Shoots which had been produced in tissue culture were stimulated to form roots by transferal to a culture medium similar to the shoot multiplication medium, but lacking the cytokinin. This medium contained 0.1 mg/l IBA (indolebutyric acid) which stimulated rooting of up to 97% of the M26 shoot tip cuttings within 6 weeks (3). This technique has also been successfully applied in the rooting of cultured shoot tips of five apple scion cultivars (4) and a similar technique was described for tissue cultured almond clones (6).
The research described in this article represents the application of these tissue culture techniques to the plant Amelanchier laevis. Amelanchier is a deciduous small tree in the Rosaceae, and is similar to the apples, cherries, and plums in many morphological and physiological respects.
Figure 1. Amelanchier laevis in flower in early spring.
Figure 2. Amelanchier is noted for its outstanding red/orange fall color.
The objectives of the current investigation were to describe a system for the in vitro propagation of Amelanchier, determine the rate of multiplication, and devise a system for rooting the cuttings which would be produced.
The culture medium contained the inorganic salts and organic components according to Murashige and Skoog (5). Cultures were initiated on agar gelled medium in 30 ml French square bottles containing 1 mg/l benzyladenine (BA). Following 10 weeks of growth on the initial medium, shoot cultures were transferred to 125 ml bottles containing a similar medium except the growth regulator concentration was 0.1 mg/l napthaleneacetic acid (NAA) and 2.5 mg/l BA. Cultures are currently being maintained on the medium containing 0.1 mg/l NAA and 2.5 mg/l BA in 500 ml glass jars capped with glass Petri dishes and sealed with saran wrap. All cultures were grown at 26 C under 500 foot candles of warm white fluorescent light with a daylength of 16 hours.
Rooting experiments were conducted using one-fourth strength Murashige-Skoog medium solidified with agar and containing 0.1 mg/l indolebutyric acid (IBA). The 500 ml jars are used for the rooting phase.
Figure 3. Mass of shoots which have proliferated from a single shoot tip of Amelanchier after 4 months in tissue culture.
Multiple shoots are seen to arise from a basal mass, which actually represents small, unexpanded shoots rather than callus. Little, if any, callus has been observed in any of the cultures examined to date.
Shoot multiplication appears to proceed primarily by lateral shoot growth, although formation of adventitious shoots cannot be ruled out. When cultures are examined closely, the active growth of lateral shoots can be observed (Fig. 4, arrows).
Figure 4. Rapid multiplication of Amelanchier occurs due to active growth of axillary shoots. Actively growing lateral shoots are indicated by the arrows.
In some cases, lateral shoots have been observed in active growth as close as one or two expanded internodes distance from the shoot tip. The conditions of the tissue culture environment thus appear to circumvent apical dominance and result in a mass of shoots each producing more shoots.
Cultures resulting from seven individual shoot tips have been sacrificed to date either as a result of contamination or in order to conduct rooting experiments. These have been in culture for a total of 4 months. A detailed count revealed more than 280 shoot tips present in the seven cultures. The mass from one shoot tip contained 105 individual shoot tips, while the remainder of the cultures contained fewer shoots.
Those shoot tips which had elongated to between 1.5 and 2.0 cm in length were used in subsequent rooting experiments (Fig. 5).
Figure 5. Shoots were removed for rooting when they exceeded 1.5 cm in length. This shoot tip cutting from tissue culture was photographed with a 3 cm paper clip for scale comparison.
Of the 280 shoots produced in the seven cultures, 110 of these had elongated sufficiently to be deemed suitable for rooting experiments. Rooting was attempted by placing the shoot tips in one-fourth strength Murashige and Skoog medium containing 0.1 mg/l IBA. The culture vessels were 500 ml glass jars with glass petri dish covers so that the lighting for the cultures was maximized. Roots at least 1 mm in length were observed on one shoot tip cutting after only 8 days in the rooting medium. Eight of the 40 shoots in the experiment had at least one root by 14 days.
The ability to mass propagate Amelanchier through tissue culture adds further evidence that plants within the Rosaceae should respond similarly to the in vitro environment. The conditions described herein are very similar to those described for apples (1, 3, 4) and plums and cherries (2). The rate at which multiplication occurs (even considering the very slow initial phase), approximately a 40-fold increase in 4 months, suggests that propagation via tissue culture could be a commercially feasible method for Amelanchier provided a suitable system for growing on the rooted plantlets were developed. Improvement of the rooting methodology and the development of a system for the accelerated production of Amelanchier from tissue cultured plantlets are the goals of the next phase of this research.
2. Jones, O.P. and M.E. Hopgood. 1979. The successful propagation in vitro of two rootstocks of Prunus: the plum rootstock Pixy (P. insititia) and the cherry rootstock F 12/1 (P. avium). J. Hort. Sci. 54:63-66.
3. Jones, O.P., M.E. Hopgood and D. O'Farrell. 1977. Propagation in vitro of M26 apple rootstocks. J. Hort. Sci. 52:235-238.
4. Jones, O.P., C.A. Pontikis and M.E. Hopgood. 1979. Propagation in vitro of five apple scion cultivars. J. Hort. Sci. 54:155-158.
5. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
6. Tabachnik, L. and D.E. Kester. 1977. Shoot culture for almond and almond-peach hybrid clones in vitro. HortScience 12:545-547.