Protocols for Micropropagation of Woody Trees and Fruits

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K. PRUSKI
2.2.3. Explant Excision and Sterilization
1. Collect the dormant buds (winter) or active buds (early spring) or actively
growing shoot tips, 1–2 cm long (spring to early summer) by cutting off
short twigs and take the twigs to the laboratory.
2. Remove buds with sharp scalpel, place them in a small paper bag in a small
container (i.e. a baby food jar) and place the container under the stream of
tap water for one hour; if shoot tips are used, remove outer leaves and
proceed as with to bud explants.
3. Peel off outer scales from the buds. If shoot tips are used, proceed to step 4.
4. Surface-sterilize by agitation (orbital shaker) in 0.5% sodium hypochlorite
(10 X diluted commercial bleach, Javex) containing 0.1% Tween-20
(surfactant) for 10 min.
5. Rinse the explants three times in sterile distilled water.
6. Trimmed to approximately 3 mm length.
7. Place the explants on 10–12 ml medium in culture tubes (25 × 150 mm)
and close with plastic caps.
8. Incubate cultures for 4 weeks in a growth-room under conditions described
in the next section (2.1.2).
2.2.4. Culture Medium, Its Preparation and Culture Conditions
The basic culture medium contained MS salts and vitamins (Murashige & Skoog,
1962) (MSMO medium – Murashige and Skoog Minimal Organic) supplemented with
sucrose at 30 g l –1 is used for growing cultures of Mongolian and Nanking cherry.
The components are listed in Table 1. Vitamins and organic compounds other than
plant growth regulators (PGR) are generally not changed in the different stages and
are as in Table 1. Different auxins and cytokinins at various concentrations are used
depending on the stage of micropropagation. One cytokinin, 6–benzylaminopurine
(BA) is used in establishment of cultures, generally at 4.4–8.8 µM, together with an
auxin, indole-3-butiric acid (IBA), or naphtalene acetic acid (NAA), at 0.5 µM. In
multiplication stage the concentration of BA in culture media is generally higher, 8–20
µM. Also another cytokinin, thidiazuron (TDZ) can be used at 0.5–1.0 µM concentration.
At the rooting stage, IBA alone (at 5–10 µM) or in combination with NAA
(at 2.7 µM) is used and cytokinin is omitted. Prior to rooting stage, proliferating
cultures of both P. fruticosa and P. tomentosa are placed on PGR-free media for the
elongation and growth of both shoots and possible root initials.
Culture conditions follow a common pattern for most of the woody plant
species. Culture media are adjusted to pH 5.5–5.7 prior to autoclaving, and are
solidified with agar at 0.6–0.7%. Various cultures containers are used to grow the
cultures. For initiation of cultures, test tubes are used, while for multiplication the
Magenta G7 containers (50 ml of medium) or baby food jars (30 ml of medium)
with Magenta caps are the most common. Cultures are incubated usually for 4 weeks
in environmental chambers at 24/22°C day/night temperature, 16-h photoperiod at
150 µEm –2 s –1 mixed fluorescent and incandescent illumination. Subcultures of plant
material to fresh medium are made every 3–4 weeks. If kept too long without
transfer, for example over 6 weeks, senescence process starts and the cultures go to
dormancy after 7–8 weeks.