Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF MATURE TREES 239
Table 1. The origin of elm species.
Species Natural area Number
of
clones
Altitude
(m)
Average
annual
rainfall (mm)
U. glabra Ore Mountains 10 800–900 680
Giant Mountains 5 750–800 720
U. minor Lowland of Labe river 3 170–200 450
Lowland of Labe river 6 250–300 450
South Moravia 6 150–160 350
U. laevis South Moravia 6 150–160 350
Lowland of Labe river 2 190–200 450
Lowland of Labe river 7 180–200 450
2.2. Induction of Organogenesis
The excised apical meristems from dormant buds were placed onto agar-solidified
(6 g l –1 ) Murashige & Skoog (1962; MS) basal medium supplemented with benzylaminopurine
(BAP), β-indolebutyric acid (IBA), glutamine and sucrose (Table 2).
medium used for various morphogenetic programs in micropropagation of elm.
Program Time of Growth
culture substances
(mg l –1 Organics
)
(mg l –1 Sucrose
) (g l –1 Table 2. Growth substances, organics and sucrose concentrations supplemented to basal MS
Strength
) of MS
Induction of 4–6 w BAP (0.2) Glutamine (10.0) 30.0 Full
organogenesis
IBA (0.1)
Shoot
3–4 w BAP (0.5) Glutamine (200.0) 30.0 Full
multiplication
IBA (0.1) Casein-hydrolysate
(200.0)
Rooting of
shoots
1 w NAA (1.0) 0 10.0 One-third
3–4 w 0 0 10.0 One-third
The pH of media was adjusted to 5.8 with 1 M KOH. The buds were cultured at
24°C under 16-h photoperiod with white fluorescent light (36W/33 Philips tubes,
Eindhoven, The Netherlands; irradiance of 30 µmol m –2 s –1 ) for 4–6 weeks. Organogenic
activity of apical meristems appeared approximately after 2 weeks of cultivation.
The differentiation of new adventitious shoots was initiated from the meristemoids
formed in calli on the basis of primary explants and/or from axillary buds (Figure 2A).
The average number of newly regenerated shoots was 1–2 in U. glabra and U. laevis
and 3–4 in U. minor where the additive shoot were also formed from axillary buds.
The average loss of primary explants, due to contamination and/or overgrowth of
callus, was detected in all clones between 10% and 40%.