Protocols for Micropropagation of Woody Trees and Fruits

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A. ONAY ET AL.
Levels of survival, the average shoot length and the average shoot number were
assessed after 5 weeks of culture. The position of the explants cultured did not
influence the survival rate but the highest length of shoots was obtained with the
apical tips explanted from the pruned shoot trunks. Explants from the pruned shoot
trunk produced most shoots and means were greater than the explants excised from
other positions.
1. Collect the actively growing shoot tips (3–5 cm long) from a pruned trunk
of mature pistachio by cutting with a sharp scalpel and cover immediately
the cut edge with a moistened cotton material.
2. Bring the shoot tips to the laboratory, chop off the leaves (with petioles)
and surface sterilize or dip the defoliated shoot tips into the sterile distilled
water (SDW) if they were not used for initiation of cultures.
Explant sterilization and initiation of cultures. In fact, it is difficult to establish
in vitro cultures from lignified stem sections from mature pistachio trees during the
dormant season. We have been conducting research on forcing shoots for years.
Shoots were forced in the greenhouse under a 24 h photoperiod until they were large
enough to excise, disinfest surface and place in vitro.
1. Remove the stem section of the explants (use only shoot tips, 15–20 mm
long) and sterilize them.
2. Treat with 20% commercial bleach with 14% available chlorine for 35 min
on a shaker at 150 rpm.
3. Gently wash with SDW at least 3 times for 5 min.
4. Cut the basal end of the rinsed explants.
5. Shake the explants with SDW at least twice for 1 h on horizontal type
shaker at 150 rpm with SDW.
6. Inoculate 3–5 mm apical tips on scion production medium (SPM) (Table 2).
7. Incubate cultures in a growth room under the light (at 40 µmol m –2 s –1 photo-
synthetic photon flux density; fluorescent lamps, 75W) and a photoperiod
of 16 h at 25 ± 2°C for 4 weeks.
8. Excise the sprouting apical tips with minimal mother tissue attached. Note
that shoot tip region consist of apical meristematic bud surrounded by a
number of leaf primordia encased by fleshy green-white tissue of a leaf
sheath.
9. Transfer the regenerated shoots to the shoot culture maintenance medium
(CMM) (Table 2).
10. Incubate until the explants are at 3–4 leaf stage. An early subculture is
recommended if the apical tips start releasing the polyphenols in the culture
medium.
11. Use freshly sprouted apical tip or healthy growing shoot tips (4–6 mm long)
as scions (Figure 1A).