《兰花人工授粉与无菌播种》

ORIGINAL PAPERA classroom exercise in hand pollination and in vitro asymbiotic orchid seed germinationPhilip J.Kauth ÆTimothy R.Johnson ÆScott L.Stewart ÆMichael E.KaneReceived:20December 2007/Accepted:7March 2008/Published online:26March 2008ÓSpringer Science+Business Media B.V.2008Abstract 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 demon-strate 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 30to 40days after pollina-tion,prior to capsule dehiscence.Spathoglottis kimballiana seed capsules are surface sterilized,seeds excised,and then sown on P723Orchid Seed Sowing Medium.Germination occurs quickly and large seedlings ready for greenhouse acclimatization develop within 4–6months.Keywords Classroom laboratory ÁTeaching exercise ÁOrchid pollination ÁOrchid seed cultureIntroductionThe 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 methodol-ogies are limited as teaching tools because clear descriptions of hand pollination methods for generat-ing 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 class-room exercise for orchid seed germination,but hand pollination techniques are not provided.Hand polli-nation is critical to instructors since a reliable seedP.J.Kauth (&)ÁT.R.Johnson ÁM.E.KaneDepartment of Environmental Horticulture,University of Florida,P.O.Box 110675,Gainesville,FL 32611,USA e-mail:pkauth@ufl.eduS.L.StewartPhytoTechnology Laboratories,14335West 97th Terrace,Lenexa,KS 66215,USAPlant Cell Tiss Organ Cult (2008)93:223–230DOI 10.1007/s11240-008-9365-1source is required to incorporate orchid seed germi-nation into classroom exercises.This paper presents a complete methodology to incorporate asymbiotic seed germination in the classroom,from flower pollination through subse-quent 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 informationIn 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 beenstudied since the late 1800s,is the co-culture of orchid seeds and their symbiotic fungi.Lewis Knud-son 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 pollinationevent,Fig.1Flower morphology of Spathoglottis parsonii .(a )Components of a flower;scale bar =1cm.(b )Location of the anther cap;scale bar =1cm.(c )Gynostemium with pollinia and anther cap;scale bar =1mm.(d )Pollinia with individual pollinium;scale bar =1mm (e )Ventral view ofgynostemium with anther cap and pollinia removed;scale bar =1mm (f )Gynostemium with pollinia attached after hand pollination;scale bar =1mmpollinators deposit pollinia onto the stigmatic surface (Fig.1e,f).A successful pollination event depends on pollen andflower age.Spathoglottisflowers remain open for several weeks,while inflorescences continually flower for several months(pers.observation).Polli-nating young,fully openflowers is recommended since pollen is most receptive1–8days afterflowers are open(see Proctor1998;Shiau et al.2002;Lo et al.2004for data on other species).Likewise,using youngflowers 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). ProceduresPlant materialSpathoglottis 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 a4:1:1v/v/v ratio of organic compost(peat),sand,and perlite(Bel-trame2006).The orchids should be grown in30–70% shade under a natural photoperiod with a daytime temperature between20–26°C.Plants should be fertilized bi-weekly with either a balanced fertilizer at150ppm 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 induceflowering.Second,the column,anther cap, and pollinia are noticeable without a microscope. Third,seed capsule formation is nearly100%after pollination.Fourth,Spathoglottis are receptive to both self-and cross-pollination.Finally,capsules mature in30–60days compared to75–120days for Phalaenopsis capsules and150–195days for Vanda capsules(Arditti et al.,1982).Hand pollination procedureIdentify 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 sameflower or anotherflower by gently placing the pollinia onto the stigmatic surface(Fig.2e, f).Remove the pollinia from anotherflower 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 monitorflower senescence,capsule development,and capsule dehis-cence.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 proce-dures.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 at4–10°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 preparationOrchid seed germination media are available from companies such as Phyto Technology Laboratories, LLC(Shawnee Mission,KS,USA)and Sigma-Aldrich (St.Louis,MO,USA).Many commercially prepared media are complete,requiring only the addition of water before autoclaving and dispensing.Media such as P723Orchid 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 1993for methods on preparing various media from stock solutions).Prepare one liter of P723medium by adding 32.74g of powder to 1000ml of distilled water in a 2-liter Erlenmeyer flask.Adjust the medium pH to 5.7with 0.1N NaOH and 0.1N HCl.Autoclave one liter of medium for 40min at 117kPa and 121°C,and allow to cool for 30min after autoclaving.Dispense 25–30ml of medium into 9cm Petri dishes;one liter of medium makes 30–40Petri 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 Mix-ture (PPM)into germination media can suppress bacterial or fungal contaminants temporarily (Plant Cell Technology,Washington,DC;www.ppm4plant-Fig.2Spathoglottisparsonii flower morphology and hand pollination sequence.(a )Flowerprofile.(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 =1.5cm ).Microwaving germination media may be suitable as well (see Phyto Technology Laboratories Orchid Media Selection &Use Guide).However,using PPM or a microwave to sterilize media are question-able compared to an autoclave or a pressure cooker.Seed culture proceduresIn preparation for capsule surface disinfection,prepare graduated cylinders,wash bottles,and 500–1000ml distilled water per student.Sterilize the graduated cylinders and wash bottles for at least 10min and the water for 40min at 117kPa 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 10–15min.While the capsule is rinsing,prepare the disinfection solution by mixing 50ml sterile water and 50ml of bleach (6%sodium hypochlorite)in a sterile gradu-ated cylinder.To facilitate surface disinfection,add one or two drops of Tween 20or 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 thecapsuleFig.3Seed germination procedure.(a )Addition of bleach to a wash bottle for capsule disinfection.(b )Shaker table for agitating wash bottles duringdisinfection.(c )Decanting of liquid from a wash bottle.(d )Removal of the pedicel from the capsule afterdisinfection.(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 dishfor 30s.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 10min (Fig.3b).If using a shaker table,agitate the bottle at 100rpm (Fig.3b).After 10min,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 pro-cess,decant the solution (Fig.3c).Add sterile water to the container,rinse the capsule for 2min,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 con-structed by cutting an opening in one side of a largeclear 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 longitu-dinally 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 fluores-cent lights in a 16-hour photoperiod atroomFig.4In vitro germination and development of Spathoglottis seeds.(a )Two week old cultures;scale bar =1mm.(b )Protocorms with leaves after four weeks in vitro culture;scale bar =1mm.(c )Seedlings transferred to larger culture vessels six months after initial seed inoculation;scale bar =1cm.(d )Acclimatized seedling six months after sowing;scale bar =1cm.(e )One year old plant;scale bar =10cmtemperature(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 seed-lings to a larger culture vessel containing80–100ml P723such as a MagentaÒGA7culture box(Magenta Corp.,Chicago,IL,USA)or Phyto Tech Culture Box TM(Phyto Technology 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.Seed-lings grow quickly under greenhouse conditions,and develop into medium size plants within1year that mayflower(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 the6years 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 seed-lings.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 orchidflowers and asymbiotic seed germination.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. ReferencesArditti J(1967)Factors affecting the germination of orchid seed.Bot Rev33:1–97Arditti J,Ernst R(1993)Micropropagation of orchids.John Wiley and Sons,New YorkArditti 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,p274 Beltrame E(2006)Spathoglottis-inside and out.Orchid Rev 114:68–71Bergman FJ(2006)Sowing orchid seed:an easy approach to new rewards.Orchids75:526–531Dressler RL(1981)The orchids:natural history and classifi-cation.Harvard University Press,Cambridge,MALo 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-Plant40:528–535Fig.5Timeline of seed germination to adult plantMudge KW,Chu CC(1992)Novel laboratory exercises in plant tissue culture:in vitro asymbiotic germination of orchid seeds.HortTechnology4:315–317Proctor HC(1998)Effect of pollen age on fruit set,fruit weight,and seed set in three orchid species.Can J Bot76: 420–427Rasmussen H,Johansen B,Andersen TF(1989)Density dependent interactions between seedlings of Dactylorhiza majalis(Orchidaceae)in symbiotic in vitro culture.Physiol Plant77:473–478Shiau YJ,Sagare AP,Chen UC,Yang SR,Tsay HS(2002) Conservation of Anoectochilus formosanus Hayata by artificial cross-pollination and in vitro culture of seeds.Bot Bull Acad Sin43:123–130Stewart SL,Kane ME(2007)Symbiotic germination and evi-dence for in vitro mycobiont specificity in Spiranthes brevilabris(Orchidaceae),and its implications for spe-cies-level conservation.In vitro Cell Dev Biol Plant 43:178–186。