Plant Tissue Culture Concepts and Laboratory Exercises (2000)

Chapter thirteen — Laboratory Exercise

Micropropagation and in vitro flowering of rose
Michael E. Kane

Figure 13.1 Micropropagation and in vitro flowering of the miniature rose. (a) Left: two-node explants (left) are cut from young shoots of potted plants and the leaf blades are removed prior to surface disinfestation. Right: Petiole bases are removed after surface disinfestation to expose lateral buds before inoculation. Scale = 5 mm. (b) Production of axillary shoots and in vitro flowering of the miniature rose following an 8-week subculture. (c) In vitro-produced flowers are smaller and have fewer petals than these produced on plants grown in the greenhouse. Scale bar = 10 mm.

 

Procedure 13.1 Stage I establishment and Stage II shoot multiplication from nodal explants of rose.
Step Instructions and comments
1 Cut several newly produced shoots from the donor plant. Remove leaf blades with a scalpel leaving attached only a small basal section (about 3 mm) of each petiole. Cut shoots into two-node explants about 15 mm in length (Figure 13.1a). It can be noted that the smoothly cutinized epidermis, typical of many miniature roses cultivars, should facilitate effective surface disinfestation of the explants. Nodes exhibiting bud break should not be used.
2 Rinse nodal segments in tap water for 15 min and then surface disinfest in 50% ethanol for 1 min followed by a 12-min agitated soak in 20% commercial bleach and three 5-min rinses in sterile deionized water.
3 In the transfer hood, trim off the bleached ends of each two-node explant and the base of each petiole to expose the lateral bud (Figure 13.1a).
4 Transfer a single two-node explant into each of ten culture tubes containing REM. Partially embed the basal end vertically into the medium.
5 Label each tube and maintain in an incubator or under a fluorescent lignite on a bench top at 25°C and provided with approximately 30 mol•m-2•sec-1 for 16h. To prevent abnormal shoot development and premature leaf abscission, sealing films such as Parafilm should not be used.
6 After 4 weeks, students should determine: (1) the percentage of visibly contaminated cultures: (2) mean number of axillary shoots produced per explants; and (3) percentage of flowering cultures. Data can be collected without removing the plants from the culture vessels.
7 In the transfer hood, Stage I shoot clusters are aseptically divided into defoliated two-node secondary explants with five explants inoculated into each of GA-7 vessels containing 60 mL freshly prepared REM. Cultures are maintained under the conditions described above and observed weekly.
8 After 7 to 8 weeks, students should determine: (1) mean shoot number and length produced per explant and (2) percentage of explants producing flowers. The data collected by each student or student team at 4 and 7 to 8 weeks should be shared to provide more representative responses.

Anticipated results

Many miniature rose cultivars display vigorous growth when established in vitro; the actual response is highly cultivar dependent. Axillary shoots should develop from the cultured primary nodal explants by 2 weeks’ culture. By week 4, two to three axillary shoots are produced from each nodal explant. In vitro flowering seldom occurs on shoots produced on primary nodal explants during the first culture period. This suggests that competency for flowering is acquired only following subculture.

Nodal explants subcultured into GA-7 vessels should exhibit vigorous shoot development (Figure 13.1b). Shoots produced in larger vessels, such as GA-7 magenta vessels, will exhibit a greater frequency of flowering. Floral buds are produced by week 6 and the flowers open by week 8 (Figure 13.1b). Approximately 30% of the shoots produce open and very fragrant flowers. Flowers produced in vitro are much smaller and are composed of fewer petals than those produced on plants grown in the greenhouse (Figure 13.1c). Flowers become senescent within 2 weeks of opening. Recurrent flowering does not occur unless shoots are subcultured. Infrequently, vegetative shoots develop from the center of open flowers in vitro. If time constraints exist or demonstration of in vitro rose flowering is the only objective, previously established stock shoot cultures can be used as the source of explants