fairly large and easy for students to manipulate during laboratory. The learning outcomes for this laboratory exercise are that a student will understand the role seed coverings play in maintaining seed dormancy, be able to explain the concept of embryo
Servet Caliskan, Sharon T. Kester, and Robert L. Geneve
Ron G. Goldy and Dana F. Moxley
A laboratory exercise is outlined and discussed for embryo culture of bean, corn, and pea embryos. Fresh, inexpensive material is generally available for these crop species throughout the year. The exercise gives students experience in embryo excision and exposure to some benefits of embryo rescue. Embryos from the three species are identified easily and can be removed without magnification, and data can be obtained within 3 weeks after culture. Further investigations using embryos are suggested.
Sharon A. Duray and Fred T. Davies Jr.
A laboratory exercise is outlined and discussed for seed priming, or osmoconditioning. The exercise was developed using an easily constructed and inexpensive seed-priming system. A variety of horticultural seeds can be used to give students experience and exposure to some of the benefits of seed priming. Seed germination data usually can be obtained within 6 to 8 days, depending on the species used. The laboratory may be modified to stress various features of seed priming, including priming agents, optimal concentrations, and ranges of germination temperatures.
John E. Preece and Carl A. Huetteman
This exercise was developed for a plant propagation course to demonstrate, in a short time, the four stages of micropropagation, the effects of cytokinin concentrations, and the differences between adventitious and axillary shoots. Greenhouse-grown stock plants were brought into the laboratory, and 4- to 5-cm-long tips of runners were surface-dis-infested for 15 min in 0.5% NaClO with 1 ml of Tween 20/liter, followed by two 5-min rinses in sterile water. Working in the open laboratory near the bases of pairs of lit Bunsen burners, students placed either single-node or shoot tip explants (2 cm long, five replications) onto MS medium with 0, 1, or 10 μM BA. Cultures were in-cubated in parafilm-sealed culture tubes on open laboratory benches. Axillary shoots grew regardless of concentration of BA, and explants on medium with 10 μM BA produced the most callus and adventitious shoots. Microshoots were rooted and ac-climatized under mist in the greenhouse. This exercise can be performed in an open laboratory without the use of laminar flow hoods, specialized sterilizing equipment, or supplemental lighting.
Thomas E. Marler and Haluk M. Discekici
A laboratory exercise is outlined in which breath is used as the source for elevating CO2. Single-plant enclosures are constructed by placing containers with stem cuttings within clear bags for maintaining high humidity during root initiation. These enclosures provide a restricted atmosphere in which elevated CO2 is adequately confined. The materials are inexpensive and readily available. The procedure is rapid, with results obtained in as few as 7 days. The increase in canopy and/or root growth as a result of elevated CO2 may be observed visually by younger audiences, or may be measured and analyzed in a manner dependent on the age group. The exercise generates an abundance of discussion and may be used to lead into many lectures on plant physiology, horticultural crop production, or global concepts of CO2 and the plant kingdom.
Sharon A. Duray and Fred T. Davies Jr.
Plant propagation instructors are challenged to develop laboratory exercises that demonstrate the theoretical aspects of seed germination. Seed priming or osmoconditioning is a relatively new technigue that has been shown to improve seed performance in horticultural crops. An esaily constructed seed priming system was designed using a pair of 2-liter glass jars, 2 aquarium pumps and air tubing. Eight sets of 40 seeds were each wrapped in coffee filters and laced in aerated treatment solutions consisting of 50 mmole K H2P O4 or an untreated control of distilled water. All seeds were treated or 0, 1, 3 or 5 days. Upon completion, seeds were rinsed, dried and placed into petri dishes containing moist filter paper to observe germination. A good test species for this exercise is Vinca rosea which typically has a poor germination percentage and rate. Seeds primed for 3 and 5 days significantly enhanced both germination percentage and rate in Vinca.
Michael E. Kane and Craig K. Chandler
Many horticultural crops are infected with bacterial, fungal, or viral pathogens that reduce yield and/or quality. Recovery and maintenance of pathogen eradicated crops, such as strawberry (Fragaria × ananassa Duch.), have been possible following the isolation and culture of apical meristems or meristem-tips in vitro. A laboratory exercise has been developed to provide experience in the procedures required for the isolation, surface disinfection, and in vitro establishment of meristem-tip explants excised from strawberry stolons. Stolons are obtained from greenhouse-grown strawberries (`Sweet Charlie') maintained in hanging baskets under a 14-h photoperiod. Stolons are cut into single-node segments and terminal tips. The leaf blades are removed and the nodal sections are rinsed and then surface-disinfected by successive agitation in 70% ethanol and 1.05% sodium hypochlorite, followed by three rinses in sterile deionized water. In the transfer hoods, each student attempts to isolate meristem-tips and shoot tips of various sizes under high magnification provided by a stereomicroscope. Explants are inoculated onto Murashige and Skoog basal medium (Murashige and Skoog, 1962) supplemented with 30 g/liter sucrose, 80 mg/liter adenine sulfate, 1.0 mg/liter benzyladenine, 1.0 mg/liter indole-3-acetic acid, and 0.01 mg/liter gibberellic acid (GA3) and solidified as 45°slants with 1.25 g/liter Phytagel and 3.0 g/liter TC agar. Growth responses are monitored weekly. After 6 weeks, students record the percentage of visibly contaminated cultures and number shoots produced per explant. The relationship between initial explant size and in vitro growth is also determined. Students index their cultures for the presence of cultivable bacteria and fungi using sterility test media.
Mark P. Bridgen, Masood Z. Hadi, and Madeleine Spencer-Barreto
A laboratory exercise on direct and indirect organogenesis from leaf explants is presented for students of plant tissue culture or plant propagation. Torenia fournieri, the wishbone flower, is used for this laboratory exercise because the in vitro production of adventitious shoots from Torenia is easy to control, seeds are easy to obtain, and plants are easy to grow. Direct shoot organogenesis results from leaf explants without an intervening callus phase, and indirect shoot organogenesis is possible after 4 to 6 weeks of callus production from leaf explants. The basal medium for all forms of organogenesis contains Murashige and Skoog (MS) salts and vitamins, 30 g sucrose/liter, and 7 g agar/liter at pH 5.7. To obtain direct shoot organogenesis, leaf explants should be placed on the MS basal medium with 1.1 μM (0.25 mg·liter-1) 6-benzylaminopurine (BAP) and 0.25 μM (0.05 mg·liter-1) indole-3-butyric acid (IBA). If leaf explants are placed on MS medium with 2.3 μM (0.5 mg·liter-1) 2,4-dichlorophenoxyacetic acid (2,4-D), callus formation will occur. Callus can be subcultured onto a MS medium with 8.88 μM BAP (2.0 mg·liter-1) plus 2.5 μM IBA (0.5 mg·liter-1) for indirect shoot organogenesis to occur.
David R. Hershey and Susan Sand
A Venturi-type proportioner (VP), trade name Hozon, can be used for an inexpensive, hands-on laboratory exercise that demonstrates the effect of water pressure on dilution ratio and water flow. Using electrical conductivity (EC) meters to determine solution concentration allows students to discover that the dilution ratio increases with water pressure, from 1:10 at 15 psi to 1:15 at 55 psi. The greater dilution at higher pressure can be explained by measuring the water flow, which is 2.3 gal/min (8.7 litersžmin-1) at 15 psi but 3.5 gal/min (13.2 litersžmin-1) at 55 psi. Experiments relating water pressure to dilution ratio provide experience in use and calibration of VPs and EC meters, as well as graph preparation and interpretation.
Michael E. Kane, Nancy L. Philman, and Matthew A. Jenks
Only a few plants are suitable for reliably demonstrating rapid direct and indirect shoot organogenesis in vitro. A laboratory exercise has been developed using internodes of Myriophyllum aquaticum, an amphibious water garden plant. Stock shoot cultures are established and maintained in vitro from nodal explants cultured on agar-solidified medium consisting of half-strength Murashige & Skoog salts (MS) and 30 g·liter-1 sucrose. Students use these cultures as the source of internode explants. Explants are cultured on agar-solidified full-strength MS with 30 g·liter-1 sucrose, 100 mg·liter-1 myo-inositol, and 0.4 mg·liter-1 thiamine·HCL and factorial combinations of 0 to 10 μM 2iP and 0 to 1.0 μM NAA. Adventitious shoot development occurs directly from the explant epidermis within 4 days and is promoted in media supplemented with 2iP alone. Cytokinin-supplemented media amended with NAA induce organogenetic callus formation, but reduce 2iP promotion of direct shoot organogenesis. After 4 weeks, shoot organogenesis on the various media is quantified and can be analyzed statistically. Chemical names used: N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP); α-naphthaleneacetic acid (NAA).