Influence of scarification method on seed germination of the terrestrial orchid Anacamptis laxiflora (Lam.)

Because orchid seeds are microscopic and lack endosperm, they establish an association with symbiotic fungi called mycorrhiza (21). In nature, this association is necessary for germination as it supplies nutrients to the seeds, including C. Reproducing the mycorrhizal association in the laboratory is possible (11). However, because it adds complexity, many practitioners opt to supply the nutrients via a sterile nutrient medium. Numerous recipes exist for nutrient media for terrestrial orchid propagation (11). Because the majority of terrestrial plants require the same nutrients for growth and development (22), most recipes share a few base ingredients in various proportions.

In this study, a modified version of “SM-organic” medium was used, a time-tested formula by veteran practitioner Svante Malgrem published by Rasmussen (11). The term “organic” refers to the N source, an amino acid mixture sold under the trade name Vaminolac® (Fresenius Kabi, Uppsala, Sweden). In Greece, Vaminolac® cannot be sold without prescription as it is considered a medicine. Therefore, it was substituted by Amina-Fe (Humofert, Metamorfosi, Greece), a liquid fertilizer containing amino acids and chelate Fe. Because Amina-Fe more than covers the Fe requirement in Malgrem’s recipe, the Fe salt was omitted. Further, Ca₃(PO₄)₂ was replaced by CaHPO₄. The use of Danish agar necessitated increasing the amount from the original recipe to achieve proper gelling. The formula is listed in Table 1. After mixing, pH was adjusted to 5.5–6.0 with a few drops of 0.1 M H₂SO₄ solution. Finally, the medium was transferred to a 1 L volumetric flask and stored in a plastic bottle at 4°C.

A commercial formulation of household bleach (Klinex®, Unilever, London), was used as stock solution. The nominal concentration of the commercial product (4.8% w/w) had been previously verified (10). The stock solution was diluted to 0.5%. A drop of Tween® 20 (Merck, Darmstadt, Germany) was added to reduce surface tension.

Presoaking the seeds in a sucrose solution was the initial step in the PRESOAK method. According to Hicks (16), sucrose encourages the growth of microorganisms, making them more susceptible to sterilization. There is little information whether the concentration of the solution has an effect. Some practitioners use a dilute solution, whereas others use up to 50% saturated solution. Hicks (16) prepared a solution by adding approximately the same amount of sucrose and seeds in his test tubes. For this study, a 10% sucrose solution was used.

The autoclavable materials (nutrient medium, potato cubes, polypropylene funnels, filter papers, Erlenmeyer flasks for draining the suspension, deionized water, beaker for disposing liquid waste, spatula, pair of pliers, absorbent paper) were wrapped in aluminum foil and sterilized in a Tuttnauer 2340 autoclave (Tuttnauer USA, Hauppauge, NY) programmed on a 30 min cycle (for cold departure) or 25 min cycle (for hot departure) at a temperature of 121°C and pressure of 1.2 bar. The autoclavable materials were transferred to the laminar flow cabinet, except the nutrient medium that was taken out just before sowing to keep it warm. Working time to solidification of the medium was about 20 min.

The interior surfaces of the laminar flow cabinet (Esco® EQU/04-EBC-2A, Singapore) were disinfected with 70% v/v ethanol. The non-autoclavable materials were disinfected with 70% v/v ethanol and placed in the cabinet: culture vessels (100 mL urine samplers made of polypropylene, individually wrapped in sterile packaging), 0.5% NaClO solution, droppers, lighter, marker, pre-printed self-adhesive labels, alcohol burner, and two test tubes per treatment, one with seeds, one without. All materials were exposed to a germicidal UVC lamp for 50 min. The workspace outside the cabinet was concurrently disinfected with a germicidal UVC lamp. Afterwards, the airflow in the cabinet was turned on for 15 min to purge airborne contaminants as per manufacturer’s instructions (Esco®. Class II Type A/B3 Biohazard Safety Cabinets. User Operation Manual.). The sterile seal of the culture vessels was broken, and each was labelled with a pre-printed self-adhesive label.

The seeds were disinfected in 15 mL Sarstedt® (Newton, NC, USA) polypropylene test tubes with conical base and screw cap as follows:

Each culture vessel was filled with approximately 16 mL of sterile nutrient medium and a 1 cm potato cube, as potato has been shown beneficial to asymbiotic orchid propagation (23,24,25). The seeds were scraped with a spatula from the filter paper and distributed evenly within the culture vessels, approximately 4 mg of seed per vessel. The tips of the steel tools were heat sterilized with the alcohol burner between sowings. Afterwards, the vessels were incubated in a dark cabinet at an ambient temperature of 22°C. At 41 d after the last sowing, all vessels were visually examined for the development of fungal and bacterial colonies. For consistency with earlier studies (10,19), the vessels were examined again for germination at 189 d after the last sowing.

The parameter for the CENTRIFUGE method was not statistically significant (Table 3). Jevšnik and Luthar (12) had used this method to obtain 100% disinfection efficiency. They attributed their success to enhanced rehydration of the testa, and, by extension, the embryo, although this is more critical with older seeds (Tomaž Jevšnik, personal communication, 2019). We modified their technique using 15 mL test tubes versus micro-centrifuge tubes and 0.5% NaClO solution versus 16.6 g/L of sodium dichlorocyanurate. Aside from those differences, Jevšnik and Luthar (12) worked on epiphytic orchid seeds and a single treatment duration of eight minutes. It may be possible that eight minutes is the optimal treatment duration for the species they were working on. It is not clear though whether they would have achieved the same results without centrifugation.

Generally, the germination period varies from a few weeks to several months according to species, culture media and the method used (32). We observed that germination of centrifuged seeds was severely delayed. At 41 d, only 18.8% of sown vessels had germinated, as opposed to 50.0% for the NaClO and 54.2% for the PRESOAK methods (Fig. 3). Usually, seed germination is affected by three main environmental factors: temperature, light, and water (33). The centrifugation was performed at 4°C. However, since the seeds had been stored at –20°C for about a year, such a short exposure to cold would be irrelevant. Perhaps more importantly, the CENTRIFUGE treatment applied a gravitational force on the seeds. There is little information about the effect of increased gravitational force on germination. Most studies researching the impact of gravitational force on plants are old and focus on post-germination development (34,35). However, in 1965, Siegel et al. studied the effect of gravitational force of 100 g (Gravitational acceleration units, not to be confused with grams.) for three days at 30°C on the germination of seeds of different species (36). Germination was inhibited compared to the control. Norris et al. (37) studied the effect of centrifugation on germination and seedling growth of oat (Avena sativa L.) seeds and confirmed that centrifugation inhibits germination and seedling growth. Moreover, the inhibition of seedling growth increased with the duration of soaking time in water before centrifugation and with the increase in centrifugal force. The force used in their study ranged from 12,000 to 28,000 g and was applied for 15 to 60 min. When soaked for less than an hour, and then centrifuged for 15 min at 12,000 g, growth was stunted for 34% of the seeds, whereas 5% did not germinate at all. Raghavan (38), experimenting with the sensitive fern (Onoclea sensibilis L.), observed a delay in germination when exposed to hypergravitational forces of low to moderate intensity. Further, he demonstrated that centrifugation induces stress in the seeds which equipment, (iv) it adds undue complexity to the process, (v) the test tubes must be moved from the laminar flow cabinet to the centrifuge and back, increasing the risk of infection, and results in the synthesis of shock proteins. Another hypothesis is that germination was delayed due to physical seed deformation caused by increased gravity.

Based on our results, the CENTRIFUGE method cannot be recommended for scarification of terrestrial orchid seeds because: (i) the gain in disinfection/scarification time is not statistically significant, (ii) the slight gain in disinfection/ scarification time could be due to the longer rinse time rather than the centrifugation itself, (iii) centrifugation requires more (vii) centrifugation unduly delays germination, although given enough time the proportion of germinated vessels catches up with the other methods.

The parameter for the PRESOAK method was statistically significant (Table 3). Pretreating the seeds in a 10% sucrose solution for six hours improves subsequent disinfection in the 0.5% NaClO solution. Spores of microorganisms hide in the porous surface of the seed (12). The large diversity of microorganisms on the seed surface may reduce susceptibility to disinfecting agents (39). Moreover, because they survived for a long time on a dry surface, microbes exhibit slow growth and reduced metabolism during rewetting, reducing the efficacy of antimicrobial agents. Finally, the specificity of antimicrobial agents varies by species of microorganism. Although sodium hypochlorite (NaClO) is a wide spectrum disinfectant, it has been found inefficient against yeast biofilm (39). For all these reasons, it is helpful to test ways to increase the susceptibility to disinfectants for a wide range of microorganisms.

The sucrose solution causes bacterial and fungal spores to germinate, making them vulnerable to the disinfecting solution (12). Furthermore, the cell growth phase influences the expression and regulation of genes involved in stress tolerance and resistance to biocide (40). The more susceptible growth phase could be the stationary phase or the exponential phase (40, 41, 42). In our study, the sucrose was used as a recommended carbohydrate source (16), with the understanding that the optimum carbohydrate source and concentration may vary according to microorganism (bacteria: 41,43,44; fungi: 45,46). Different carbohydrates sources act on different bacterial processes such as germination, elongation or division (43). Similarly, fungal processes such as sporulation, germination and growth are affected (46).

Supposably, one could test different concentrations of sucrose or glucose (e.g., from 0.5% to 50%) to determine if the presence of sugars, even in small quantities, is enough to enhance the disinfection. Additionally, testing different pretreatment times (e.g., 1 to 12 h) could help estimate the optimal soaking time in the sugar solution. A key question is whether the additional delay imposed by the presoaking step is worth the enhanced disinfection efficacy.

From a practical standpoint, comparing disinfection/scarification methods for a particular germination tolerance may be more useful than comparing methods across a range of tested times. A working professional in a commercial or research facility may be interested to know what length of disinfection is likely to result in a predetermined germination rate, along with an estimate of certainty of achieving that rate. The problem of inverse prediction is one of solving Eq. (1) for X₁, given P, X₂, and X₃. For example, if the predetermined tolerance for germination of culture vessels is 90%, the NaClO method predicts about 59 min (Fig. 4). The 95% confidence interval is calculated as 53 to 67 min. The CENTRIFUGE method predicts about 58 min, resulting in a savings of only one minute; the predicted value even lies within the 95% confidence interval of the NaClO method. Accordingly, the PRESOAK method will trim about nine minutes off the disinfection time. We think that the added complexity and delay of the CENTRIFUGE and PRESOAK methods hardly justify time savings of only a few minutes. While statistical measures are useful to compare scarification methods, they should be co-evaluated along with other, more practical considerations.

Figure 4

Having said that, we must point out that A. laxiflora seeds have a thin testa compared to other terrestrial orchids, so that the addition of a presoaking stage in sucrose solution may not confer a substantial advantage. Perhaps the advantage would be greater on species with a thick testa, such as Himantoglossum robertianum. Indeed, seeds of H. robertianum take longer to disinfect (10,19). Also, in our experience, immature capsules of terrestrial orchids used for the technique of “green podding” are harder to disinfect than mature seeds, possibly because microorganisms are protected in the folds of the dusty capsules. In such cases, the PRESOAK method may be worth considering.