Protocols for Micropropagation of Woody Trees and Fruits

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V.M. KULKARNI ET AL.
2.2.2. Initiation of Embryogenic Callus Cultures through Scalp Cultures
In this method, highly proliferating shoot cultures (initiated from shoot-tips) are
used as the starting explants (Dhed’a et al., 1991; Schoofs, 1997). The shoots are cut
0.5 cm above the apical meristem and cultured on BM1B medium and observed for
the highly proliferating cluster of tiny shoots at the base of the explant. These
cultures can be subcultured on the same medium till cauliflower like meristem (CLMs)
cultures are obtained. These can be subcultured and maintained on the same medium.
Good quality scalps (defined as a clump of white, compact, cauliflower-like proli-
ferating meristems) are selected from these cultures using a dissection microscope.
The scalps usually contain a high proportion of meristematic domes, are isolated
from the clusters and cultured on BM2Z medium for embryogenic callus development.
Swelling of the explants, development of corm tissues and dedifferentiation of
leafy portions into watery callus is commonly observed during the initial few weeks.
This is followed by the development of heterogeneous globules and formation of a
friable embryogenic callus bearing many translucent proembryos.
A friable embryogenic callus mass bearing many translucent proembryos
(Figure 3D,E), obtained either from immature male inflorescence or scalp cultures,
is ideal for initiating ECS.
2.2.3. Initiation of Embryogenic Cell Suspension Cultures
Cell suspensions can be obtained by culturing about 100 mg of embryogenic calli
with proembryos in 25 ml capacity conical flask containing 8 ml of BM2L medium
(Cote et al., 1996; Ganapathi et al., 2001) in case of male flowers and BM2Z
medium in case for scalps. These are maintained on a gyratory shaker at 80 rpm
under a photoperiod of 16 h light at 45 µmol photons m –2 s –1 and a temperature of 26
± 1°C. A good cell suspension culture containing actively dividing and densely
packed cytoplasmic cells can be recovered after 3–4 months (Figure 3F,G). During
this period the suspensions are subcultured every 10 days by replacing 4 ml of used
medium with the fresh BM2L medium. Hereafter, the suspensions are cultured in
125 ml capacity conical flasks containing 25 ml BM2L medium. At each subculture,
0.5 ml packed cell volume (PCV) + 4.5 ml of the spent medium are added to 25 ml
of BM2L medium. In order to control release and oxidation of the phenolic
compounds, ascorbic acid at 10 mg/l is often added to the BM2L or BM2G media.
For embryo development, 0.5 ml of PCV of cell suspension is aspirated onto
glass fibre filters and is transferred to BM3 medium. The embryo development can
be observed within approximately 3–6 weeks. The globular stage embryos are
visible initially. The conversion of the somatic embryos into plantlets (Figure 3I)
can be achieved on BM4 medium (1–2 monthly passages). If required these shall be
followed by transfer onto BM5 medium till fully grown plantlets with well developed
shoot and roots are obtained (4–6 weeks).
2.2.4. Greenhouse Hardening of Emblings
The somatic embryogenesis derived plantlets (emblings) are hardened (Figure 2F)
essentially in the same way as described above in Section 2.1.3 in the case of shoottip
derived plantlets. These can further be field planted for evaluation (Figure 2G).