Protocols for Micropropagation of Woody Trees and Fruits

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limited and hence in vitro rooting using Agrobacterium rhizogenes was reported by
Das et al. (1996). Boggetti et al. (1999) and Mneney and Mantell (2002) have also
attempted micropropagation in cashew. Thimmappaiah and Shirly (1999) have been
able to regenerate cashew completely using explants from juvenile source and the
protocol standardized in our laboratory is described in this chapter. Our success with
regeneration from explants of mature tree source has been limited owing to poor
growth, elongation and rooting of shoots (Thimmappaiah et al., 2002a). Micrografting
as a technique to rejuvenate cashew and overcome rooting has also been described
(Thimmappaiah et al., 2002b). Earlier attempts on micrografting in cashew were
reported by Mantell et al. (1997) and Ramanayake and Kovoor (1999).
Regeneration of cashew through embryogenesis from cotyledon (Hegde et al.,
1994; Jha, 1988; Sy et al., 1991), immature embryos (Cardoza & D’Souza, 2000;
Nadgauda, 2000; Cardoza & D’Souza, 2002; Shirly & Thimmappaiah, 2005) have
been reported but complete regeneration and establishment of plantlets are yet to be
achieved.
2.1. Materials
2. EXPERIMENTAL PROTOCOL
1. Mature cashew seeds, in vitro raised seedlings, defoliated terminal shoots.
2. Sodium hypochlorite (NaOCl) (4% available chlorine), mercuric chloride,
concentrated hydrochloric acid, rectified spirit, sterile distilled water,
Tween-20.
3. Laminar flow hood, stainless steel sterile surgical blades, scalpel handle,
spirit lamp, sterile Petri plates, forceps, tissue paper, rimless test tubes (25 ×
150 mm), screw cap bottles (200 ml capacity), baby food jars (Sigma).
4. Tissue culture chamber, portable autoclave, microwave oven, environmental
shaker.
6
5. Thidiazuron (TDZ), naphthalene acetic acid (NAA), N -benzyl adenine
(BA), indole-3-butyric acid (IBA), polyvinylpyrrolidone (PVP-360), Lglutamine,
casein hydrolysate, activated charcoal (AC).
6. Media: Murashige & Skoog (1962) (MS) and Raj Bhansali (1990) (RBM)
basal media (Table 1) and their formulations (Table 2).
2.2. Methods
THIMMAPPAIAH ET AL.
Gogate & Nadgauda, 2003) and nucellus (Ananthakrishnan et al., 1999; Gogte &
The technique of regeneration involves 1) raising of in vitro seedlings or seedlings
in greenhouse as a source of explants, 2) explant preparation and sterilization, 3)
initiation of shoot cultures on medium, 4) axillary shoot bud proliferation on
multiplication medium, 5) shoot bud elongation phase on hormone free medium, 6)
root induction phase and 7) hardening and potting stages.