Anther and Microspore Culture and Production of Haploid Plants
INTRODUCTION
Haploid and double haploid plants attract the interest of
geneticists, plant embryologists, physiologists, and breeders. Their genetic characteristics make them an elegant
experimental system for genetic studies as well as an integral part of breeding programs, especially in generating
pure lines. Haploid plants can be induced by the male as
well as female gametophytes. This article focuses on haploid plants derived from the male gametophyte, i.e.,
anther and microspore cultures, concisely discussing the
origin of haploid plants and techniques applied to their
production, after a historical overview.
HAPLOIDS
The development and viability of pollen play a key role
in the fertility of plants. Besides its importance in sexual
reproduction, pollen can be used for haploid plant production. Haploid plants are genetically characterized as
plants containing only one set of chromosomes. The haploid state occurs due to the reduction of zygotic (diploid)
chromosome number to the gametic (haploid) number during
meiosis. In nature, haploid plants appear via abnormal
fertilization, i.e., chromosome elimination or mispairing
during the crossing-over. Haploid plants are sterile and
therefore doubling of the chromosome set is required
to produce fertile plants, which are called double haploids
(DHs) or homozygous diploids. Two basic genetic features make DHs distinct for genetic studies and breeding.
The best-known application of haploids is the F1 hybrid
system for the production of homogeneous hybrid
varieties. The DH lines are also used for targeted genetic
manipulation, mutant breeding, and selection, which
considerably reduces the time required for the production
of new cultivars.
Historical Overview
In 1922, Blakeslee and co-workers first discovered the
appearance of natural haploid embryos and plants, which
were derived from gametophytic cells of Datura stramonium. To date, naturally occurring haploid plants are
described in about 100 species of angiosperms. In 1954,
Tucker, for the first time, observed that mature pollen
grains of a gymnosperm Ginkgo biloba can be induced to
proliferate in culture to form a haploid callus, but the direct
formation of embryolike haploid structures from another
culture of Datura innoxia was first reported by Guha and
Maheswari in 1964.
Their experiments clearly demonstrated the feasibility of induction of haploid structures
from anther tissues. In 1967, Bourgin and Nitsch succeeded in producing the first haploid plants from cultured
anthers of Nicotiana sylvestris and Nicotiana tabacum.
Later in 1974, the first description of microspore culture
was also made by Nitsch. Since then, the techniques of
microspore and anther cultures were optimized for a wide
range of economically important dicotyledonous and
monocotyledonous plants.
Origin of Haploids
Androgenesis
Two basic strategies are applied to the induction of haploids from higher plants: in vivo and in vitro induction by
various physical, chemical, or biological stimulants. The
first method for haploid production, developed by Kasha
and Kao in 1970, is based on chromosome elimination
in hybrid embryos. This methodology exploits the fact
that when two unrelated plant species are crossed, the
chromosome sets of both parents fail to pair during the
crossover stage of meiosis. For example, with crosses between common barley (Hordeum vulgare) and its wild
ancestor (Hordeum bulbosum), the chromosomes of H.
Anther and pollen culture represent the major techniques for in vitro induction of haploid plants. The development of haploid plants can be induced from pollen
via embryogenesis. The formation of embryos from the
androgenic (male) tissues is called androgenesis. In this
case, the microspores are switched from their normal gametophytic fate to sporophytic development.
Different
physical and chemical stimuli have been studied for the
induction of androgenesis. The most efficient and widely
applied techniques include
1) cold pretreatment of spikes;
2) starvation—the cultivation of dissected
anthers in media without carbon source; and
3) incubation under higher temperatures.
Genotype, physiological state of donor plants, stage of
microspore development, culture media, and culture conditions are also important determinants of androgenesis. The anthers containing microspores in the mid- or
late-uninucleate stage are most suitable for the induction of
androgenesis.
Of the different media components, the
carbon source, its concentration, and the ratio of nitrate
and ammonium ions (NH4 +) are important in achieving
embryogenesis and the development of green plants from
microspores.[7,8] Based on these findings several basal
media were developed for androgenic cultures. Phytohormones, especially the content of cytokinins and auxin;
aeration and permanent supply of fresh, well-buffered
medium; increased osmotic pressure; and temperature are
all critical for successful establishment of androgenic
cultures. Regeneration of haploid plantlets from androgenic cultures can be achieved by direct embryogenesis
from microspores or via organogenesis.
ANTHER CULTURE
The technique of another culture is relatively simple and
efficient and requires minimal facilities. The
androgenesis can be induced by pretreating whole inflorescences (cold pretreatment) or by pretreating dissected
anthers (high temperature, starvation). Anthers are cultured on a solid or in a liquid medium on a rotary shaker at
50–60 rpm. The cultures are kept at 24–27C.
First,
anthers are cultured on the callus induction medium for
about 2 weeks in darkness and subsequently transferred to
a regeneration medium containing phytohormones and
organic substances at 16-hour photoperiods (2000–8000
lux) for shoot regeneration. Developing plantlets are then
transferred to a rooting medium containing a lower concentration of carbohydrates and other nutrients. The regeneration frequency of androgenic cultures is usually
very high. In barley, it ranges from 4.8 to 50 green plants
per single anther.
Plant breeding companies routinely use another culture
for the production of haploid plants. The only disadvantage of this technique is the regeneration of plants with
different ploidy due to the presence of both gametophytic
and sporophytic cells in the culture.
MICROSPORE CULTURE
The technique of microspore (pollen) culture was developed more recently than anther culture. In this technique,
pollen grains are separated from the anther tissues and
cultured in a liquid medium. Microspores provide
haploid single cells that can be utilized for various biological studies. Different techniques are applied for the isolation of
microspores.
The most efficient is technique of
microlending, in which small pieces of the inflorescence are
put in a blender and quickly cut to release microspores
into the isolation solution. The crude preparation is filtered through a sieve and the microspore suspension is centrifuged to separate microspores. The plating density
(the number of viable microspores per volume of medium)
is an important factor in the induction of androgenesis.
The optimal population density depends on the genotype,
the quality of donor material, and the isolation technique.
The induction of androgenesis occurs either while they are
still inside the spikes (cold pretreatment) or directly after
the isolation (high temperatures, starvation). Microspores
are cultured in a liquid induction medium on a rotary
shaker.
They are kept in the dark at 24–27C for 3–4
weeks. The emerging calli of visible size are transferred to
a solidified medium. Further cultivation of microspore-derived cultures is similar to those derived from the dissected anthers.
The isolated microspore culture offers the possibility
of combining selection procedures with the advantages
of a haploid system. The nutritional requirements of
the isolated microspores are much more complex than
those of dissected anthers. The use of isolated microspore
culture finds wide application in different fundamental studies and provides greater opportunities for cells.
CONCLUSION
Pollen (microspore) and another culture can be used for the
production of haploid and diploid plants. Haploid plants
are valuable material for breeding new varieties and
biotechnological applications. Fertile diploid plants
represent an essential source for producing pure inbred
lines, which are homogeneous and show no segregation.
Haploid and diploid plants considerably accelerate and
simplify breeding and selection processes.
Using the
method of haploidization it is possible to obtain homozygosity for genes in cases where this is normally difficult
to achieve, for example for self-incompatible alleles.
Haploid cell cultures are also useful material for mutation analysis and cell modification. Microspores as single cells
represent an ideal system for in vitro cell selection and
genetic manipulation. Microspores can be used for microinjection, electroporation, particle bombardment and cocultivation with Agrobacterium tumefaciens resulting in
transgenic haploid and homozygous plants.