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17
Haploid Cultures
Sandra M. Reed
C
HAPTER
17 C
ONCEPTS
• Haploids may be produced from male gametophytes (androgenesis) or female gametophytes
(gynogenesis).
•
In vitro
androgenesis involves culture of anthers or isolated microspores.
• Gynogenesis may be induced in unfertilized ovule or ovary culture.
• Success of haploid cultures depends on genotype, media, and cultural conditions.
• Stage of microspore development at time of culture is critical to the success of androgenesis.
• Chromosome doubled haploids (diploids) are useful in breeding projects.
INTRODUCTION
The ability to produce haploid plants is a tremendous asset in genetic and plant breeding studies.
Heritability studies are simplified, because with only one set of chromosomes, recessive mutations
are easily identified. In addition, doubling the chromosome number of a haploid to produce a
doubled haploid results in a completely homozygous plant. Theoretically, the genotypes present
among a large group of doubled haploids derived from an F
1
hybrid represent in a fixed form the
genotypes expected from an F
2
population. Use of doubled haploids in breeding programs can thus
greatly reduce the time required for development of improved cultivars. To be most useful, a large
number of haploids from many different genotypes are required.
Haploids have been available for genetic studies for many years. Prior to the 1960s, they were
mostly obtained spontaneously following interspecific hybridization or through the use of irradiated
pollen, but usually only infrequently and in very small numbers. Haploid methodology took a giant
step forward 40 years ago when Guha and Maheshwari (1964) found that haploid plants could be
obtained on a regular basis and in relatively large numbers by placing immature anthers of
Datura
innoxia
Mill. into culture. This work was rapidly expanded using tobacco (
Nicotiana tabacum
L.),
which became the model species for anther culture experiments. To date, androgenic haploids have
been produced in over 170 species; several good reviews provide lists of these species (Maheshwari
et al., 1982; Bajaj, 1983; Heberle-Bors, 1985; Dunwell, 1996). While efforts have been more
limited, haploids have also been obtained from
in vitro
culture of the female gametophyte in over
30 species (Keller and Korzun, 1996; Lakshmi Sita, 1997). Gynogenesis has been successfully
applied to several species in which androgenesis is generally ineffective, such as sugar beet (
Beta
vulgaris
L.), onion
(Allium
cepa
L.), and the Gerbera daisy (
Gerbera jamesonii
H. Bolus ex Hook).
226
Plant Development and Biotechnology
Although much of the terminology used in this chapter has been discussed in previous chapters,
the
in vitro
induction of haploids involves a few specialized terms. A haploid is a plant with the
gametic or
n
number of chromosomes. Doubled haploids, or dihaploids, are chromosome doubled
haploids or 2n plants. Androgenesis is the process by which haploid plants develop from the male
gametophyte. When anthers are cultured intact, the procedure is called anther culture. Microspore
culture involves isolating microspores from anthers before culture and is sometimes referred to as
pollen culture. Haploids are derived from the female gametophyte through a process referred to as
gynogenesis.
In vitro
gynogenesis involves the culture of unfertilized ovules or ovaries. While both
androgenesis and gynogenesis may occur
in vivo
, the usage of the terms in this chapter will refer
to the
in vitro
induction of haploids via these two mechanisms.
This chapter will begin with general discussions of androgenesis and gynogenesis, followed
by a review of the factors that affect the successful production of androgenic and gynogenic
haploids. Finally, some of the basic procedure used for the
in vitro
production of haploids will be
summarized. Excellent discussions of
in vitro
haploid production, along with specific protocols for
a number of crop species, can be found in Jain et al., 1996a; 1996b; 1996c; 1996d; 1997.
ANDROGENESIS
D
EVELOPMENT
OF
H
APLOIDS
Haploid plants develop from anther culture either directly or indirectly through a callus phase.
Direct androgenesis mimics zygotic embryogenesis; however, neither a suspensor nor an endosperm
is present. At the globular stage of development, most of the embryos are released from the pollen
cell wall (exine). They continue to develop, and after 4 to 8 weeks, the cotyledons unfold and
plantlets emerge from the anthers. Direct androgenesis is primarily found among members of the
tobacco (Solanaceae) and mustard (Cruciferae) families.
During indirect androgenesis, the early cell division pattern is similar to that found in the
zygotic embryogenic and direct androgenic pathways. After the globular stage, irregular and
asynchronous divisions occur and callus is formed. This callus must then undergo organogenesis
for haploid plants to be recovered. The cereals are am