Agriculture Reference
In-Depth Information
2.2.3 Ovule Culture
Embryos are difficult to excise. To prevent damaging the embryos during the exci-
sion process, they are sometimes cultured while still inside the ovule. This tech-
nique is referred to as ovule culture or in ovulo embryo culture (Reed
2005
). The
unfertilized ovary is surface sterilized and the ovules are taken and placed into the
culture. Excision of ovule, followed by culture on specific media may be either ex-
tremely easy to accomplish, as in case of large-seeded species in which only a single
ovule is present, or time-consuming and intricate, as in small-seeded polyovulate
species. Two types of ovule support systems have been developed. The filter pa-
per support system involves culturing of the ovules on top of filter paper placed
over liquid medium, whereas the vermiculite support technique demands placing
the ovules on a sterile vermiculite/liquid media mixture (vermiculite support) with
the micropylar side down. Unpollinated ovule culture has been used for haploid
production in sugar beets and onions. Since there is usually only one egg cell per
ovule, ovule culture has much less potential than microspore culture (Kasha and
Maluszynski
2003
). In
Nicotiana rustica
cv Rustica ovules with placenta were iso-
lated from flower buds and were cultured on N
6
medium supplemented with growth
regulators (Katoh and Iwai
1993
).
2.3 Wide Hybridization
Haploids can also be induced by a process of selective chromosome elimination that
follows certain interspecific pollinations. This phenomenon was discovered first in
barley with crossing between
Hordeum vulgare
and
H. bulbosum
(Kasha and Kao
1970
) and is now used routinely in wheat and other cereal breeding programmes;
haploids were induced in these species following pollination with maize pollen.
High frequency gynogenic haploids of
Triticum aestivum
have been raised by cross-
ing them with
H. bulbosum
followed by embryo culture (Barclay
1975
; Zenketler
and straub
1979
; Inagaki
1990
). The process involves a phase of embryo rescue in
vitro, usually followed by chromosome doubling with colchicine. The term “em-
bryo rescue” refers to an vitro techniques the purpose of which is to promote the de-
velopment of an immature or weak embryo into a viable plant (Reed
2005
). Embryo
rescue has been widely used to raise plants from hybridization in which failure of
endosperm development causes embryo abortion. In embryo rescue procedures, the
artificial nutrient medium serves as a substitute for the endosperm, thereby allowing
the embryo to continue its development. Embryo rescue techniques are among the
oldest and most successful in vitro procedures. One of the primary uses of embryo
rescue has been to produce interspecific and intergeneric hybrids. While interspe-
cific incompatibility can occur for a wide variety of reasons, one common cause is
embryo abortion. The production of small, shrunken seed following wide hybrid-
ization is indicative of a cross in which fertilization occurs but seed development
is disrupted. Embryo rescue procedures have been very successful in overcoming
