Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO PROPAGATION OF FRAXINUS SPECIES 187
After 1 month on proliferation medium, white ash 2-node segments typically
produce an average of 5.8 new axillary shoots which is more than double the 2.3
shoots from green ash nodal segments (Van Sambeek et al., 2001). In addition, the
longest axillary shoot after 1 month in subculture tend to be slightly longer on white
ash than on green ash nodal explants although there can be substantial variation
among clones within an open-pollinated family or species. Genotypic differences in
axillary shoot proliferation rates among cultures arising from open-pollinated seed of
different trees also have been reported by others for green and European ash (Tabrett
&
Hammatt, 1992; Kim et al., 1997).
3.2. Micropropagation by Regeneration of Adventitious Buds and Shoots
For adventitious shoot regeneration, cut seeds (either immature or mature) are
prepared and best placed on agar-solidified MS medium supplemented with 10 µM
TDZ and 0.1 or 1 µM 2,4-D. Organogenesis occurs in the callus formed where the
cut ends of the cotyledons and hypocotyls touch the medium (Bates et al., 1992;
Preece & Bates, 1995). If cotyledons are detached from the embryonic axis,
organogenesis is reduced and shoot development will be slower than if cotyledons
remain attached to the embryonic axis. If organogenic cultures are transferred to the
ASP medium, buds are more likely to elongate into shoots that can be excised, rooted
under mist, and acclimatized to a normal greenhouse environment. We have also
achieved adventitious shoot regeneration on the unwanted callus that forms on
tissues touching the medium during the in vitro establishment and axillary shoot
proliferation stages of both white and green ash (Navarrete et al., 1989; Van
Sambeek et al., 2001). Adventitious shoots typically have a thinner more transparent
stem and narrower more succulent unifoliate leaves than the stem and leaves on the
developing axillary shoots in these cultures. Kim et al. (1997) also reported formation
of organogenic callus around the nodes on their green ash subcultures in
contact with the culture medium.
Tabrett and Hammatt (1992) reported high rates of adventitious shoot regeneration
on excised hypocotyls from immature and mature seed of European ash when
cultured on MS supplemented with 20 µM TDZ and 0.5 µM IBA. Hypocotyls from
immature seed tended to have higher rates of regeneration with fewer necrotic
cultures. When using seed that had been dried and stored, the best regeneration rates
from excised hypocotyls occurred on MS supplemented with 0.5 µM TDZ and 0.5
µM IBA. Higher levels of TDZ tended to result in more cultures that produced
vitrified (hyperhydrous) adventitious shoots. Most excised hypocotyls exposed to a
primary medium with TDZ for 2 to 5 weeks and then transferred to DKW supplemented
with 20 µM BAP produced adventitious shoots that could be rooted.
We have also achieved embryogenesis on white ash by placing cut seeds on MS
medium containing a high auxin to low cytokinin ratio (Preece et al., 1987; Bates
et al., 1992; Preece & Bates, 1995). The highest rates of embryogenesis have been
observed when cut white ash seed where cultured either on MS containing 10 µM
2,4-D with 0.1 or 1 µM TDZ or on DKW medium supplemented with 5 µM BAP
and either 1 or 5 µM 2,4-D. The number of germinants showing embryogenesis on
the DKW medium could be increased to 20% by transferring germinants with callus