Protocols for Micropropagation of Woody Trees and Fruits

374 L. TIAN AND S.I. SIBBALD
younger or over-mature fruit is used. Store fruit at 4°C, and process (see below) as
soon as possible after collection, preferably within 2 months. Longer storage will
result in an overgrowth of microorganism contamination.
Remove the flesh (mesocarp) from the endocarp (stone) with a paring knife.
Wash the endocarp containing the seed with 0.05% sodium hypochlorite (1%
commercial bleach) for approximately 10 minutes with stirring, and rinse several
times with water until no further flesh is removed and the water remains clear.
Allow the endocarps to air dry on absorbent paper on the lab bench for 2 days before
storing the endocarps in plastic mesh bags at 4°C. The endocarps containing the
seeds can be stored under this condition and be used for micropropagation as needed
over the year.
2.1.2. Surface Disinfection
Crack stored endocarps with a hammer to release seeds. Care must be taken to avoid
damaging the seed. Surface-disinfect seeds in a flask containing a solution of 0.5%
sodium hypochlorite with 0.005% Tween 20 for 15 minutes while stirring. Under
aseptic conditions, remove the bleach solution and wash the seeds three times with
sterile deionized water to remove any remaining bleach. Soak seeds overnight in
sterile water at room temperature to soften the tissue for easier dissection.
2.1.3. Explant Dissection
The following day in a laminar flow hood, cut seeds in half crosswise in a Petri dish,
remove the seed coat (Figure 1A) and split open the cotyledons to reveal the
embryonic axis (Figure 1B). Remove the embryonic axis using a scalpel (#11 blade
preferred) and dissect into three segments: epicotyl, hypocotyl and radicle. Discard
the radicle since it generally fails to produce shoots. Cut the hypocotyl into three
slices as shown in Figure 1C. Epicotyls are used for propagation via axillary bud
development and hypocotyls are used for propagation via adventitious shoot induction
(Scorza et al.,1995; Tian et al., 2004; 2005; Sibbald et al., 2006).
2.2. Culture Medium
Three different types of media can be used for plum micropropagation, including
MS medium (Murashige & Skoog, 1962), B5 medium (Gamborg et al., 1968) and
woody plant medium (WPM) (Lloyd & McCown, 1981). The current protocol is
based on the use of Murashige & Skoog’s medium.
Shoot induction medium (Table 1) contains MS salts, 100 mg/L myo-inositol,
0.4 mg/L thiamine HCl, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.5
mg/L indolebutyric acid (IBA), 25 g/L sucrose, 7 g/L Bactoagar (Difco) (Mante
et al., 1991). Adjust pH to 5.9 with the addition of HCl (or KOH if the pH is too
low). Place the medium in 1L glass bottles and autoclave at 121°C for 30 minutes.
After cooling to approximately 45°C, add 1.66 mg/L filter-sterilized thidiazuron
(TDZ). Pour approximately 35 ml of the medium into 25 × 100 mm petri plates and
allow to solidify.