Agriculture Reference
In-Depth Information
a suitable alternative for improving desirable gene(s) in a directed manner without
the insertion of undesirable DNA fragments. The establishment of stable and re-
generative tissue culture and transformation systems is a prerequisite for barley and
other cereal crops. Different explants, immature embryos (Breiman,
1985
), ma-
ture embryos (Lupotto
1984
), apical meristems (Cheng and Smith
1975
), anthers
(Kao and Horn
1982
), microspores (Köhler and Wenzel
1985
), cell suspensions
(Kott and Kasha
1984
) and protoplasts (Lazzeri and Lörz
1990
) have been used
for this purpose. In practical breeding, to develop homozygous transgenic plants
with a stable transgene is very interesting and important (Vyroubalová et al.
2011
).
Androgenesis is an elegant system for genetic transformation (Jähne et al. 1994;
Stöger et al.
1995
), and could provide a practical alternative for the production of
transgenic doubled haploid plant species, in which regeneration from somatic cells
is difficult, especially in the recalcitrant cereals. This protocol may be used to over-
come genotypic limitations of doubled haploid formation in cultivars that had pre-
viously been found to be recalcitrant in anther culture (Sopory and Munshi
1996
).
To date, haploid offspring have been obtained from over 200 species, including all
the major crops and hundreds of cereals (Floss et al.
2009
). By using this system
(transgenic doubled haploid plant formation), it has been possible to produce ho-
mozygous transgenic crop plants with targeted genes and crops (Kumlehn et al.
2006
, Aionesei et al.
2006
; Fukuoka et al.
1998
). This chapter has been focused on
combination of in vitro androgenesis and genetic transformation technology using
different stress tolerance genes for the production of fertile transgenic/ homozy-
gous plants rapidly.
2 Methods
Various approaches are employed in order to obtain fertile transgenic plants using
androgenesis and genetic transformation systems in cereal crops. The successful
development of transgenic plants necessitates a reliable tissue culture regenera-
tion system, gene construct(s), suitable vector(s) for transformation and efficient
procedures to introduce desired genes into target plants. Generally, some steps are
common for almost all crop species which are mentioned here in the flow chart
below.
2.1 Androgenesis (Anther and Microspore Culture)
General techniques of anther culture, media and other steps by the protocol of
Schmid (
1990
) are followed. For isolated microspore culture, microspore isolation
procedure, media and other steps by the protocol of Kunz et al. (
2000
) and Huang
(
1992
) are followed.
