Protocols for Micropropagation of Woody Trees and Fruits

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Table 2. Media formulations used at different stages of micropropagation.
Media Culture Culture Shoot bud Rooting Hardening
additives initiation multiplication elongation
Basal
medium
¾ MS 3/4 MS ½ MS ½ MS ½ MS
TDZ — 0.1mg/l — — —
NAA — 0.1 mg/l — 2.5 mg/l —
IBA — — — 2.5 mg/l
Sucrose 30 g 30 g 30 g 30 g 30/15/0 g
AC 2 g — 2 g — 1 g
PVP-360 — 1.0 g/l — — —
2.8. Hardening, Acclimatization and Potting
THIMMAPPAIAH ET AL.
1. In the first stage of hardening the in vitro rooted shoots are cultured on
liquid half-strength MS medium containing 1 g/l AC and a reduced percentage
of sucrose (1.5%) for 2 to 3 weeks (Figure 1E).
2. Transfer the shoots into the same medium without sucrose for another 2–3
weeks by siphoning out the old medium. This facilitated shoot elongation
and secondary root formation. One of the roots becomes prominent at this
stage and assumes the role of tap root when planted in field.
3. Prior to potting the plantlets are taken out of the container carefully and
rinsed gently in SDW to remove traces of media and then immersed for 20
min in 0.2% bavistin (carbendazim 50% WP) solution for preventing
contamination of plantlets in pots.
4. In the second stage of hardening, the in vitro hardened plantlets are trans-
ferred to 4” plastic pots containing sterile pot mixture of sand and soil rite
(2:1 v/v) (autoclaved for 2 h at 121°C) (Figure 1F) and plantlets are covered
with 200 gauge transparent polythene bags for maintaining humidity.
5. The potted plantlets are irrigated initially for 10 days with only SDW and
then with 1/10 MS macrosalt solution (pH 6.8–7.0).
6. The humidity is gradually reduced by punching holes and increasing the
number of holes in polythene bags and finally by fourth or fifth week the bags
are completely removed.
7. Over-irrigation needs to be avoided and sometimes drenching of pots with
0.2% bavistin is required to prevent any residual contamination if any in
plantlets. The survival of plantlets during this stage varies from 80 to 100%.
8. After hardening in pots for 4–6 weeks in laboratory, the plantlets are
carefully transferred to 20 cm earthen pots containing mixture of red earth,
sand and compost or FYM (1:1:1 v/v) and kept in greenhouse under partial