Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF PINEAPPLE
Table 2. Nutrient media compositions used for pineapple micropropagation.
Medium Macroelements
BSM
Microelements
RM 1 1 1 NAA 0.54
IBA 1.97
BSM: Nitsch’s (1951) or MS (1962) macro- and microelements, vitamins;
SMM: MS (1962) macro- and microelements, vitamins;
HFMS: MS (1962) macro- and microelements, vitamins;
RM: White’s (1954) macro- and microelements, vitamins;
Gelation of solid media was with gelrite (0.2%); Fe EDTA was added separately
according to Murashige and Skoog (1962) medium and the pH was adjusted to 5.8
prior to autoclaving; NAA, naphthalene acetic acid; IBA, indole butyric acid; Kn,
kinetin; BAP, benzyl amino purine.
2.4. Culture Vessels and Culture Conditions
Carbon source
(%)
All culture vessels (test tubes and flasks) used in the protocol should be made of
Borosil glass, 25 × 150 mm size. Non-absorbent cotton should be used for plugging
the tubes prior to autoclaving at 121°C, 1 kg/cm for 15 min. All cultures should be
incubated at 25 ± 2°C in 16/8 h dark/illumination (12.1 µMm –2 s –1 , Philips) cycle.
Phytagel (Sigma) 0.2% is appropriate for solidifying culture media – BSM, SMM
and HFMS. RM is devoid of phytagel and therefore the shoots (3–4 cm) have to be
placed over filter paper bridges for support and for the induction of rooting. Carbon
source used was sucrose (1–2%) as described in the text and Table 2. The pH of all
media should be adjusted to 5.8 using 1N NaOH or 1N HCl, prior to autoclaving.
Soilrite (Soilrite mix, TC is a mixture of 75% Irish peatmoss and 25% expanded
Perlite, obtained from M/s Chougule Industries Ltd., Mumbai) or autoclaved fine
soil may be used to harden the in vitro raised plantlets.
2.5. Rooting of Shoots, Recovery of Plantlets and Acclimatization
Growth
adjuvants (µM)
1 1 2 BAP 4.44
SMM 1 1 2 NAA 9.67
IBA 9,84
Kn 9.29
HFMS 1 1 2 –
Each elongated shoot can then be isolated and subcultured on liquid ‘rooting
medium’ (RM) comprised of White’s (1954) basal medium supplemented with
NAA (0.54 µM) and IBA (1.97 µM) and 1% sucrose (Table 2), for the induction of
roots (Figure 2F). Plantlets with actively growing roots can be carefully removed
from culture tubes and washed with tap water and transferred to paper cups containning
either autoclaved soilrite or soil (Figure 2G). All plantlets were transferred to
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