Protocols for Micropropagation of Woody Trees and Fruits

52 C. HARGREAVES AND M. MENZIES
(Menzies et al., 1985; Menzies & Aimers-Halliday, 1997). More recent advances in
the area of juvenility maintenance include the development of cryogenic methods
in combination with somatic embryogenesis (Hargreaves & Smith, 1992, 1994;
Hargreaves et al., 2002). Methods for organogenic material have been more difficult
to develop though significant progress has been made with zygotic embryos and
recently, shoot tip meristems (Hargreaves et al., 2004, 2005).
Somatic embryogenesis was originally seen as the way forward in terms of
delivering clonal forestry strategies to commercial forest operators, because the
technique results in tissue that can be easily cryopreserved (thus ensuring juvenility
while field testing takes place) and has the potential to produce millions of plants
quickly (Durzan & Gupta, 1988; Attree & Fowke, 1991). However, major problems
have been low genotype capture and a high labour cost of in vitro procedures (Park
et al., 1998; Timmis, 1998).
Genotype capture using organogenesis methods with zygotic embryos (mature
and immature) or seedling/stool bed material is high and the in vitro culture period
short. Media formulations include few plant growth regulators, and less skilled
observation through developmental stages is required than with somatic embryogenesis
(Hargreaves et al., 2005).
Propagation techniques for radiata pine which utilise organogenic approaches
are showing great versatility for a range of applications, including arresting of
juvenile growth via cool storage and more recently developed cryogenic methods.
These also extend to the preservation of genetic resources in the case where native
populations may be under threat from human interference, including climate change
and disease. The potential is promising for amplifying valuable seed that may be both
limited in supply and less fit, in the case of historic seed collections and where new
and novel hybrids may have been bred (Hargreaves et al., 2007). Other uses of the
approach are to facilitate the safe (contamination free, pre-screened) dissemination of
proven/novel genetic material internationally as shoot cultures. This material arrives
in a ready-to-amplify condition.
This chapter sets out to describe the current methods for organogenic multiplication
and cryogenic storage of radiata pine. Details are also given for the transition
of material from the laboratory to the nursery and field.
2.1. Explant Preparation
2.1.0. Culture Media
Constituent name Constituent
formula
2. EXPERIMENTAL PROTOCOL
Shoot
initiation
Shoot
multiplication
Medium* Medium*
(1/2 LP5)
(LPch)
Root
initiation
Medium**
(GD)
Major elements mg l –1 mg l –1 mg l –1
Ammonium sulphate (NH4)2SO4 200.00
Ammonium nitrate NH4NO3 200.00 400.00