Protocols for Micropropagation of Woody Trees and Fruits

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D.T. NHUT ET AL.
its economical importance, propagation of passion fruits in general and yellow
passion fruit in particular is of interest of many researchers and breeders.
Passion fruit is a perennial plant that can be propagated by seeds, cuttings, airlayering
or grafting. Most commercial passion fruit producers worldwide use
seedlings to establish plantations, because they do not spread the woodiness virus
(Nakasone and Paull, 1998). However, seed propagation results in undesirable
variability, inadequate and seasonal supply (Isutsa, 2004). Propagation using cuttings
and grafting is occasionally practiced, but these methods risk spreading the
woodiness virus (Nakasone and Paull, 1998). Although this species is resistant to
Fusarium wilt (Gardner, 1989) and nematodes (Drew, 1997), other fungi, viruses
and bacteria, are responsible for significant losses in passion fruit production.
Therefore, a reliable method for in vitro passion fruit propagation would have
considerable benefits. In vitro regeneration of passion fruit has been obtained from
axillary shoots or internodal segments (Robles, 1978, 1979), shoot apices or nodal
segments cultivation (Kantharajah & Dodd, 1990; Drew, 1991; Faria & Segura,
1997a; Biasi et al., 2000; Monteiro et al., 2000; Reis et al., 2003) or from adventitious
buds developed from leaf discs (Dornelas &Vieira, 1994; Appezzato-da-Glória et
al., 1999), hypocotyls, leaves and cotyledons (Dornelas & Vieira, 1994; Faria and
Segura, 1997b), leaf discs (Monteiro et al., 2000; Otahola, 2000; Becerra et al.,
2004; Trevisan & Mendes, 2005), mesophyll and cotyledon-derived protoplasts
(Dornelas & Vieira, 1993; d’Utra Vaz et al., 1993; Otoni et al., 1995), and internodal
segments (Biasi et al., 2000). Several studies indicated that various juvenile tissues
from Passiflora spp. can be used as explants to obtain adventitious shoots (Kantharajah
& Dodd, 1990; Dornelas & Vieira, 1994; Kawata et al., 1995). Moreover, the regeneration
after protoplast fusion and micropropagation (Kawata et al., 1995) has also
been described. A mature endosperm culture was reported for P. foetida (Mohamed
et al., 1996). Embryo and endosperm cultured from seeds of several Passiflora
species mainly collected in the wild has also been attempted (Guzzo et al., 2004).
Optimized protocols have been established mainly by using different combinations
of plant growth regulators (Dornelas & Vieira, 1994; Kawata et al., 1995; Faria &
Segura, 1997b), and different salt solutions (Faria & Segura, 1997b; Monteiro et al.,
2000). Though many reports on in vitro culture of passion fruit have been published,
there are no reports on the use of TCL technology to micropropagate this species.
Therefore, in this chapter, a novel protocol on shoot formation of P. edulis f.
flavicarpa via TCL technology is presented.
2. THIN CELL LAYER TECHNOLOGY IN MICROPROPAGATION
OF P. EDULIS F. FLAVICARPA
Schematic representation of the protocol for micropropagation of yellow passion
fruit is described in Figure 1.
2.1. Preparation of tTCL for Shoot Regeneration
Nodal segments of 3-year-old plant grown in the greenhouse, Dalat Institute of
Biology are used as primary explants. After removal of leaves and axillary buds, the