Protocols for Micropropagation of Woody Trees and Fruits

2.1. Explant Preparation
MICROPROPAGATION OF MEDITERRANEAN CYPRESS
2. EXPERIMENTAL PROTOCOL
2.1.1. Explants from Juvenile Material
Mature seeds and seedlings are used as source of juvenile plant material. In our
experience, seeds are collected from both open and controlled-pollinated trees growing
in an experimental orchard, belonging to the Italian CNR, located near to Florence
(central Italy, 43° 45′ N, 11° 10′ E) and established with the aim to select cankerresistant
clones. The Mediterranean cypress embryos reach full maturity in late
summer of the second year after fertilisation. Hence, the cones have to be harvested
at this period and stored in air porous bags and well ventilated rooms. When cone
moisture is below 10% (preferably in climatic chamber at an average temperature of
35°C), cones open and the seeds can be easily collected. Seed germination ability
of Mediterranean cypress is generally lower than 50%, primarily due to the high
percentage of empty seeds (Giannini et al., 1999). The seeds can be stored for up to
10 years at 2–4°C in air-tight containers. An easy way to germinate mature seeds,
without stratification and preventing risks of contamination, consists of placing disinfected
seeds (see below) over filter paper, moistened with sterile water, inside Petri
dishes maintained at a constant temperature of 18–19°C under a 16-h photoperiod
(80 µmol m –2 s –1 ). After 5–6 weeks, seedlings 20–30 mm in length, are utilised for
in vitro establishment of shoot cultures.
2.1.2. Explants from Adult Material
In our work, both very old trees (about 150-year old, from natural growing conditions)
and 20- to 40-year old trees (canker-resistant clones from experimental orchards) have
been tested for in vitro culture during 15 years of experimentation. Apical shoots (not
bearing male or female cones) are commonly used as a source of primary explants,
collecting them from lateral branches of the lower third of the crown. Indeed, preliminary
observations show that in vitro morphogenetic competence of shoots collected
from basal branches (lower third of the crown) results significantly greater than that
of the upper ones. Primary explant collection is performed just before the onset of
vegetative growth, around the end of winter growth cessation. However, as cypress
growth is potentially a continuous and undetermined process (depending on limiting
environmental conditions, such as low temperature and/or drought) and bud burst
cannot be taken into account to determine the onset of growth, repeated collections
of plant material can be advised during mid winter and early spring. In addition, “reinvigoration”
of plants has pursued by side-veneer grafting scions from 30-year old
trees onto 1-year old rootstocks. Grafted plants are then grown in pots, under greenhouse
conditions, and used as another source of adult explants.
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