Liquid Culture Systems for in vitro Plant Propagation

562 S. M. Nalawade et al.
leaves on the medium supplemented with 1.0 mg l -1 zeatin (Figure 2B),
however, root development was arrested. The development of roots occurred
in embryos with well-developed cotyledonary leaves when they were
cultured in half-strength liquid MS medium supplemented with 1.0 mg l -1
zeatin riboside for three weeks.
Our initial attempts to transfer the tissue culture plant to soil immediately
after conversion in liquid medium containing 1.0 mg l -1 zeatin riboside were
not successful. Therefore, to further develop roots, shoots and tuberization in
vitro, converted somatic embryos were cultured on medium supplemented
with 6% (w/v) sucrose and different levels of either ABA, growth retardants
(paclobutrazol, ancymidol), PEG-4000, or GA3. Similar to our study, higher
sucrose concentration (Khuri and Moorby, 1995) and plant growth retardants
(Seabrook et al., 1993; Vreugdenhil et al., 1994) have been used for potato
microtuber formation in vitro. ABA and PEG as an osmoticum enhanced
the accumulation of storage reserves in somatic embryos of white spruce
(Leal et al., 1995; Roberts et al., 1990).
Of all the treatments tested, the medium with 6% (w/v) sucrose alone
promoted tuber, root, and shoot development (Figure 2 C). The plantlets
with well-developed roots, shoots and tubers from the converted somatic
embryos were hardened for two months in growth chambers.
Recurrent somatic embryos developed directly in the region of the
junction of the cotyledonary leaf and root of converted somatic embryos
when cultured on ABA- containing media (Figure 2 D). It has been reported
that ABA has an important role in early embryonic events (Senger et al.,
2001). Among the various ABA treatments tested, the maximum number of
secondary embryos were observed on medium containing 8 mg l -1 ABA. In
the medium supplemented with higher ABA concentrations (8 - 16 mg l -1 ), a
decrease in the number of somatic embryos with browning of the primary
somatic embryo was observed. The somatic embryos induced on lower
concentrations (0.5 and 1.0 mg l -1 ) of ABA favored precocious conversion.
Embryos induced on 4 - 16 mg l -1 ABA showed development of secondary
embryos. The development of somatic embryos directly without an
intervening callus phase was confirmed by scanning electron microscopy
and histology. Histological study revealed that the somatic embryos arose
directly from the epidermal layer of cells. Generally, the development of the
secondary embryos is not synchronized and the development is also up to the
pre-embryonic mass or globular embryo stages. (Raemakers et al., 1995;
Fernando and Gamage, 2000).