog, 1962). Light (1989) suggested, over a multi-year experiment, that climatic variation in pod development may affect the optimum time for collection for seed germination. Both Zhang et al. (2010a) and Deng et al. (2012) reported the most suitable embryonic age of C. macranthos for asymbiotic germination to be 56 DAP, the maximum GP was 31.4% and 68.08%, respectively. The seeds did not germinate when the embryonic age was less than 28 DAP or higher than 84 DAP. Cypripedium acaule mature seeds are extremely difficult to germinate (Henrich et al., 1981, Lauzer et al., 1994, Light, 1989, Linden, 1992, Oliva & Arditti, 1984, St. Arnaud et al., 1992, Stoutamire, 1964, Withner, 1953). Without exception, these authors all stated that immature seeds from green capsules of C. acaule germinated better than mature seeds. For example, St. Arnaud (1992) reported that 30-DAP seeds did not germinate, while 72% of 90-DAP seeds had viable embryos, but only 3.4% germinated; 60-DAP seeds, after 4 months, had an 18.9% mean GP, the highest GP from capsules being 37.1%. After 8 months the mean GP was 25.1% although the highest level of GP from a capsule was 72.8% (Table 2). The best time to germinate C. caleolus seed is when it is fully developed (with fully developed embryo), but still white or ash coloured and loose in the capsule, at 50–60 DAP (Light, 1989). This is because, at this stage, the waterrepellent properties of the testa have not fully developed and the embryos could rapidly imbibe water (Ramsay & Stewart, 1998). Wagner & Hansel (1994) reported the pattern of C. caleolus seed formation in which each stage had a different GP. In Van Waes and Debergh (VWD) medium (van Waes & Debergh, 1986b), by 30 DAP when the proembryo consisted of only a few cells, 40% GP could be achieved. The living integument cells play an important nutritive and activating role for the growing embryo and the artificial culture medium may contribute by helping the integument cells to survive. Therefore, an appropriate composition of the culture medium is assumed to be essential for the successful germination of immature orchids. At this stage of development (30 DAP), vacuum infiltration of seeds with liquid nutrient medium did


5

not promote germination. Germination was highest in seeds collected at 40 DAP, when the promotive effect of vacuum infiltration became visible, resulting in almost 80% germination while the control value was about 60%. With advancing seed maturity, the readiness to germinate decreased continuously because the inner integument increasingly becomes impermeable to water and inhibits germination. The 50-DAP seeds that were vacuum infiltrated had a mean GP of up to 56% (corresponding control value was only 24%). Vacuum infiltration not only enhanced and accelerated germination, but also promoted organ differentiation, specifically seedling formation. In the last phase of seed maturation, in addition to the inhibiting effect of the testa, physiological dormancy mechanisms in the embryo became obvious. At about 60 DAP, the GP dropped to below 20%, with small differences between infiltration and control seeds. Infiltration with nutrient solution simulated the germination of dormant embryos causing them to swell faster, the protocorm stage was earlier and more roots formed. Lucke (1982) also reported the optimum time for germination of C. caleolus to be 42–49 DAP. De Pauw & Remphrey (1993) reported that in 1989, the period after pollination in which seed were collected had a significant effect on germination in three Cypripedium species. In C. reginae and C. calceolus var. parviflorum, germination peaked for seed collected at 56 DAP. After 64 DAP there was a sharp decrease in germination. However, in C. candidum, peak germination occurred when seed was collected at 42 DAP. Thereafter, germination decreased sharply. There was an increase in germination when seed was collected at 84 DAP relative to 70 DAP, but only on VWD medium and not on Harvais medium (Harvais, 1982) or on modified Norstog medium (Norstog, 1973). But in another year (1990), C. candidum had the same tendency as other Cypripedium species in which germination peaked for seed collected at 56 DAP, followed by a sharp decrease at 70 DAP. There was little difference in germination between seed collected at 70 DAP and 84 DAP. Asymbiotic germination of ripe orchid seeds in vitro often fails due to mechanical and physiological mechanisms of dormancy. During seed maturation, the integuments become increasingly impermeable to water (Stoutamire, 1974; Van Den 1987). Additionally, the testa and embryo may contain substances that inhibit germination even in hydrated seeds (Harvais, 1980; van Waes & Debergh, 1986a, b). The accumulation of abscisic acid (ABA), a widespread germination inhibitor in dormant seeds, may be possible. Lee (2003) found that endogenous ABA content of C. formosanum was low at 60 DAP, but increased rapidly during 120–150 DAP. A high level of ABA accumulated on the surface wall of the embryo proper and the shrivelled inner integument of mature C. formosanum seeds by using an immunohistochemical technique, which coincided with a rapid decrease in seed germination. However, mature seeds are usually used in experiments because they can be stored longer than green capsules under refrigeration (Chu & Mudge, 1994). The mature seed of C. debile are relatively easier to germinate than immature seeds, possibly related to their cytological changes. First, the suspensor cell protruded beyond the micropyle opening of the inner testa, making

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the inner testa insubstantial. Second, Nile red staining indicated that the deposition of cuticular material on the testa was fragmentary, which resulted in the less hydrophobic nature of the testa, making it easier for the mature seeds of C. debile to obtain water and nutrients for germination (Table 1; Hsu & Lee, 2012).


Methods to break dormancy In a severely cold environment, ABA and hydrophobic substances, including cuticular substances, polyphenols, and lignin, may help seeds to survive in a stringent climate. Under natural conditions, seasonal changes in temperature, wetting and drying, or mechanical abrasion and digestion by mycorrhizal fungi may be important factors in damaging the testa and carapace of Cypripedium seeds (Lee et al., 2005). Refrigeration (prechilling) The superior germination typical of immature seed suggests that a type of dormancy may exist in mature seed. Cold treatment may be required to break dormancy (Ballard, 1987; Chu & Mudge, 1994; Fast, 1982; Stoutamire, 1974; Tomita & Tomita, 1997). In experiments with C. calceolus var. pubescens, not only was final GP improved by prechilling at 5 C for 8 weeks, but the rate and degree of synchronization of germination were also improved (Chu & Mudge 1994). Seeds incubated for 100 days at 24 C without prechilling had a lower GP (44.8%) on agar medium than with prechilling (91.7%). Ramsay & Stewart (1998) reported that C. calceolus could adapt to a cool climate and that a cold period was not necessary to break dormancy and for germination to occur. Nevertheless, this was not necessarily the case, and it did appear to be beneficial to the long-term health of seedlings if there was a chilling period so that the growing period of the plants was synchronous with the natural seasons. C. reginae seeds have been shown to germinate without cold treatment (Curtis, 1936; Harvais, 1973; Henrich et al., 1981; Linden, 1980; Oliva & Arditti, 1984). However, Stoutamire (1974) and Ballard (1987) reported cold treatment to be essential for C. reginae seed germination. The effects of cold treatment (4 C for 2 months) on the germination of C. candidum seeds were variable and depended on both the medium and time of collection. In general, cold treatment advanced the overall pattern, so that germination peaked at 49 DAP, as opposed to 56 DAP. At this peak, germination was higher on modified Norstog medium with cold treatment than on the same medium at room temperature. With Harvais medium, germination was considerably lower after cold treatment than at room temperature. At 84 DAP, as the seeds approached maturity, germination of cold-treated seeds was greater than of those held at room temperature, but only on modified Norstog and VWD media, and not on Harvais medium (De Pauw & Remphrey, 1993). Chilling C. macranthos seeds at 4 C prior to culture at 20 C was another factor that stimulated germination. Even chilling for 2 weeks promoted germination, and chilling for 12 weeks enhanced it the most: the frequency of germination was 67% after 3 months of culture at 20 C. However, the promotive effects of chilling on germination were reduced by holding seeds at 20 C for 3 and 6 weeks prior to a 4 C


chilling treatment (Miyoshi and Mii, 1998). In C. acaule, treatment for 3–5 months at 5 C and then transfer to 25 C resulted in 70% germination (Coke, 1990). The physiological effect of cold stratification is either a decrease in the level of endogenous ABA or an increase in the content of endogenous cytokinin, both of which can stimulate germination (van Staden et al., 1972). Adequate germination and growth without the use of a cold treatment has also been reported. Some reports found that vernalization did not enhance germination (Ballard, 1990; Harvais, 1973, 1982; Henrich et al., 1981; Lauzer et al., 1994; Linden, 1980; Ling, 1990; Light, 1989; Stoutamire, 1974; van Waes & Debergh, 1986b). For example, Ballard (1990) reported a variable and different response of C. acaule seeds to temperature treatment during incubation in which maximum germination (22%) was achieved independent of chilling in comparison with exposure to 5 C for 1–6 months. Lauzer et al. (1994) reported that rinsing with cold water for up to 14 days before disinfection, or cold stratification of disinfected seeds on germination medium for up 6 months, did not improve germination. A cold treatment (4 C for 2 months) decreased the GP of C. calceolus and C. calceolus var. pubescens (De Pauw & Remphrey, 1993). Softening the testa Germination of ripe Cypripedium orchid seeds can be improved by softening the testa with hypochlorite solution or cell wall-degrading enzymes and by leaching out the inhibiting substance with water (Bae et al. 2009, 2010; Ballard, 1990; Huang and Hu, 2001; Lauzer, 1994; Linden, 1992; Piao et al., 2011; Table 3; Sokoliski et al., 1997; St. Arnaud et al., 1992; van Waes & Debergh, 1986b). Miyoshi & Mii (1998) reported that the duration of the treatment of C. macranthos seeds, with a solution of hypochlorite prior to sowing, was one of the critical factors that affected germination. Approximately 70% of seeds germinated when they were treated with either a solution of sodium hypochlorite (NaClO) that contained 0.5% available chlorine for 60 min, or with one of calcium hypochlorite [Ca(ClO)2] with 3.2% available chlorine for 7 h. Lauzer et al. (1994) reported that increasing the duration of disinfection of C. acaule seeds with NaClO from 40 min to 2 h prompted a significant increase in germination from 3.9% to 10.4%, although there was no germination following longer periods of disinfection (5 and 24 h). Moreover, prolonged exposure to NaClO caused damage to the embryo, thus decreasing germination. Many orchid scientists use a Ca(ClO)2 solution for surface sterilization, because it is more gentle on orchid tissue than NaClO, but Ca(ClO)2 also seems to be more gentle to pathogens and so a longer sterilization period is required. In addition, Ca(ClO)2 easily loses strength when stored as a solid. For these reasons, NaClO is usually used for sterilising and softening the testa in experiments (Lauzer et al., 1994). Sokolski et al. (1997) reported that the GP of the full mature seed of C. reginae was significantly greater and development was faster following longer exposure treatment times (20, 28 or 36 min) than the 12 min exposure in 0.5% NaClO solution after 27 days of culture, and when the full mature seed of C. reginae was soaked in 0, 0.12, 0.25 or 0.5% NaClO

Seed maturity (DAP)

Mature

Mature

Mature (180)

Mature (120)

Mature (91)

Mature (90)

Mature (120)

Mature

Species

C. acaule

C. flavum

C. formosanum

C. guttatum

C. macranthos

C. macranthos var. rebunense

C. macranthos

C. macranthos

0 min 10 min 20 min 40 min 80 min 160 min

0 77.3

0 30 min 45 min 60 min

0 min 10 min 20 min 40 min 80 min

0 40 min 2h 5h 24 h

0.5% NaClO

3.2% Ca(ClO)2

1.0% NaClO

0.12 h 1h 2h 3h 4h 5h 6h 7h 7 min 15 min 30 min 60 min

0 5 15 30 45 60

Control 0 1% NaClO for 50 min, then maintained at 4 C for 3 months in the dark

0.5% NaClO

Control 1.0% NaClO 30 min

1.0% NaClO

0.5% NaClO

0.6% NaClO

Treatment methods

Table 3. Effect of treatment methods on seed germination of Cypripedium species.

9a 18 b 20 bc 33 c 44 d 59 e 63 ef 69 f 18 b 27 c 42 d 72 f

0 0 19.6 b 27.8 a 17.3 b 9.1 c

210 8.9

4.2 3.8 4.0 2.6 2.2 7.8

84

0.1 0.1 0.6 0.8

69 84 80 40 0

3.6 3.9 10.4 0 0

Germination (%)

90

84

HP þ 5.0 mM Zeatin 210

120

POM medium

120

112

180


References

Miyoshi & Mii (1998)

BM1 liquid medium þ 1.0 mM BA

(continued )

Bae et al. (2009)

POM medium

Shimura & Koda (2004)

Modified Harvais þ 1.0 mg/L BA

Piao et al. (2011)

Zhang et al. (2010b)


Bae et al. (2009)

Huang & Hu (2001)

Lauzer et al. (1994)

Harvais

MS þ 1.0 mg/L Kn þ 0.1 mg/L NAA þ 1.2 mg/L PE

Basal medium and medium additives


DOI: 10.3109/07388551.2013.841117

Seed biology of Cypripedium 7

Mature

30

C. macranthos

C. calceolus

Mature (180)

C. formosanum

Ultrasonic treatment

Ultrasonic treatment with ‘‘Sonifer 250’’

Vacuum infiltration (5 kPa)

Chilling at 4 C

1.0% NaClO

weeks weeks weeks weeks weeks

0 30 min 45 min 60 min

0 (embryos inside the seed coat) 3.5 min (embryos inside the seed coat) 3.5 min (embryos outside the seed coat) 7.0 min (embryos inside the seed coat) 7.0 min (embryos outside the seed coat)

Control Treatment Control Treatment Control Treatment Control Treatment

0 2 4 8 12

90 min 7 min 15 min 30 min 60 min

Treatment methods

0.1 0.8 0.14 0.05

7.4

0.4

16.2

1.7

3.5

40 40 60 80 24 56 520 520

26 a 40 b 53 67 d 64 d

36 d 39 d 60 e 59 e 0a

Germination (%)

120

180

100–120

90



MS þ 1.0 mg/L Kn þ 0.1 mg/L NAA þ 1.2 mg/L PE

BM1 medium

BM1 liquid medium þ 1.0 mM BA

Basal medium and medium additives

Lee et al. (2005)

Lauzer et al. (1994)

Wagner & Hansel (1994)


References

S. Zeng et al.

BM1, Van Waes and Debergh (1986b) medium; Ca(OCl)2, calcium hypochlorite; CW, coconut water; DAP, days after pollination; BM1, Basic culture medium 1 (Van Waes and Debergh, 1986b); Kn, kinetin; MS, Murashige and Skoog (Murashige and Skoog, 1962) medium; NAA, a-naphthaleneacetic acid; NaOCl, sodium hypochlorite; PE, potato extract; POM, Phytomax Orchid Maintenance medium (POM, Sigma, USA; Bae et al., 2009)

Mature

C. acaule

60

50

40

Seed maturity (DAP)

Species

Table 3. Continued


8 Crit Rev Biotechnol, Early Online: 1–14



DOI: 10.3109/07388551.2013.841117

solution for 12 min, the GP of the seed was higher and development progressed faster at higher concentrations after 21 or 28 days of culture, but were not significantly different after 35 or 42 days of culture. Lee et al. (2005) reported that by soaking mature seeds (180 DAP) of C. formosanum in 1% NaOCl the GP could be increased slightly, while soaking from 45 to 60 min resulted in better germination (0.6 and 0.8%, respectively) than the control (0.1%), although soaking seeds with 1 N NaOH or 1 N HCl did not promote seed germination. The GP of C. macranthos seeds increased by soaking them in 0.5% NaOCl for 160 min, but the GP did not increase with 0.5% NaOCl for 10, 20, 40, or 80 min. Huang and Hu (2001) reported the GP of C. flavum seeds was highest (84%) by soaking with 0.5% NaOCl for 10 min, but the seeds did not germinate by soaking in 0.5% NaOCl for 80 min. The frequency of protocorm formation in C. guttatum and C. macranthos on Phytomax Orchid Maintenance (POM) medium was 77.3 and 27.8%, respectively, by soaking with 1.0% NaOCl for 30 min, while the seeds of both species did not respond or germinate without NaOCl treatment (Bae et al., 2009). The testas of Cypripedium orchid seed are composed of lignin and cellulose and not cutin (Baskin & Baskin, 1998; Carlson, 1940). Bae et al. (2010) reported that the brown testa of C. macranthos turned white or transparent in the presence of 1.0% NaOCl for 30 min and that the zygotic embryos inside the testas could be seen. Scanning electron microscopy (SEM) revealed that NaOCl treatment resulted in small perforations of the testa that affected its rigidity by decomposing cell wall materials. Ultrastructural observation by Transmission Electron Microscopy (TEM) revealed that the cell wall of the testa, made up of three layers, remained intact without NaOCl treatment. However, after 1.0% NaOCl pretreatment for 30 min, cell walls had disintegrated with loosely aggregated fibrillary cell wall components in all three layers and some fragments were liberated from cell walls that spread to the inside of the testa, which might facilitate the absorbance of nutrients into seeds to support the growth of zygotic embryos. Treatment with ultrasound and vacuum infiltration The use of ultrasound has been used to increase the germination of mature seeds of C. acaule (Lauzer et al., 1994) and C. formosanum (Lee et al., 2005). Lauzer et al. (1994) reported that embryos of treated seeds released from the testa and treated with ultrasound with a ‘‘Sonifer 250’’ for 3.5 min allowed a third of the embryos to become completely free from the testa and prompted 16.2% germination of the released embryos, in comparison with 3.5% in seeds without ultrasound treatment, and only 1.7% for embryos inside the testa. However, following ultrasound for 7 min, GP was 7.4% for released embryos, and only 0.4% for embryos inside the testa (Table 3). Lee et al. (2005) reported 30 min of ultrasonic treatment with a Branson 8210 of mature seeds (180 DAP) of C. formosanum increased germination (0.8%) compared with the control (0.1%), but longer periods of ultrasonic treatment for 45 or 60 min resulted in lower germination (0.14 and 0.05%, respectively).

9

Vacuum infiltration with nutrient solution promoted the germination of C. calceolus when lignification began in the testa. The treatment was ineffective for very young (30 DAP) and fully mature (60 DAP) orchid seeds. Samples were infiltrated using an intermittent vacuum (5 kPa) under a bell jar connected to a water vacuum pump. A vacuum was applied to young, fragile seeds three times for 5 min and to older seeds twice for 10 min. By 30 DAP, 40% germination was possible. The living integument cells play important nutritive and activating roles for the growing embryos, vacuum infiltration of seeds with liquid nutrient medium did not promote germination in this development stage. By 40 DAP and 50 DAP, following treatment, the mean GP was nearly 80% and 56%, the control values for untreated seeds were about 60% and 24%, respectively. By 60 DAP, the GP dropped to below 20%, with small differences between infiltrated and control seeds (Wagner & Hansel, 1994; Table 3).

Factors influencing germination of Cypripedium seed Media Basic media A large number of medium types were tested in Cypripedium seed germination (Table 4). The components of the main basal media used during seed germination of Cypripedium species have been listed in Supplementary 2. Cypripedium prefers a low salt medium and nitrogen source plays a critical role in germination (Fast 1982; Piao et al., 2011; van Waes & Debergh, 1986b). C. reginae seed did not germinate on agar made up with sterile distilled water or on mineral medium alone (Harvais, 1973). Germination and growth of C. reginae were compared on four media including B5, MS, Pf3.6 at pH 5.5 and Pf3.6 at its natural pH 4.5. Pf3.6 at pH 4.5 was best while all cultures on MS died (Harvais, 1982). De Pauw & Remphrey (1993) sowed Cypripedium seeds on Harvais, van Waes and Debergh (VWD) and modified Norstog (1/2 Norstog macroelements) media. There were no significant differences in GP between media for C. reginae. There was no significant difference between media at 64 DAP in C. calceolus var. parviflorum, and at this stage, the GP of the three Cypripedium spp. was highest than at other stages. However, at 70 DAP and 84 DAP, GP was highest on 1/2 Norstog macroelements compared with the other two media (Harvais and VWD). At 42 DAP, the GP of the three Cypripedium spp. was highest and protocorm development was faster on 1/2 Norstog macroelements for all three species and at all periods of collection. Lee et al. (2005) reported that seed GP (from 48.6 to 59.5%) of C. formosanum collected at 90 DAP was not significantly different on four tested media [Harvais, modified MS, Thomale GD (Thomale, 1957), modified Norstog]. At 120 DAP, even if germination was higher on Harvais medium, seed GP declined (ranging from 2.7% to 7.3%). Seed GP was poor (0–0.04%) when seeds were collected at 150 DAP. The authors inferred that the composition of the media might be a critical factor affecting the GP of C. formosanum, although the timing of seed collection was crucial. Oliva & Arditti (1984) reported seed germination of nine species, hybrids or varieties of Cypripedium on three media

10

S. Zeng et al.


Table 4. Effect of basal medium on seed germination of Cypripedium species.


Media

Germination Germination (%) period (days)

Species

Modified Norstog C. reginae (64 DAP seeds) VWD Harvais Modified Norstog C. calceolus var. Parviflorum (64 DAP seeds) VWD Harvais Modified Norstog C. candium (42 DAP seeds) VWD Harvais Harvais C. flavum Modified Harvais VWD Zak MS BM1 (Van Waes and Debergh, 1986b) C. macranthos var. rebunense HP MS C. macranthos VWD Harvais Modified Harvais Hyponex I Modified MS C. macranthos Modified VWD Modified Harvais Modified KC

61 47 56 34 42 34 45 46 30 75 884 80 60 40 7.3 8.9 8.67 38.67 68.00 23.33 0 25.96 68.06 60.22 28.50

References

140

De Pauw & Remphrey (1993)

112

Huang & Hu (2001)

60

Shimura & Koda (2004)

64

Piao et al. (2011)

49

Deng et al. (2012)

Harvais, Harvais (1982) medium; HP, Hyponex-peptone medium (Shimura and Koda, 2004); JA, Joseph Arditti medium (designated by Jo et al., 2001); KC, Knudson’s C medium (Knudson, 1946); MS, Murashige and Skoog medium (Murashige and Skoog, 1962); VWD, Van Waes and Debergh medium (Van Waes and Debergh, 1986b); Zak medium (Arditti, 1982)

(full or half-strength and modified Curtis; Norstog; Hyponex) and obtained the following results: (1) The best overall germination of mature seeds of Cypripedium was on Hyponex medium. (2) C. reginae germinated well on Hyponex and modified Curtis media. (3) Germination of C. californicum and C. montanum was enhanced by full or half-strength Curtis medium when the pH was 7.0–7.5. (4) C. calceolus seeds germinated more rapidly on Curtis medium (Curtis, 1943). (5) Immature seeds of C. acaule and C. pubescens var. parviflorum germinated well on Norstog, Hyponex and fullstrength Curtis media. Huang & Hu (2001) reported the effect of five basic media, including modified Harvais, Harvais, VWD, MS and Zak, on C. flavum seed germination. The former three were suitable for seed germination with a GP of about 90, 75 and 80%, while the GP of the latter two media was about 40% and 60%. Piao et al. (2011) reported the most suitable medium for C. macranthos germination was Harvais medium among five tested media (modified Harvais, Harvais, VWD, MS, Hyponex), although Deng et al. (2012) reported modified VWD to be better than modified Harvais, modified KC and modified MS. Plant growth regulators Seed germination of Cypripedium species is usually enhanced by the inclusion of PGRs in the medium (Table 5). Orchid seeds have no endosperm and no cotyledons and lipid droplets in the embryo are the primary storage material (Arditti & Ernst, 1984). Cytokinins (CKs) may play an important role in lipid mobilization, and thus the requirement for CKs in the

germination medium may be related to the seed’s utilization of lipid (De Pauw et al., 1993). De Pauw et al. (1995) reported improved GP of C. candidum in the presence of N6-benzyladenine (BA) and 6-(a,a-dimethylamino)-purine (2iP) up until 0.8 mg/L, while germination in the presence of kinetin (Kn) was not significantly different from the control (i.e. without CKs). De Pauw et al. (1995) also reported protocorm morphology and development were significantly affected by the different CKs in the media. Miyoshi and Mii (1998) reported 58–70% GP of C. macranthos seeds after the addition of 1 mM CK including BA, thidiazuron (TDZ), zeatin, 2iP or Kn, in comparison to 17% in BM1 liquid medium without CKs or 20% in medium with 1.0 mM N-(2-chloro-4-pyridyl)-Nphenylurea (4PU). However, Bae et al. (2010) noted that the mature seeds of C. macranthos could germinate on POM medium without any PGRs following pretreatment with 1.0% NaClO for 15–60 min (Table 3). Harvais (1982) reported that GA3 up to 5 mg/L had no effect on germination of C. reginae and early development until shoot formation, when it caused marked abnormal shoot elongation with spindly leaves. Lauzer et al. (1994) reported that 10 mg/L GA3 decreased the seed germination of C. acaule. Aminopurines are not only the major PGRs affecting germination, they also play important roles in development and morphogenesis. Auxins could counteract the adverse effects of supraoptimal levels of CKs on germination. For C. reginae, all cultures with a CK (1 mg/L Kn): auxin [0.1 mg/L of a-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D)] ratio of 10:1 were markedly better


DOI: 10.3109/07388551.2013.841117

11

Table 5. Effect of plant growth regulators on seed germination of Cypripedilum macranthos.


Seed maturity (DAP)

Media

Mature

BM1 liquid medium

Mature (56)

VWD þ 10% CW þ 0.5 g/L AC

Plant growth regulators None 4PU 1.0 mM BA 1.0 mM TDZ 1.0 mM Zeatin 1.0 mM 2iP 1.0 mM Kn 1.0 mM Kn Kn Kn Kn

None 0.3 mg/L 0.6 mg/L 1.2 mg/L 2.4 mg/L

Germination (%)


References

17 20 58 59 49 58 70

90


9.09 14.62 19.23 22.91 30.33

49

Deng et al. (2012)

2iP, 6-(a,a-dimethylamino)-purine; 4PU, N-(2-chloro-4-pyridyl)-N-phenylurea; AC, activated charcoal; BA, N6-benzyladenine; BM1, Basic culture medium 1 (Van Waes and Debergh, 1986b); CW, coconut water; DAP, days after pollination; TDZ, 1-phenyl-3-(1,2,3-thiadianol-5-yl)urea (thidiazuron); VWD, van Waes and Debergh medium (van Waes and Debergh, 1986b)

than the control (1 mg/L Kn). Among the auxin treatments, the NAA-treated cultures were significantly better than the other treatments, with the best, largest, healthiest protocorms and plantlets forming in this order: NAA4IAA4IBA4 2,4-D4control. Huang & Hu (2001) reported Harvais medium with 0.4 mg/L Kn or 0.2 mg/L BA to be the most suitable for seed germination of C. flavum. Deng et al. (2012) reported 1.2 mg/L Kn to be the most suitable concentration for germination of C. macranthos, but Piao et al. (2011) reported that BA had a better effect than Kn for this species, 0.5 mg/L BA being the most appropriate level. Carbohydrates For germination in vitro, some terrestrial orchid species can germinate on pure water-based agar, but Cypripedium require soluble sugars (Rasmussen, 1995). Carbohydrates serve as an energy source in the medium, while osmotica have significant effects on orchid seed germination and protocorm development. Soluble sugars in asymbiotic media are usually added in concentrations between 10 and 20 g/L in the form of glucose, fructose or sucrose (Knudson, 1946; Rasmussen, 1995). Leroux et al. (1995) reported that on medium containing 2% dextrose, proembryos of C. acaule developed and formed a protocorm. On medium without dextrose, the proembryos developed slowly into a protocorm. Harvais (1973) reported seed germination of C. reginae on a medium with three sugar sources, in which 1% was better than 2%, sucrose was better than dextrose, and dextrose better than fructose. Ballard (1987) reported a 69% GP of C. reginae on medium supplemented with sucrose, 51% with dextrose, and 61% with trehalose, all of them at 20 g/L. In an experiment with sucrose, seed GP was 65% on a medium with 20 g/L sucrose, 63% with 15 g/L sucrose, and 60.5% with 10 g/L sucrose. Organic amendments Cypripedium seed germination and protocorm development are stimulated or inhibited by organic amendments (Chu & Mudge, 1994; Deng et al., 2012; DeMarie et al., 1991; Steele, 1995; Yan et al., 2006). Organic amendments include coconut

water (CW), apple homogenate (AH), banana homogenate (BH), potato homogenate (PH), casein hydrolysate (CH), pineapple extract (PE), yeast extract (YE), tryptone, peptone or pure amino acids, such as glutamine (Rasmussen, 1995). They are often acidic and contain soluble sugars, amino acids, vitamins and PGRs (Arditti, 1982). Potato extract or L -glutamic acid at 10% (w/v) (Harvais, 1973, 1982) and 100 mL/L CW drained from a fully ripe coconut (Chu & Mudge, 1994) could promote the GP of C. reginae, but CH and YE had a deleterious effect. However, DeMarie et al. (1991) reported the stimulation of germination and protocorm development of C. reginae by YE. Muick (1978) reported that by adding CW, BH, yeast and sugar, a sufficient amount of mycelia developed, which caused C. calceolus seed embryos to germinate. In C. calceolus var. pubescens, 1/4-strength MS, supplemented with 10% CW, led to synchronous germination (Chu & Mudge, 1994). Huang & Hu (2001) reported that 5% CW, BH or PH in Harvais medium could accelerate C. flavum seed germination, but the effect was not significantly different among 5% CW, BH or PH or without organic amendment. Harvais media supplemented with 0.5 mg/L BAP and 20 g/L PH was the most effective (Yan et al., 2006). Modified Harvais medium supplemented with PH or AH could accelerate C. macranthos seed germination, but not if supplemented with BH or CH (Piao et al., 2011). Vitamins and other components Many media recommended for orchid growth, meristem and cell culture commonly include niacin, pantothenic acid, thiamine, pyridoxine and biotin (Arditti, 1967; Harvais, 1973). Vitamins supplemented in media are usually thought to be beneficial during the heterotrophic phase of growth. Harvais (1973, 1982) reported that i-inositol improved germination, while niacin and i-inositol were useful for the general health of C. reginae protocorms and plantlets; pantothenic acid, thiamine and pyridoxine improved shoot development and leaf broadening, the best result being obtained with pantothenic acid, then thiamine, and least of all, pyridoxine. Harvais (1982) reported that a mixture of 50 mg/L L-glutamic acid and 1 mg/L Kn stimulated the germination and growth of C. reginae seeds.

12

S. Zeng et al.


Activated charcoal The first attempt to darken a culture medium for orchid-seed germination seems to have been made in an effort to germinate native American Cypripedium (Curtis, 1943). Darkening with activated charcoal had a clearly positive effect on the growth of other Cypripedium spp. seedlings (Bae et al., 2009; Ernst, 1974, 1975; Yan et al., 2006). However, Chu & Mudge (1994) noted that activated charcoal did not stimulate the seed germination of C. calceolus var. pubescens. Activated charcoal not only absorbs toxic substances, including phenolics, but also creates partial darkness and stabilizes the pH to some extent (Rasmussen, 1995). Darkness is similar to the underground environment of Cypripedium in its natural habitats, and thus benefits seed germination. In addition, activated charcoal may also improve aeration and could add microelements, establish polarity, affect substrate temperature, and may gradually release certain adsorbed products, such as nutrients and PGRs (Johansson et al., 1990), which would benefit seed germination and seedling growth. However, inevitably, activated charcoal absorbs certain compounds (including certain vitamins and PGRs), which may be harmful to seed germination and seedling growth (Ernst, 1974; Yam et al., 1990). pH In most studies of Cypripedium seed germination, germination was possible at a pH of 5.0–6.0, with the pH of most media usually being between 5.5 and 5.7 (Harvais, 1982; Chu and Mudge, 1994; Bae et al., 2009, 2010). For C. macranthos, pH 5.5–5.6 was more suitable for seed germination, than other pHs in the range of 4.9–5.5 or 6.1– 7.7. However, for C. flavum, GP was not significantly different at any pH between 4.9 and 6.7, although if the pH exceeded the lower or upper limit of this scope, GP decreased. Oliva & Arditti (1984) reported that C. californicum and C. montanum could germinate on full or half-strength Curtis medium with pH 7.0–7.5. Harvais (1982) reported germination and growth of C. reginae was the better on Pf3.6 at pH 4.5 than Pf3.6 at pH 5.5. Culture temperature The best GP for many orchid species was usually achieved between 22 and 25 C, but in most studies on Cypripedium, seed germination was only possible below 20 C (Stoutamire, 1974), and 23–26 C was usually used in experiments after a 4 C pre-treatment or no treatment (Bae et al., 2009, 2010; Chu & Mudge, 1994; De Pauw & Remphrey, 1993; Lauzer et al., 1994; Shimura & Koda, 2004). After seed germination, there was better germination and growth of C. reginae at 25 C than at 15 C (Harvais, 1973). Light Germination is impeded or prevented by illumination in terrestrial orchid species and thus darkness is often required for germination in vitro (Ballard, 1987; Rasmussen et al., 1990). Harvais (1973) found that C. reginae seeds germinated better in the dark than in a light regime. Although some germination and growth did take place in the light, the


protocorms died before reaching the first-leaf stage. Oliva & Arditti (1984) found that illumination seemed to have had no significant effect on the germination of C. acaule seeds, which could germinate in the light or darkness. St. Arnaud et al. (1992), however, found that light positively influenced seed germination and protocorm survival of this species. Protocorms must reach a specific size before they are transferred to light. Independent of the initial size of protocorms, protocorm survival was always over 95% in darkness, but in light, survival was dependent on the initial size of the protocorm: after 6 months, the survival percentage increased from 6.7% with smaller protocorms (stage 1) to 60% with largest protocorms (stage 4). Light has been shown to inhibit seed germination in C. reginae, C. irapeanum, C. acaule, C. californicum, and C. candidum (Ballard, 1987; Harvais, 1973; Linden, 1980; Muick, 1978; Stoutamire, 1974). Harvais (1973) showed better germination in the dark than in the light, and even though some germination and growth took place in the light, the protocorms died before reaching the first-leaf stage. Protocorms that germinated in the dark were transferred to light after 4 or 8 weeks, but all protocorms died soon after. However, if the protocorms that germinated in the dark were transferred to light after 12 weeks, despite high mortality, those protocorms that survived were quite healthy and thrived well. Yet, by the end of this experiment, the protocorms grown continuously in the dark were less developed than the surviving protocorms in the light. Harvais (1973) reported that germination of C. reginae in the dark was much better than in the light, and even though some germination and growth did take place in light, the protocorms died before reaching the first-leaf stage. All protocorms that germinated in the dark died when they were transferred to light 4 or 8 weeks after germination. However, the protocorms that survived were quite healthy and thrived well even though the mortality of protocorms transferred was high 12 weeks after germination.

Liquid culture Harvais (1982) found that C. reginae seeds in liquid culture media could germinate, although protocorms and green plants in liquid culture died. Chu & Mudge (1994) found that final GP, rate of germination and degree of synchronization of C. calceolus var. pubescens in liquid suspension culture (LSC) were as good as the effects of prechilling (8 weeks at 5 C). Final GP (160 days of after sowing) of prechilled seeds incubated in LSC was nearly twice (more than 90%) as much as GP (45%) of non-prechilled seeds on agar media. LSC without prechilling resulted in 80% seed germination; moreover, LSC could substitute prechilling (8 weeks at 5 C). Hsu & Lee (2012) noted that liquid culture promoted the germination of mature seeds of C. debile. Dilution of phytotoxic exudates or testa inhibitors in LSC may improve the GP of orchid seeds (Arditti, 1967).

Micropropagation of Cypripedium As Cypripedium explants are often recalcitrant in tissue culture, there are not more than ten studies to date on the micropropagation of Cypripedium. The explants used in these studies only included root tips, nodes of in vitro seedlings and


DOI: 10.3109/07388551.2013.841117

seed- or protocorm-derived callus. Owing to constraints of length of the paper, the summary of micropropagation of Cypripedium is in Supplementary 3.


Shoot growth and rooting Shoot growth and rooting of Cypripedium are significantly affected by medium choice. Bae et al. (2009) reported that the growth of shoots and roots was more effective in POM medium than on MS medium (100% and 80%, respectively). Roots were significantly longer in protocorms cultured on POM medium than on 1/3 POM medium. Bae et al. (2009) on 1/2 Harvais media supplemented with 0.6 g/L AC, after 4 weeks, 100% of plantlets rooted. Vernalization can lead to the development of a new rhizome segment instead of leaf development (Stewart & Mitchell, 1991). Bae et al. (2009) reported that cold treatment (4 C) was necessary to break the dormancy of C. guttatum protocorms, especially to stimulate shoot and root growth.

Greenhouse acclimatization and field establishment The transplanting of Cypripedium seedlings in vitro is usually difficult because of the inevitable formation of dormant buds. Bud dormancy in C. acaule reportedly could be overcome by 3–4 months postchilling at 4 C (Ling et al., 1990). The survival percentage of C. flavum was 60% in a greenhouse at 18–22 C and 80% humidity (Yan et al., 2006). Ramsay & Stewart (1998) reported that when C. acaule seedlings were potted in Seramis compost (4:1 mixed pine and leaf mould) in northern England, approximately 50% of the seedlings produced leaves in May that remained green until October, 30 of these plants producing dormant buds until the following year. Only 10% of the plants in loam compost (sieved loam collected from the wild site mixed with grit, leaf mould and pine bark) produced leaves which did not persist during summer. Chu & Mudge (1994) reported that when 11-monthold C. calceolus var. pubescens seedlings were transplanted to a greenhouse into sphagnum moss or a mixture of course sand and perlite (1:1), 80% of seedlings showed no browning and survived. Frosch (1986) noted that 32 in vitro C. reginae plants that were planted were able to flower normally. The rhizome of Cypripedium spp. requires a constant dormancy period of at least three months to maintain healthy growth in the following season. When in full dormancy, the species requires a cold, constant winter and can then tolerate severe frosts. If dormancy is interrupted by warm spells that stimulate growth, Cypripedium spp. might die (Plummer, 2000).

Conclusion The germinative ability of Cypripedium seeds can depend on the capsule, origin, season or growth stage. Many factors, including physical and nutritional, affect the germination of Cypripedium seed, and up to now, none of the reported protocols of Cypripedium propagation can be used for all Cypripedium species.

Declaration of interest This study was supported by the Guangdong Key Technology Research and Development Program (2011B020304004; 2010B060200037).

13

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