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Rice Genomics and Genetics 2013, Vol. 4, No. 4, 14-27
http://rgg.biopublisher.ca
16
Dry somatic embryos lack the vigour normally
associated with seedling from normal seeds. The
reason for this is not yet been specified, although there
are several possibilities. The dry somatic embryos
may lack storage proteins or some other critical
components required after germination by the
seedlings. The storage protein levels have been
increased with some improvement in vigour (Lai et al.,
1992; Lai and McKersie, 1993; Lai and McKersie,
1994a, Lai and McKersie, 1994b). Somatic embryos
store starch and sucrose, whereas, seeds store a
hemicellulose in the cell wall of the endosperm called
galatomannan (McCleary and Matheson, 1974;
McCleary and Matheson, 1976). The sucrose reserves
in the dry somatic embryos are rapidly depleted after
imbibitions (Lai et al., 1995). In some instances, there
may be injury to the somatic embryos if the proper
dying procedure is not followed (Lecouteux et al.,
1993). In comparison to true seeds, water uptake
during imbibitions of dry somatic embryos is quite
rapid, because the somatic embryos lack a testa, there
is no barrier to water uptake. Imbibation injury is,
therefore, another possible cause of poor seedling
vigour in synthetic seeds.
2.2 Hydrated synthetic seed
Hydrated seeds are produced in those plant species
where the somatic embryos are recalcitrant and
sensitive to desiccation. Encapsulation of somatic
embryos in hydrogel capsules produces hydrated
synthetic seeds. The most used method to induce
artificial seed is isnotropic gelation of sodium alginate
by calcium ions. Redenbaugh et al. (1984) developed
the technique of hydrogel encapsulation of individual
somatic embryos of alfalfa. Since then encapsulation
in hydrogel remains to be the most studied method of
artificial seed production.
2.3 Double layered synthetic seed
Technology has been developed to prepare
double-layered synthetic seeds (Kinoshita, 2002). For
somatic embryo encapsulation, sodium alginate is
largely used; however, this is excessively permeable
with loss of the nutritive substances and/or
dehydration risk during conservation and transport
causing detrimental effects on the synthetic seed
conversion and on the plantlet growth. In order to
overcome these problems, Micheli et al. (2002)
developed double coat
encapsulation and
encapsulation coating procedure in M.26 apple
rootstock. The inner layer contained a large quantity
of sucrose. To prevent the diffusion of sucrose from
artificial seeds to non-sterilized substrate, artificial
seeds were enveloped in a dialysis membrane. The
enveloped artificial seeds germinated quickly in
non-sterilized vermiculite.
3 Production Technologies
3.1 Gelling agent
A number of substances like, potassium alginate,
sodium alginate, carregeenan, agar, gelrite, sodium
pectate, etc. have been tested as hydrogels (Table 1).
Sodium alginate has a low cost and good gelation and
biocompatibility characteristics.
Smidsrod and
Skjak-Braek (1990) suggested alginate for preparation
of artificial seeds. Though many coating materials
have been tried for encapsulation of somatic embryos,
sodium alginate obtained from brown algae has been
considered as the best and is being popularly used at
present. Alginate has been chosen for case of capsule
formation as well as for its low toxicity to the embryo.
Alginate capsulated embryos can resist unfavourable
field conditions without desiccation. The rigidity of
the gel beads protects the fragile embryo during
handling.
Table 1 Gelling materials and complexing agent for encapsulation of plant propagules for production of synthetic seeds
Gelling agent
Complexing material
Substance
Concentration (%)
Substance
Concentration (mM)
1. Sodium alginate
0.5~5.0
Calcium salt
30~100
2. Sodium alginate with gelatin 2.0
Calcium chloride
30~100
3. Carrageenan
0.2~0.8
Potassium chloride
500
4. Locust Beam Gum
0.4~1.0
Ammonium chloride
500
5. Gelrite*
0.25
-
-
6. Agar*
0.8~2.0
-
-
Note: *Temperature is lowered down for solidification