A Laboratory Exercise to Demonstrate Meristem-tip Culture of Strawberry

Abstract

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.