The
A crucial step during the
The agitation of seeds is usually conducted within test tubes. Some practitioners have modified the standard technique; Jevšnik and Luthar (12) obtained germination percentage rates between 60 and 90% by centrifuging the seeds after disinfection in microcentrifuge tubes. Ponert et al. (13) used a syringe to mix the seeds with the disinfection solution and obtained good results also. Some authors (12,15,16) report a method called presoaking, whereby the seeds are pretreated in a sucrose or honey solution before being disinfected.
Svante Malmgren is a veteran practitioner who has propagated more than 200 terrestrial species of orchids and orchid hybrids
Previous studies (10,19) attempted to determine the optimal disinfection time for two terrestrial species,
The aim of this study was to test three disinfection/scarification methods on seeds of
To test the influence of the disinfection method (that also serves as scarification) on seed disinfection and germination, combinations of three methods and nine durations (5, 15, 25, 35, 45, 55, 65, 75, and 85 min) were tested on seeds of
Each experimental treatment consisted of one duration and one disinfection/scarification method. Each experimental run consisted of 16 culture vessels per treatment that served as experimental replications plus four “blanks”, i.e. culture vessels without seeds. The blanks were subject to the same treatment as the vessels with seeds, including simulated sowing with imaginary seeds. The purpose of the blanks was to reveal possible shortcomings of our sowing technique that were not due to seed-borne contaminants.
Mature seeds were collected in May 2018 on the island of Cephalonia, Greece.
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.
Formula for modified “SM-organic” nutrient medium
Ingredients | Quantity |
---|---|
Bottled water to make Vikos® (Ioannina, Greece) | 1 L |
CaHPO4 | 99 mg Equivalent to 75 mg Ca3(PO4)2 in the original formula |
KH2PO4 | 75 mg |
MgSO4.7H2O | 75 mg |
Soluvit® Fresenius Kabi, Uppsala, Sweden | 10 mL |
Amina-Fe Humofert, Metamorfosi, Greece | 0.92 mL Equivalent to 0.5 mL Vaminolac® (Fresenius Kabi, Uppsala, Sweden) plus 10 mg FeSO4 in the original formula |
Kinetin | 5 mg |
Saccharose (sucrose) | 10 g |
Activated charcoal | 1 g |
Danish agar | 12 g |
Pineapple juice | 25 mL |
0.1 M H2SO4 for pH adjustment | 15–20 drops |
Potato (Solanum tuber tuberosum) | 1 cm3 per culture vessel |
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:
NaClO: the first method consisted of scooping 60 mg of seeds into the test tube and filling it nearly to the top with 0.5% NaClO disinfecting solution. A second test tube serving as blank was filled only with disinfecting solution. Both tubes were capped and shaken vigorously a few times to remove any air bubbles in contact with the seeds. The shaking was repeated every 15 min thereafter till the end of the disinfection period. Afterwards, the suspensions were decanted into Erlenmeyer flasks fitted with a polypropylene funnel lined with filter paper to recover the seeds. The filter paper was soaked in the same disinfecting solution as the seeds. After filtering the suspensions, each filter paper was rinsed with seven mL of sterile deionized water using a dropper.
CENTRIFUGE: the second method was identical to the first, except two minutes before the end of the disinfecting period the tubes were placed in a SIGMA 3-16PK centrifuge (Osterode am Harz, Germany) for two minutes at 3,077 g (Gravitational acceleration units, not to be confused with grams.) and a temperature of 4°C. The speed and the temperature of the centrifuge were configured according to Jevšnik and Luthar (12). After centrifugation, the supernatant was decanted into an empty beaker. Afterwards, the seeds were rinsed three times as follows: the tubes were filled with sterile deionized water nearly to the top and centrifuged using the same settings. The supernatant was decanted between rinses. Afterwards, the tube contents were emptied into an Erlenmeyer flask fitted with polypropylene funnel lined with filter paper to recover the seeds.
PRESOAK: six hours before disinfection, the tubes were filled with 10% sucrose solution nearly to the top, capped, and shaken vigorously to remove any air bubbles in contact with the seeds. At the end of the presoak, the suspensions were decanted into Erlenmeyer flasks fitted with a polypropylene funnel lined with filter paper to recover the seeds. After filtering the suspensions, the seeds were scraped from the filter paper with a spatula and transferred into one of the two clean test tubes (the other serving as blank). Thereafter, the same procedure as for the NaClO method was applied.
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 infection and germination probabilities were modelled based on the duration of soaking and the scarification method applied. The infection or germination variable (Y) is binary: a vessel is either infected or not; it germinates or not. The duration of soaking and the scarification method are continuous and categorical variables, respectively. Logistic regression can be used to characterize the relation between a dependent variable (necessarily taking the values 0 and 1) and one or more explanatory variables that can be categorical or continuous (26, 27, 28). If the NaClO method is used as a reference, the probability of infection or germination is modeled as a function of three regressors (duration of soaking in NaClO, plus two “dummy” or indicator variables representing the two alternative methods of scarification):
where P is probability, Y = nominal response, X1, X2, X3 = regressors, and β0, β1, β2, β3 = fitted parameters. The duration of soaking is X1. “Dummy” or indicator variables X2, X3 are defined in Table 2. If
Combinations of “dummy” or indicator variables that uniquely identify each scarification method for the purpose of logistic regression analysis. The NaClO method is specified as reference, in which case X2 = X3 = 0
Variable | NaClO | CENTRIFUGE | PRESOAK |
---|---|---|---|
X2 | 0 | 1 | 0 |
X3 | 0 | 0 | 1 |
Essentially, parameters β2, β3 represent the added probability of success (in terms of the log odds-ratio) when an alternative scarification method is used other than NaClO.
The R programming language (29) was used to conduct the analysis. The level of significance for statistical tests was set a priori at α = 0.05.
The infection rate for the 108 “blank” vessels was 1.9%, which shows that our technique was valid and that nearly all contamination came from the seeds. As far as the vessels with seeds, we observed that all vessels that were not infected, germinated. This greatly simplified subsequent analysis, because once germination probability is modelled, the percent probability of infection can be estimated as 100 minus percent probability of germination. Ultimately, what matters most is the probability of germination, because it determines how many useable containers with seedlings are available for further culture.
Proportions of germinated vessels for each method are compared in Fig. 1. The fitted model is statistically significant. The statistical measures are in Table 3. Germinated seeds at 189 d after sowing are depicted in Fig. 2.
Logistic regression results and parameter estimates for log odds-ratios from Eq. [2]. The regressors are duration of soaking in NaClO and scarification method. The NaClO method was used as the reference
Model | -LL | DF | χ2 | p > χ2 |
---|---|---|---|---|
Difference | 142.21 | 3 | 284.42 | <.001 |
Full | 145.08 | |||
Reduced | 287.29 | |||
R2 | 0.50 | |||
N | 432 |
Term | N | Estimate | z | OR | 95% C.I. | ||
---|---|---|---|---|---|---|---|
Intercept ( | 144 | -3.8866 | 0.447 | -8.694 | <.001 | 0.021 | [0.008 – 0.047] |
Duration ( | 432 | 0.1023 | 0.009 | 10.790 | <.001 | 1.108 | [1.089 – 1.130] |
Centrifuge ( | 144 | 0.1969 | 0.363 | 0.543 | 0.587 | 1.218 | [0.598 – 2.492] |
Presoak ( | 144 | 0.9947 | 0.373 | 2.664 | 0.008 | 2.704 | [1.315 – 5.709] |
LL = log likelihood; DF = degrees of freedom; χ2 = chi-square;
The equations that were used to model germination probabilities in Fig. 1 are derived from Eq. (1) substituting the parameter estimates from Table 3. To model infection probabilities, the sign of the parameters can simply be reversed, i.e., they would be β0 = 3.8866, β1 = –0.1023, β2 = –0.1969, and β3 = –0.9947. From Fig. 1 it follows that the longer the chemical treatment, the lower the predicted infection rate and the higher the predicted germination rate. At 85 min, the predicted infection rate approached zero for all methods and the predicted germination rate approached 100%.
Infected and germinated vessels were counted at 41 and 189 d (Fig. 3). At 41 d, there was not much difference in the proportion of germinated vessels between the NaClO and the PRESOAK method. On the contrary, the proportion of germinated vessels for the CENTRIFUGE method was substantially lower, meaning that centrifugation severely delayed germination.
The results of the logistic regression highlight the absence of significant difference between the NaClO method that was used as a reference and the CENTRIFUGE method (p = 0.587); however, there is a significant difference between the NaClO and the PRESOAK method (p = 0.008).
With a weak NaClO solution (0.5%) and disinfection/scarification times ranging from 5 to 85 min, a good fit of the cumulative probability curve for germination of
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 (
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.
Having said that, we must point out that
We conclude that (i) the longer the chemical treatment of seeds, the lower the infection rate, and the higher the germination rate, (ii) there is no significant difference in germination rate between the NaClO and the CENTRIFUGE method, (iii) centrifugation delays germination, although given enough time the proportion of germinated vessels catches up, (iv) the PRESOAK method is superior to the NaClO method, because the sucrose solution stimulates the germination of spores on the surface of the seeds, making them more susceptible to the disinfectant. However, the savings in disinfection time hardly justify the extra preparation time and complexity. Seeds with thicker testa as well as whole immature capsules could potentially benefit more from pretreatment in sucrose solution.