Protocols for Micropropagation of Woody Trees and Fruits

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2.2. Disinfection of Plant Material
M. MHATRE
Explant – Leaf Bases of In Vitro Shoots. The basal ends of in vitro produced shoots
isolated from stock cultures can also be used as explants after peeling them and
placing them with their ventral sides in contact with the defined multiplication
medium. The basal section can be cultured (one per tube) in the medium for the
induction of multiple shoots. This process would take 6 weeks and the shoots thus
obtained can be multiplied and maintained on the same medium. Elongation of
shoots can be done on hormone-free medium and rooted on liquid rooting medium
(Soneji et al., 2002b).
Dormant axillary buds (one at the base of each leaf of the crown) of pineapple can
be carefully excised with minimum unavoidable surrounding tissue (Figure 1, step 1;
Figure 2A) and collected in a 5 × 5 cm pre-sterilized damp (dampened with tap
water) muslin cloth, tied into a bundle with a string and placed into a pre-sterilized
conical flask and stoppered. The bundle containing the excised axillary buds can
then be washed thoroughly with soap and tap water. Following this, surface
sterilization of the buds should be carried out in a laminar air flow unit. The axillary
buds can be sterilized in 0.1% mercuric chloride for 2 min and rinsed several times
(~ 4–5 washes) in sterile tap water (Figure 1, step 2). Each axillary bud (one per
culture tube) can be placed vertically for sprouting with its basal end slightly
embedded in the bud sprouting medium (Figure 2B).
2.3. Shoot Regeneration and Maintenance
Media. The surface sterilized dormant axillary buds of pineapple should be cultured
on ‘bud sprouting media’ (BSM) comprised of: hormone-free N (Nitsch’s, 1951) or
MS (Murashige & Skoog, 1962) basal medium, containing BAP (4.44 µM). The
buds will sprout and give rise to 1–1.5 cm, 1–2 shoots (Figure 2C) after 4–6 weeks
in culture (Figure 1, step-3). A single shoot should then be transferred to ‘shoot
multiplication medium’ (SMM) – comprised of MS basal medium supplemented
with NAA (9.67 µM), IBA (9.84 µM) and Kn (9.29 µM) for further proliferation
(Figure 2D). In this medium each shoot can be made to proliferate to give rise to 10–
20 shoots. SMM can be used also as a maintenance medium on which a tuft of 4–5
(3–4 mm) shoots can be subcultured and maintained as a cyclic proliferating stock
culture leading to 50–60 shoots per culture (Figure 2D). SMM can also be used as
liquid medium (without addition of phytagel) in flasks for further proliferation of
shoots (Figure 2E). However, shoots produced in flasks tend to be elongated and can
be directly rooted on rooting medium. A tuft of 4–5 (1–2 cm) shoots proliferated
on SMM solid medium, can be subcultured on ‘hormone-free MS’ basal medium
(HFMS) for shoot elongation. It is necessary for each shoot to attain a height of 4–5
cm before they can be isolated and induced to root. Shoot elongation would take 4–5
weeks on HFMS medium (Table 2).