Protocols for Micropropagation of Woody Trees and Fruits

PROPAGATION OF MONGOLIAN CHERRY AND NANKING CHERRY
2. Flow hood, orbital shaker, sterile Petri dishes, Erlenmeyer flasks, glass test
tubes (15 × 150 mm) with plastic caps, Magenta GA7 vessels, pipettes,
forceps, scalpels, stainless steel surgical blades.
3. Fully controlled environment tissue culture growth-room.
4. Culture media (MSMO – see Table 1), Bacto agar.
5. Plant Growth Regulators (PGRs): 6-benzylamino purine, Thiadizuron
(TDZ), indole-3-butiric acid (IBA), indole-acetic acid (IAA), naphthalene
acetic acid (NAA), gibberellic acid (GA3).
6. Terminal dormant buds, shoot tips, regenerated apical or axillary shoots.
2.2. Methods
The protocol would require: 1) the establishment of in vitro cultures from either
dormant or active buds, or shoot tips; 2) multiplication of shoots, 3) rooting either
in vitro or ex vitro, 4) acclimatization and 5) transplanting to the field.
2.2.1. Explant Preparation
To obtain true-to-type propagated plants via tissue culture methods, care must be
taken in both the choice of explant and the method of multiplication. Most often
shoot tips and meristems, as well as dormant (winter) or active (spring) terminal
buds are the explants of choice due to their genetic stability. Explant size is not that
important for micropropagation purposes as for obtaining disease-free plants. A
general approach is: the smaller the explant, the less the possibility of bacterial and
fungal contamination. In our laboratory, cultures of P. tomentosa (a black fruit
clone) were established from terminal buds and dissected up to 3 mm by removing
the outer scales (Figure 2A) showed no sign of contamination after over 1 year of
continued culture, while cultures obtained from stem pieces with lateral buds
developed bacterial contamination after several months in culture. Once established,
explants of both Mongolian and Nanking cherries form small rosettes and these are
later moved to the multiplication stage.
2.2.2. Growing Conditions of Mother Plants
Explants of Mongolian cherry and Nanking cherry used in our work were taken
from 10-year-old plants grown in the shelterbelt row at Crop Diversification Centre
North, Edmonton, Alberta, Canada (53° N latitude, 113° W longitude). The plants
were derived from rooted cuttings taken from F1 seedlings selected for fruit size and
flavor. Terminal (dormant) buds were dissected from current year growth in
December. December collection was chosen to avoid winterkill conditions that
occur on the Canadian prairies between January and March due to frequent freezing
and thawing periods (Pruski et al., 2000). In subsequent experiments, active young
buds collected in early May and actively growing young shoot tips collected in early
June were also used as explant sources with various success (Pruski, unpublished).
Actively growing stem tips of P. fruticosa field grown trees (Liaoning, China) were
also successfully used as explants by Ping et al. (2001).
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