Agriculture Reference
In-Depth Information
a hindrance towards complete access of the microspores. There is a need to ensure
direct embryogenesis to eliminate an intermediary callus phase that can promote
gametoclonal variation among regenerants. The large number of successful reports
on haploid production through anther culture stands as a witness of the immense
benefits that this system offers. However, the presence of extraneous tissue in the
form of anther wall makes this system complex for genetic studies where precision
is inevitable.
As haploid plants in crop species obtained from microspore cultures through
embryogenesis rather than from callus, the problem of extensive culture induced
variability found in earlier reports on anther culture can be significantly done away
with. With improvement of frequencies from microspores, the genotypic compe-
tence has also been reduced so that the system can be used in breeding programs.
The feature of a high frequency of spontaneous chromosome doubling in some
crops which results in completely fertile doubled haploid plants without subsequent
doubling treatments, is another advantage of microspore culture. In breeding, the
instant production of true breeding lines in diploid or allopolyploid species saves a
number of generations in the breeding program. High density cultures of synchro-
nized microspores can be set up containing more than thousand embryos per ml
of culture media. Although a newer technique in comparison to anther culture, the
potential of microspore culture has been realized quite early and efficient proce-
dures have been successfully developed to produce doubled haploids in microspore
cultures of tobacco, rapeseed, pepper, wheat, barley, rice, etc (Maluszynski et al.
2003a
; Touraev et al.
2001
).
2 Methodologies of In Vitro Haploid Production
2.1 Androgenesis
In androgenesis, immature pollen grains are induced to follow the sporophytic mode
of development with the application of various physical and chemical stimuli. There
are two methods for in vitro production of androgenic haploids, viz anther culture
and pollen culture.
2.1.1 Anther Culture
Anther culture imparts an easy and one step protocol for haploid plant production
(Chaturvedi et al.
2003
).There is no single specific condition or protocol for induc-
ing in vitro androgenesis in all plants since different species and even different
cultivars within a species show diverse requirements. The following methods were
adopted by Chaturvedi et al. (
2003
) to initiate in vitro androgenesis in neem plants
(
Azadirachta indica
A. Juss.):
