A classroom exercise in hand pollination and in vitro asymbiotic orchid seed germination

Plant Cell Tiss Organ Cult (2008) 93:223 230 DOI 10.1007/s11240-008-9365-1 ORIGINAL PAPER A classroom exercise in hand pollination and in vitro asymbiotic orchid seed germination Philip J. Kauth Æ Timothy R. Johnson Æ Scott L. Stewart Æ Michael E. Kane Received: 20 December 2007 / Accepted: 7 March 2008 / Published online: 26 March 2008 Ó Springer Science+Business Media B.V. 2008 Abstract While many scientific reports on orchid seed germination provide germination protocols, few provide concise descriptions of plant selection, hand pollination, and asymbiotic seed culture for use in classroom exercises. Another major limitation for conducting orchid seed germination exercises is the availability of seeds or flowers to pollinate. In this paper, we outline an efficient and reliable classroom exercise using the orchid Spathoglottis to demonstrate hand pollination and subsequent asymbiotic seed germination. Flowers of S. parsonii are hand pollinated and subsequent seed capsule development is carefully monitored. Hand pollination of the orchid flower provides an opportunity to discuss floral morphology and associated reproductive biology. Capsules are harvested 30 to 40 days after pollination, prior to capsule dehiscence. Spathoglottis kimballiana seed capsules are surface sterilized, seeds excised, and then sown on P723 Orchid Seed Sowing Medium. Germination occurs quickly and large seedlings ready for greenhouse acclimatization develop within 4 6 months. P. J. Kauth (&) T. R. Johnson M. E. Kane Department of Environmental Horticulture, University of Florida, P.O. Box 110675, Gainesville, FL 32611, USA e-mail: pkauth@ufl.edu S. L. Stewart PhytoTechnology Laboratories, 14335 West 97th Terrace, Lenexa, KS 66215, USA Keywords Classroom laboratory Teaching exercise Orchid pollination Orchid seed culture Introduction The rising popularity of orchids has created a demand for high quality plants as well as industry workers trained in orchid hybridization and in vitro asymbiotic seed germination techniques. Asymbiotic techniques involve germinating orchid seeds in vitro on a defined medium containing carbohydrates, vitamins, minerals, and a solidifying agent. Since orchid seed germination requires training and practice, high school and college instructors have expressed interest in learning these techniques to incorporate them into science class curricula. In addition, instructors and students can use these methods to study the unique biology of orchid flowers and seeds. Many publications on asymbiotic germination describe efficient methods; however, these methodologies are limited as teaching tools because clear descriptions of hand pollination methods for generating seed capsules are often absent. Lo et al. (2004) provided a protocol for pollinating orchid flowers and subsequent seed germination, but the pollination procedures are not descriptive enough for teaching methods. Mudge and Chu (1992) provided a classroom exercise for orchid seed germination, but hand pollination techniques are not provided. Hand pollination is critical to instructors since a reliable seed

224 Plant Cell Tiss Organ Cult (2008) 93:223 230 source is required to incorporate orchid seed germination into classroom exercises. This paper presents a complete methodology to incorporate asymbiotic seed germination in the classroom, from flower pollination through subsequent seed germination and development. After completing this exercise students will be able to: (1) describe orchid flower morphology and associated reproductive biology (2) recognize seed capsule development and maturation (3) produce seedlings using asymbiotic seed culture procedures. Background instructional information In nature, orchid seeds utilize mycorrhizal fungi during germination as sources of carbohydrates, nutrients, and water (Stewart and Kane 2007). In vitro symbiotic germination, which has been studied since the late 1800s, is the co-culture of orchid seeds and their symbiotic fungi. Lewis Knudson discovered asymbiotic germination in the 1920s (Arditti 1967), and this is the preferred method of producing orchids for commercial purposes. Orchid flowers are trimerous, possessing three petals and three sepals (Fig. 1a). The third petal is modified into a labellum or lip typically found oriented toward the bottom of the flower. Potential pollinators use the labellum as a landing platform, directing them to the gynostemium. A distinguishing feature of the Orchidaceae is the gynostemium or column, which is the fusion of the style, stigma, and stamens (Dressler 1981). The anther cap and pollinia are located at the front of the gynostemium, while the stigma is located directly behind the anther on the underside of the gynostemium (Fig. 1b, c). The pollen grains are joined together into masses called pollinia (Fig. 1d). During a pollination event, Fig. 1 Flower morphology of Spathoglottis parsonii. (a) Components of a flower; scale bar = 1 cm. (b) Location of the anther cap; scale bar = 1cm. (c) Gynostemium with pollinia and anther cap; scale bar = 1 mm. (d) Pollinia with individual pollinium; scale bar = 1mm(e) Ventral view of gynostemium with anther cap and pollinia removed; scale bar = 1mm(f) Gynostemium with pollinia attached after hand pollination; scale bar = 1mm

Plant Cell Tiss Organ Cult (2008) 93:223 230 225 pollinators deposit pollinia onto the stigmatic surface (Fig. 1e, f). A successful pollination event depends on pollen and flower age. Spathoglottis flowers remain open for several weeks, while inflorescences continually flower for several months (pers. observation). Pollinating young, fully open flowers is recommended since pollen is most receptive 1 8 days after flowers are open (see Proctor 1998; Shiau et al. 2002; Lo et al. 2004 for data on other species). Likewise, using young flowers less than one week from opening ensures that the stigmatic surface is receptive to pollen. After two weeks, flowers close and pollen becomes brown and unreceptive (pers. observation). Procedures Plant material Spathoglottis parsonii and S. kimballiana are used for this exercise, but these techniques can be used for any orchid. Spathoglottis orchids are easy-to-cultivate tropical terrestrial orchids, and are reliable sources of seed capsules for lab exercises. These orchids are available at local plant/orchid nurseries and garden centers. They grow well in a standard soilless potting mix such as Fafard No. 2 (Conrad Fafard, Inc., Agawam, MA) or a mix containing a 4:1:1 v/v/v ratio of organic compost (peat), sand, and perlite (Beltrame 2006). The orchids should be grown in 30 70% shade under a natural photoperiod with a daytime temperature between 20 26 C. Plants should be fertilized bi-weekly with either a balanced fertilizer at 150 ppm nitrogen or a slow release fertilizer. Spathoglottis grow rapidly, and single shoots can become large specimens within six months. Plants may need to be divided and repotted on a regular basis. Spathoglottis have several attributes that allow for efficient classroom demonstrations. First, plants flower year-round under a natural photoperiod since no specific cultural requirements are necessary to induce flowering. Second, the column, anther cap, and pollinia are noticeable without a microscope. Third, seed capsule formation is nearly 100% after pollination. Fourth, Spathoglottis are receptive to both self- and cross-pollination. Finally, capsules mature in 30 60 days compared to 75 120 days for Phalaenopsis capsules and 150 195 days for Vanda capsules (Arditti et al., 1982). Hand pollination procedure Identify the column and anther cap on a fully opened flower (Fig. 2a). Gently remove the anther cap and pollinia with a tooth pick (Fig. 2b). Dislodge the pollinia and anther cap from the gynostemium by applying slight upward pressure to the bottom of the anther cap (Fig. 2c). The pollinia will adhere to the toothpick on contact. Carefully remove the anther cap from the pollinia (if still attached) without dislodging the pollinia (Fig. 2d). After removal, transfer the pollinia to the same flower or another flower by gently placing the pollinia onto the stigmatic surface (Fig. 2e, f). Remove the pollinia from another flower before cross-pollinating with the pollinia from the donor flower. During this step, apply gentle upward pressure against the stigma while retracting the toothpick to maintain contact between the pollinia and stigma. Seed capsule development and maturation time is species, hybrid, and growing condition dependent. For first time hand pollination, closely monitor flower senescence, capsule development, and capsule dehiscence. No physical signs are visible indicating Spathoglottis capsule maturity, but other species capsules may turn light green, yellow, or brown. Once capsule development time is estimated by allowing capsules to dehisce, repeat the hand pollination procedures. Harvest the capsule one to two weeks before capsule maturity by cutting the capsule from the inflorescence (Fig. 2g). Harvesting the capsule at this point will ensure that the capsule does not dehisce (Fig. 2h). To limit surface contamination during harvest, handle capsules with forceps and use a clean razor blade to cut the pedicel. Place harvested capsules in a clean paper bag and store at 4 10 C for a maximum two days, since capsules may dehisce soon after collecting. Do not store capsules in the freezer or in plastic bags. Freezing will cause permanent damage to the capsules, and plastic bags promote fungal and bacterial growth by limiting air exchange. Medium preparation Orchid seed germination media are available from companies such as PhytoTechnology Laboratories, LLC (Shawnee Mission, KS, USA) and Sigma-

226 Plant Cell Tiss Organ Cult (2008) 93:223 230 Fig. 2 Spathoglottis parsonii flower morphology and hand pollination sequence. (a) Flower profile. (b) Location of the anther cap and pollinia. (c) Removal of the anther cap with the pollinia. (d) Removal of the anther cap from the pollinia. (e) Transfer of pollinia onto the stigmatic surface. (f) Gynostemium with attached pollinia. (g) Green capsule ready to harvest. (h) Dehisced capsule. Scale bars = 1.5 cm Aldrich (St. Louis, MO, USA). Many commercially prepared media are complete, requiring only the addition of water before autoclaving and dispensing. Media such as P723 Orchid Seed Sowing Medium, Knudson C, and Vacin and Went are suitable for Spathoglottis seeds. Media can also be prepared in the laboratory or classroom by mixing and storing concentrated stock solutions (see Arditti and Ernst 1993 for methods on preparing various media from stock solutions). Prepare one liter of P723 medium by adding 32.74 g of powder to 1000 ml of distilled water in a 2-liter Erlenmeyer flask. Adjust the medium ph to 5.7 with 0.1 N NaOH and 0.1 N HCl. Autoclave one liter of medium for 40 min at 117 kpa and 121 C, and allow to cool for 30 min after autoclaving. Dispense 25 30 ml of medium into 9 cm Petri dishes; one liter of medium makes 30 40 Petri dishes. If an autoclave is not available, a pressure cooker is an excellent alternative to sterilize equipment and medium (see Bergman 2006). Alternatively, adding Plant Preservative Mixture (PPM) into germination media can suppress bacterial or fungal contaminants temporarily (Plant Cell Technology, Washington, DC; www.ppm4plant-

Plant Cell Tiss Organ Cult (2008) 93:223 230 227 Fig. 3 Seed germination procedure. (a) Addition of bleach to a wash bottle for capsule disinfection. (b) Shaker table for agitating wash bottles during disinfection. (c) Decanting of liquid from a wash bottle. (d) Removal of the pedicel from the capsule after disinfection. (e) Removal of remaining floral tissue from the sterilized capsule. (f) Bisection of the capsule. (g) Bisected capsule with immature seeds exposed and removed. (h) Inoculated Petri dish tc.com). Microwaving germination media may be suitable as well (see PhytoTechnology Laboratories Orchid Media Selection & Use Guide). However, using PPM or a microwave to sterilize media are questionable compared to an autoclave or a pressure cooker. Seed culture procedures In preparation for capsule surface disinfection, prepare graduated cylinders, wash bottles, and 500 1000 ml distilled water per student. Sterilize the graduated cylinders and wash bottles for at least 10 min and the water for 40 min at 117 kpa and 121 C. Remove any dried flower parts from the capsule prior to surface disinfection. Remove surface debris by placing the seed capsule in a container covered with cheesecloth secured with a rubber band. Rinse the capsule under cool tap water for 10 15 min. While the capsule is rinsing, prepare the disinfection solution by mixing 50 ml sterile water and 50 ml of bleach (6% sodium hypochlorite) in a sterile graduated cylinder. To facilitate surface disinfection, add one or two drops of Tween 20 or another surfactant, such as liquid dish soap, to the disinfecting solution. After rinsing, place the seed capsule in a sterile wash bottle, add 70% ethanol, and agitate the capsule

228 Plant Cell Tiss Organ Cult (2008) 93:223 230 for 30 s. Decant the ethanol by slightly loosening the cover allowing the ethanol to slowly drain into a waste container. After the ethanol wash pour enough disinfecting solution into the wash bottle to cover the capsule (Fig. 3a). Cover the wash bottle and agitate for 10 min (Fig. 3b). If using a shaker table, agitate the bottle at 100 rpm (Fig. 3b). After 10 min, check the capsule. The capsule may have yellow or white bleach marks, but should still be firm. If the capsule is still dark green, agitate in the disinfection solution for five additional minutes. After the disinfecting process, decant the solution (Fig. 3c). Add sterile water to the container, rinse the capsule for 2 min, and repeat two more times. Once the capsule is rinsed, place it in a sterile Petri dish under a laminar flow hood. If a laminar flow hood is not available, a sterile transfer box can be constructed by cutting an opening in one side of a large clear plastic storage container. Disinfect the inside surface of the container with a 10% bleach solution and allow to dry. Hold the capsule with a sterile forceps and remove the pedicel and any remaining floral tissue with a sterile scalpel (Fig. 3d, e). Cut the capsule longitudinally with a sterile scalpel blade (Fig. 3f). Use a sterile spatula or inoculating loop to remove and dispense seeds from the capsule onto a Petri dish containing the germination medium (Fig. 3g, h). Do not place too many seeds on the surface of the germination media, since high seed densities inhibit germination and development (Rasmussen 1989). Seal the culture vessels with one layer of sealing film such as Parafilm (Pechiney Plastic Packaging Inc, Chicago, IL, USA) or Nescofilm (Karlan Research Products, Santa Rosa, CA, USA). Place the seed cultures under cool-white fluorescent lights in a 16-hour photoperiod at room Fig. 4 In vitro germination and development of Spathoglottis seeds. (a) Two week old cultures; scale bar = 1 mm. (b) Protocorms with leaves after four weeks in vitro culture; scale bar = 1 mm. (c) Seedlings transferred to larger culture vessels six months after initial seed inoculation; scale bar = 1 cm. (d) Acclimatized seedling six months after sowing; scale bar = 1 cm. (e) One year old plant; scale bar = 10 cm

Plant Cell Tiss Organ Cult (2008) 93:223 230 229 temperature (23 25 C). Check cultures periodically and discard contaminated cultures. Germination should occur within two weeks of seed inoculation evident by green protocorms (Fig. 4a). Leaves form on the protocorms after four weeks culture, and seedlings develop within four months of seed sowing (Fig. 4b, c). At this time, aseptically transfer seedlings to a larger culture vessel containing 80 100 ml P723 such as a Magenta Ò GA7 culture box (Magenta Corp., Chicago, IL, USA) or PhytoTech Culture Box TM (PhytoTechnology Laboratories, LLC). After two to four additional months, seedlings are ready for greenhouse acclimatization (Fig. 4d). Remove seedlings from culture vessels and wash the culture medium from the roots. Place seedlings in a soilless potting mix. Place clear plastic humidity domes or plastic bags over the seedlings for two weeks, and gradually remove them over an additional two weeks. Water seedlings when the top portion of the potting mix becomes dry. Begin fertilizing seedlings after removing the humidity dome. Seedlings grow quickly under greenhouse conditions, and develop into medium size plants within 1 year that may flower (Fig. 4e; for complete timeline see Fig. 5) This exercise represents an excellent opportunity to incorporate both technical and horticultural aspects of orchid hybridization as well as in vitro seed germination into the classroom. In the 6 years of conducting this laboratory, students often remark that this is one of their favorite exercises. Since growing orchids from seeds to mature plant is lengthy, conducting this experiment early in the semester is recommended. Following through with this exercise until seedlings become mature plants may be difficult if the course is offered semesterly. Instructors can prepare seed cultures during the previous semester to provide students an opportunity to acclimatize seedlings. However, the emphasis is seed germination and seedling development, not obtaining mature plants. Providing a complete protocol for instructors is necessary to determine the proper timeline for conducting this laboratory. This exercise teaches students diligence and patience, and rewards them with orchids raised from seed. This paper provides a detailed and easy to follow protocol for hand pollination of orchid flowers and asymbiotic seed germination. Fig. 5 Timeline of seed germination to adult plant Acknowledgements Brand names are provided as references; the authors do not solely recommend or endorse these products. The authors thank Nancy Philman and Daniela Dutra (Environmental Horticulture Dept., University of Florida) as well as an anonymous reviewer for excellent comments. References Arditti J (1967) Factors affecting the germination of orchid seed. Bot Rev 33:1 97 Arditti J, Ernst R (1993) Micropropagation of orchids. John Wiley and Sons, New York Arditti J, Clements MA, Fast G, Hadley G, Nishimura G, Ernst R (1982) Orchid seed germination and seedling culture. In: Arditti J (ed) Orchid biology: reviews and perspectives II. Cornell University Press, Ithaca, New York, p 274 Beltrame E (2006) Spathoglottis-inside and out. Orchid Rev 114:68 71 Bergman FJ (2006) Sowing orchid seed: an easy approach to new rewards. Orchids 75:526 531 Dressler RL (1981) The orchids: natural history and classification. Harvard University Press, Cambridge, MA Lo SF, Nalawade SM, Kuo CL, Chen CL, Tsay HS (2004) Asymbiotic germination of immature seeds, plantlet development and ex vitro establishment of plants of Dendrobium tosaense Makino a medicinally important orchid. In vitro Cell Dev Biol-Plant 40:528 535

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