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

Abstract

A critical step during in vitro sexual propagation of terrestrial orchids is the treatment of the microscopic seeds with a disinfecting solution that kills bacteria and fungi attached to the seeds. This treatment is necessary to prevent infection of the culture vessels. At the same time, the treatment serves to scarify the seeds, a process that disrupts seed dormancy and initiates germination. The literature is inconclusive with respect to the proper combination of disinfecting solution strength and treatment duration. Both factors should be adapted to each species to guarantee minimal infection rate without damaging the embryo. This research aims to compare three disinfection/scarification methods for seeds of Anacamptis laxiflora (Lam.): (i) soaking in 0.5% NaClO, (ii) soaking in 0.5% NaClO, then centrifugation, and (iii) presoaking the seeds in sucrose solution, then soaking in 0.5% NaClO. The seeds were soaked in the disinfecting solution for 5 to 85 min. Following scarification, the seeds were sown in modified Malmgren nutrient medium. Infected and germinated vessels were counted at 41 and 189 d after sowing. We found that the longer the chemical treatment, the lower the infection rate, and the higher the germination rate. There was no significant difference in germination rate between the NaClO and the NaClO-plus-centrifugation method; in fact, the slight savings in disinfection time effected by centrifugation were more than offset by the added complexity of the method. Moreover, we found that centrifugation significantly delays germination. The sucrose presoak-plus-NaClO method was superior to plain NaClO, as the sucrose stimulates the germination of microbial spores on the surface of the seeds, making them easier to kill. Perhaps seeds with thicker testa as well as whole immature capsules could benefit even more from the pretreatment in sucrose solution.

Asymbiotic in vitro seed germination, in vitro seedling development, and ex vitro acclimatization of Spiranthes

“…This sensitivity to sodium hypochlorite may be attributable to various factors; unfortunately, there is little information available to elucidate these in Spiranthes . Thin seed coats, the presence of air space in the integument and between the integument and the embryo, the structural and chemical composition of the integument and seed coat, the presence of different cuticular substances, and “wettability” have all been cited as factors influencing the response of orchid seed coats to chemical reagents (Molvray and Kores, 1995 ; Arditti and Ghani, 2000 ; Deconninck and Gerakis, 2021 ). One issue with such a shortened scarification period using sodium hypochlorite is that bacterial and fungal spores may not be completely killed during the process, leading to contamination of cultures before germination and initial seedling development is complete.…”

“…One issue with such a shortened scarification period using sodium hypochlorite is that bacterial and fungal spores may not be completely killed during the process, leading to contamination of cultures before germination and initial seedling development is complete. Recent research has shown that soaking seeds in a sucrose solution before chemical scarification can cause bacterial and fungal spores to germinate, making them more sensitive to the chemical regent and thereby increasing the efficacy of the sterilant, but this has not been tested with North American Spiranthes (Deconninck and Gerakis, 2021 ). Other scarification reagents should be tested, and alternative options such as ultrasonic treatment after an initial chemical sterilization or culture of immature embryos should be attempted to diversify the range of techniques for successfully germinating Spiranthes seeds (Miyoshi and Mii, 1988 ).…”

“…One issue with such a shortened scarification period using sodium hypochlorite is that bacterial and fungal spores may not be completely killed during the process, leading to contamination of cultures before germination and initial seedling development is complete. Recent research has shown that soaking seeds in a sucrose solution before chemical scarification can cause bacterial and fungal spores to germinate, making them more sensitive to the chemical regent and thereby increasing the efficacy of the sterilant, but this has not been tested with North American Spiranthes (Deconninck and Gerakis, 2021).…”