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Rice Genomics and Genetics 2013, Vol. 4, No. 4, 14-27
http://rgg.biopublisher.ca
17
3.2 Encapsulation methods
3.2.1 Encapsulation by dropping of hydrogels into
complexing agent
For encapsulation, embryos or buds are mixed with
autoclaved sodium alginate (0.5%~5%, w/v), which is
prepared in suitable tissue culture basal medium
supplemented with sucrose. Embryos or buds are then
picked up individually and dropped into sterilize
aqueous solution of 2%~3% (w/v) calcium salt
solution [CaCl
2
or (CaNO
3
)
2
] with occasional
agitation. Calcium alginate beads were formed within
15~30 minutes. Here, ion-exchange reaction occurs
and sodium ions are replaced by calcium ions forming
calcium alginate beads or capsules surrounding the
embryo/bud. The size of the bead depends upon the
inner diameter of the pipette nozzle. Compactness and
hardening of the encapsulated bead is modulated with
concentration of sodium alginate and calcium chloride
as well as duration of complexing. Optimum
concentration of sodium alginate for production of
synthetic seed ranges from 2%~3% with a complexing
solution contraining 75~100 mM calcium chloride
(Ara et al., 1999; Bhattacharjee et al., 1998; Bekheet
et al., 2002; Sparg et al., 2002; Priya et al., 2003, Roy
and Mandal, 2008).
3.2.2 Mechanized encapsulation method
Automated encapsulation process is a quick method of
artificial seed production. An encapsulation machine
can be used successfully to encapsulate somatic
embryo in order to achieve a time and hand labour
saving and to increase the accuracy. Sicurani et al.
(2001) developed mechanical excision of explants.
This technique is useful for the production of
synthetic seeds through encapsulation of diffentiating
propagules (tissue fragments with shoot primodia) in
woody species. Brischia et al. (2002) also used
mechanically manipulated explants of apple rootstock
for encapsulation. They suggested that machine
processed explants can be encapsulated for production
of synthetic seeds.
The development of synthetic seed involving the
production of artificial endosperm and self-breaking
treatment for gel as well as equipment for
encapsulation and sorting were described by
Sakamoto et al. (1992). The development and
preservation of embryogenic cell lines, somatic
embryogenesis in bioreactors and embryo-to-plant
conversion; preservation and coating were discussed
by Petiard et al. (1993) with reference to carrots and
Coffea camphora.
About 55000 and 5000
embryos/litre can be produced daily for carrots and
coffee, respectively.
Alternatively, the embryos could be mixed in a
temperature depended gel such as, gelrite, agar,
agarose, etc., placed in the well of a micro-titer plate,
and gelled as the teperature was lowered. The judicial
and intelligence coupling of artificial seed technology
with that of microcomputer in achieving automated
encapsulation and regeneration of plantlets would
tremendously increase the efficiency of encapsulation
and production of homogeneous and high quality
seeds, and will thus revolutionize the current concept
of commercial micropropagation method.
3.2.3 Semi-automated encapsulation
Somatic embryos are mixed in sodium alginate
solution (2%~5%) prepared with the appropriate
nutrient
medium.
Sodium alginate solution
impregnated with somatic embryos is dropped into
calcium chloride solution (Figure 2). Molle et al.
(1993) have suggested the use of a dual nozzle pipette
in which the embryos flow through the inner pipette
and the alginate solution through the outer pipette. As
a result, the embryos are positioned in the centre of
the beads for better protection.
Figure 2 Sodium alginate solution impregnated with somatic
embryos is dropped into calcium chloride solution