Protocols for Micropropagation of Woody Trees and Fruits

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M. MISHRA ET AL.
each plate inoculate 10 zygotic embryos. Incubate cultures at 25°C ± 2°C in the dark
for 4 weeks. Globular embryos appeared after 4 weeks of inoculation, which will
form bipolar-shape. Subculture somatic embryos on this medium (after 4 weeks), this
will result in proliferation of embryos (Figure 2 a, b, c).
2.3.2. Somatic Embryo Maturation and Germination
After 4 weeks, several creamy white to light yellow thread like embryos will appear.
Transfer the clumps of somatic embryos on half strength MS medium containing 3%
sucrose and 0.8% Difco bacto-agar, 400 mg/l glutamine, MS vitamins, 0.2 mg/l
BAP and 0.1 mg/l NAA (Regeneration medium). (Table 1) Incubate the cultures
under 16 h light (25 µmol m –2 s –1 ) at 25°C ± 2°C. Allow somatic embryos to
germinate. If callus formation starts at the base of the shoots, than transfer the
cultures on MS medium devoid of any plant growth regulators. This will check the
callus growth at the base of shoot.
Figure 2. Somatic embryogenesis in papaya. A) Immature zygotic embryo, B) Somatic embryo,
C) Germination of embryos, D) Rooting of plantlets, E) Acclimatization of plantlets, F) Acclimatized
plants in poly bags, G) Establishment of plants in field.
2.3.3. Rooting and Acclimatization
Transfer regenerated plantlets (5 cm) in transparent culture bottles containing autoclaved
vermiculite supplemented with ¼ MS liquid medium, and incubate them
under light (25 µmol m –2 s –1 ) illumination) at 25ºC ± 2ºC for 2–3 weeks. Sometimes
good rooting is a problem. In such case, add 1 mg/l IBA solution in vermiculite for
better root growth. Gradually loosen the cap of bottle as soon as plant starts touching
the cap of bottle. Transfer the plantlets into a poly house provided with 50% shade