Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF CASHEW
2.2.1. Explant
Shoot explants like nodal cuttings (1–2 nodes), shoot-tips and coteledonary nodes
with and without cotyledons excised either from 1 month old in vitro-raised seedlings
or greenhouse seedlings are used. In vitro-raised seedlings are obtained by germinating
mature seeds in vitro after sterilizing the seeds. Similarly to raise seedlings in greenhouse
the seeds are sterilized and germinated in aluminum trays containing sterile
sand or pot mixture.
Sterilization and In Vitro Germination of Seeds
1. Soften fresh mature cashew seeds in concentrated hydrochloric acid for 10
min in a fume hood.
2. Rinse seeds several times in sterile distilled water (SDW).
3. Surface sterilize the seeds in the flow hood first by immersing in 70%
ethanol (1 min).
4. Rinse in SDW (1–2 times).
5. Sterilize seeds second time in 50% sodium hypochlorite solution (4%
available chlorine) with 1 to 2 drops of Tween-20 for 20 min by constant
stirring (in a shaker).
6. Rinse at least thrice in SDW.
7. Inoculate the disinfected seeds into sterile screw cap bottles (200 ml capacity)
(5.5 × 10.5 cm OD × H) laid with moist (in SDW) absorbent cotton (surgical
grade) at bottom.
8. Germinate the seeds at 25 ± 2°C in a growth chamber in dark.
9. The seedlings emerged 25–30 days after inoculation are used as a source of
explants (Figure 1A).
Explant Sterilization
Shoot explants collected from 3 to 4-week-old greenhouse raised seedlings are
sterilized following the procedure described below.
1. Collect actively growing terminal shoots (5–8 cm long) in a clean beaker.
2. Shoots are defoliated in laboratory and segmented into one or two cuttings.
Petioles are removed close to the axils without damaging the dormant buds
in the axils.
3. Dip shoot segments in 70% ethanol for 20–30 seconds to do a preliminary
disinfection.
4. Wash the shoot segments several times in SDW after adding and rinsing in
0.01% detergent and pinch of carbendazim (50% WP).
5. Surface sterilization of explants is carried out in a sterile container in 0.1%
mercuric chloride containing a drop of Tween-20 (wetting agent) by constant
agitation for 5–7 min. Explants of cashew from nursery or greenhouse source
are prone to high contamination. Mild sterilants were found not effective.
6. Rinse in SDW minimum of three to five times.
7. Segment the explants to size (1.0–1.5 cm) in SDW and inoculate into tubes.
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