Optimization of in vitro asymbiotic seed germination protocol for Serapias vomeracea

Serapias vomeracea is a wide-spread, perennial orchid species that can be found in arid meadows, on abandoned agricultural land and heathlands up to a height of 1000 m. The plant’s distribution extends over Mediterranean-Atlantic countries (Portugal, Spain, France, Italy, the Balkans, Greece, and Turkey). However, it is also found in North Africa (Morocco and Tunisia) where it is under high pressure of inbreeding depression (1). This species is one of the most widespread tuberous orchids in Turkey. Thus it is easily accessible in many cities and regions. Ease of accessibility in the environment and the economic value of its tubers make S. vomeracea one of the most collected orchid species from its native habitats (2). The component that makes the tubers of orchids so valuable is the polysaccharide glucomannan. Glucomannan is obtained from the tubers of orchids and the Konjac plant (Amorphophallus konjac). Glucomannan is currently used as a raw material for Salep production which is a traditional hot beverage in Turkey and is also used in food supplements, especially in weight loss capsules and other nutritional supplements, due to its hydrophilic properties (2). Also, the polysaccharide glucomannan is used as a thickener in the food industry and has been used to

treat high cholesterol (3, 4). Even though tuberous orchids are not yet extinct, there is evidence of population decline due to over-collection because of increasing commercial value and rising demand from industry. Therefore, an effective strategy should be designed, not only to conserve them in nature but also to take advantage of their economic potential through scientific investigation and development of commercially viable farming. One element of this strategy would be to develop and standardize both the optimum seed surface sterilization method and an efficient in vitro seed germination protocol that can be applied to economically valuable orchids including exotic hybrid ones, on a commercial scale.

As orchids can produce countless, dust-like seeds in their ovaries, the seeds have been mostly preferred as starting materials in tissue culture studies on terrestrial orchids (5). However, orchids are slow-growing plants in nature, and their seeds require a mycorrhizal relationship to germinate (6). Thus their seed germination rates are very low in nature. In contrast, in vitro cultures can offer an alternative for faster and more efficient production of orchids, and similar plants, that are difficult to germinate and develop in nature (7). Examination of various culture media, especially those developed mainly for orchids, can offer a detailed understanding of their nutritional requirements for in vitro germination.

In surface sterilization experiments of many in vitro germination studies, disinfectants such as ethyl alcohol (EtOH), sodium hypochlorite (NaOCl), calcium hypochlorite (CaOCl), hydrogen peroxide (H₂O₂), silver nitrate (AgNO₃) and mercuric chloride (HgCl₂) have been commonly used (8). However, less toxic chemical compounds should be used in the surface sterilization treatment of orchid seeds since they have thin seed coats which make the embryo particularly vulnerable to toxic chemicals such as disinfectants (9). In this context, in addition to disinfectant selection, treatment duration is also important.

Another major factor is the method of surface sterilization used for orchid seeds. Orchid seeds are generally weighed and put into a pouch made from filter paper before they are dipped into disinfectants for surface sterilization following which they are sown using a scalpel or inoculation loop (10, 11). However, because the seeds are so small and difficult to handle a large number of seeds remain in the paper pouches.

As can be seen, there is scope for a range of improvements in the successful culturing of orchids including a selection of the optimal sterilizing agent, improved seed recovery following sterilization and selection of the optimal culture medium. Also, by investigating the optimal in vitro seed germination medium for Serapias vomeracea a better understanding of the plant’s developmental physiology would be forthcoming, which would be of benefit in the development of controlled farming of this glucomannan-rich species for industrial purposes. Therefore, this study aimed to optimize the surface sterilization protocol for S. vomeracea seeds and to select the optimal seed germination medium by comparing the commonly used media in in vitro orchid culture.

When using seeds as starting materials in tissue culture studies the most important measure of success is the percentage of the seeds which germinate. Successful germination will be due to the fact that the seeds cultured on the medium receive water and swell and take macro- and microelements from the medium (16). Therefore, it is necessary to eliminate the waxy compounds that may prevent the water from entering through the seed coat and thus to convey a hydrophilic nature to the seeds (17). It has been shown that ethyl alcohol provides a hydrophilic structure to the coats of orchid seeds by eliminating pectin, suberin and other waxy compounds present on the coat, thereby ensuring effective contact with NaOCl during surface sterilization (18). In addition, the same researchers reported that pretreatment with EtOH significantly increases germination percentages. Although the composition of hydrophobic compounds on the seed coat varies from species to species, the improved results with EtOH showed that the hydrophobic compounds on the seed coat of S. vomeracea might be too dense to be eliminated by NaOCl sterilization alone. Ponert et al. (18) also recommended placing the seeds in a syringe during surface sterilization treatment in order to prevent seed loss during sowing. In addition, it should be noted that since the microbial load of orchid seeds may differ, the durations of the surface sterilization applications may vary for each study. The seed quantity is also another important factor affecting the success and the length of the surface sterilization process. For example, Ercole et al. (19) used 25 mg of S. vomeracea seeds per centrifuge tube but subjected them to 1 ml of 1% NaO-Cl solution for 20 min. In that study, no seed viability test was employed, but the authors reported higher germination rates of up to 95% in symbiotic cultures on solid oat medium. However, the authors did not assess the developmental stages of the seeds. Furthermore, considering that 1 mg of S. vomeracea seeds would be between 180-200 individual seeds, counting 25 mg of seeds under the microscope at the beginning, middle and the end of the incubation period would be much more time-consuming than our method, and may result in counting inaccuracy due to human error.

In this study, the effectiveness of the TTC test was increased by keeping the seeds in dH₂O for 24 hours. The reason for this is likely to be due to the onset and/or acceleration of metabolic activities in the seeds receiving water. An additional effect may be because of the increased permeability of the seed coat due to the oxidation of lignin (20). In a study on Orchis coriophora, Bektas et al. (21) incubated the seeds in 5% sucrose with several drops of commercial bleach solution for 12 hours before the TTC test. Seed viability was found to be 54±2.1%. However, in a similar study on S. vomeracea, the result of seed viability was not indicated (22). Plant seeds need a carbon and energy source until they germinate and become autotrophic when cultured in vitro (23). This is more evident in non-endosperm-containing orchid seeds. Therefore, sucrose application may increase seed viability results on the TTC test by also accelerating metabolic activities.

The seeds of epiphytic and terrestrial orchids have been reported to germinate faster and contain more water when they are grown symbiotically. This is because mycorrhizal fungi increase the seed germination rate by facilitating water, carbon and nutrients intake (24). Thus, the length of germination time in asymbiotic cultures may be related to the slower water, carbon and nutrients intake (25). In this study, although solid KN medium was found to be the best overall inducer of germination, higher germination rates were generally observed from liquid media. This finding suggests that liquid media facilitates easier intake of water and other required nutrients compared with solid media. In this context, epiphytic Doritaenopsis orchid cultures have been reported to germinate faster in liquid medium than solid medium, but the overall germination rate remained the same at the end of the incubation period (26). A general observation from our study was that at the end of extended incubation periods the rhizoids of the protocorms were much shorter in liquid media, but in the solid media, the rhizoids developed normally. This may be related to the pressure and/or surface tension of the liquid culture on the protocorms and/or to easier access of the protocorms to nutrients. Rhizoids are primitive root precursors and conduits for nutrients to the protocorms. Rhizoids also play a role as carbohydrate depositories for subsequent stages of growth (27). Since the seeds float in liquid media and are not present on a single solid surface as with solid media, there are disadvantageous in using liquid media during the seed counting step. Also, it is necessary to agitate the cultures during incubation to prevent elements and the seeds precipitating in liquid media. For all these reasons, it is recommended to use solid media for culturing S. vomeracea.

Compared to LN medium used in the experiments, KN and VW media are the simplest ones in terms of microelements, vitamins, and amino acids. However, both media gave the highest seed germination results among the liquid cultures. Macroelements cover the largest portion in culture medium composition. Therefore, this difference in germination success can be attributed to the differences between the macroelement compositions of the media. LN medium contains 46% less of KH₂PO₄ than KN and VW medium. VW medium contains Ca₃(PO₄)₂ while KN medium contains NH₄H₂PO₄ which act as additional phosphate sources. Therefore, the success of germination of these liquid culture media may be due to increased availability of phosphate to the seeds. Rich phosphate content in Mishra medium was found to be responsible for increased seed germination rates in Cymbidium eburneum (28). In nature, enhanced plant development due to increased phosphate uptake as a result of mycorrhizal symbiosis was reported for Goodyera repens (29). Rodrigues et al. (30) stated that the initial phosphorus (P) demand in young orchid plants may be greater than that of adult plants. Therefore, phosphate-rich media should be considered in asymbiotic germination of S. vomeracea seeds. However, KN medium which had the highest seed germination rate, is also distinguished from the other media by the higher total nitrogen ratio, mainly coming from NH₄NO₃ and Ca(NO₃)₂. Thus, the success of germination in solid media may also be related to the presence of nitrate, which is present in much greater amounts than phosphate. Furthermore, in addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly nitrogen-enriched (31). Similar to the findings obtained in this study, Gümüş and Ellialtıoğlu (11) reported that the highest germination rate of S. vomeracea seeds (76.85%) was found when using KN medium, whereas seed germination decreased down to 47.51% after a reduction of inorganic nitrogen sources from the medium. They supplemented the KC medium with 20 g L^-1 sucrose but collected the germination data after 90 d of incubation in the dark. Bektaş and Sökmen (22) stated that the germination rate of S. vomeracea seeds in KN medium without sucrose was only 27.95±2.4% after 60 days of incubation. Furthermore, at the same incubation period, the authors reached to 73.74±1.6% germination ratio after testing Orchimax medium which is enriched by trypton to provide an additional source of reduced organic nitrogen, vitamins and nutritional agents. The medium also includes activated charcoal and MES 2-(N-Morpholino)ethanesulfonic acid buffer. However, the authors showed that absence of activated charcoal in Orchimax medium reduced the germination ratio down to 43.11±1.5%. Therefore, the main effective component is activated charcoal in this complex medium. In our study, we showed that there is no need to use such a complex medium, but simple Knudson C medium can offer an easy-to-prepare and cost-effective alternative to other complex media. In the present study, 58.77±5.51% of the seeds germinated on sucrose-enriched KN medium after 45 days of incubation. It is evident that the germination percentage of the seeds is closely related to the extent of the incubation period. If the incubation period is extended, more germinated seeds will be counted. KN medium showed high germination rates also for Calopogon tuberosus var. tuberosus (32), Dendrobium aphyllum, Rhyncostlis retusa, Cymbidium longifolium (33) and C. aloifolium cultures (34). Our results also showed that glycine amino acid and vitamins such as nicotinic acid, pyridoxine, thiamin, and myo-inositol which are completely absent from KN media and largely absent from VW media are not required for the germination of S. vomeracea seeds. However, at subsequent developmental stages, the nutritional needs of the plant would differ.

The additional low-g centrifugation treatment in our seed surface sterilization method successfully separated empty seed coats (with no embryo) from the seeds with embryos using their mass difference. This method is ideal for eliminate counting errors, especially for stage 0 and 1 seeds, since it removes the non-viable seed coats before sowing. Here, a rapid pre-treatment with EtOH is also advised to enhance the effectiveness of the TTC test to achieve more reliable results. Our study showed that the use of solid KN medium appears to be the optimum choice for S. vomeracea seed culture germination. The medium is known for its simplicity and ease of preparation while offering various modification possibilities that can enhance germination success. Based on the KN medium, the addition of several growth regulators such as auxin or cytokinin derivatives, or promoters such as oligomeric or polymeric chitosans, natural carbon sources such as banana homogenate or coconut water may enhance the effectiveness of KN medium in germinating S. vomeracea seeds, but further study would be required to confirm this. The outcome of this study suggests that our improved surface sterilization method and use of a solid KN medium may be a simple, cost-effective and reliable alternative to commonly used sterilization methods and use of other more complex media for S. vomeracea cultures.