Microspore And Anther Culture

Over the past twenty years, remarkable progress has been made in the field of cell and tissue culture techniques. Subsequently techniques like anther and microspore culture are often considered as routine methods for the generation of haploid and dihaploid (DH) plants in many species, including the genus Brassica (Lichter, 1982; Keller et al, 1987).

Wenzel et al (1977) were the first to produce B. napus plants from anther culture, and later by microspore culture (Thomas & Wenzel, 1975). However, microspore culture is favoured over anther culture as for B. napus it is ten times more efficient in terms of embryo production (Siebel and Pauls, 1989). This single-cell method also eliminates any anther/microspore interactions and is more suitable for mutant selection and gene transfer (Swanson et al, 1993).

Haploids can be regenerated from both male and female gametophytic cells through the culture of anthers, microspores, ovaries and ovules. A positive in vitro response will lead to the development of embryos and/or callus from which plants can be regenerated.

The most effective way of producing haploids has been through androgenesis, by means of isolated microspore cultures. Although such haploids have been obtained from several crop species, the practical application of haploids in breeding programmes is limited to those species that are able to generate haploid plantlets in large numbers. The two major factors limiting the effective use of microspore culture are low embryoid yield and poor plant regeneration from microspore derived embryoids.

Microspores can be used directly in mutant isolation provided that frequencies of embryogenesis or callus formation are sufficiently high. Mutant isolation studies were originally conducted on B. napus, and it is possible that anther/microspore cultures can be used to identify agronomically desirable variants without the use of mutagens or specific in vitro selection strategies.

Highly embryogenic, isolated microspore cultures offer enormous potential as recipient cells for the introduction of foreign genes by intranuclear microinjection of plasmid vectors, or via transformation using A. tumefaciens (Pechan, 1989; Potrykus, 1993). Thus the foreign gene insertions are duplicated when the cell undergoes chromosome doubling to produce a homozygous plant, so that all breeding individuals will pass on one of the foreign sequences during sexual reproduction (ie. double homozygous crossing for hybrid production).

Since the first report of isolated microspore culture in B. napus, vast progress has been made in evaluating the factors influencing embryogenesis efficiency. These include:

and are all crucial to the successful production of haploid embryoids in Brassica (Gland et al, 1988; Lichter, 1989; Hansen and Svinnset, 1993).

Development And Applications Of Haploid And DH Lines

An important and effective way of producing haploids has been through isolated microspore culture, but haploid plants also occur naturally at a low frequency (Thompson, 1969; Stringham & Downey, 1973). Haploidy can be used to achieve homozygocity in one simple step (spontaneous or colchicine induced chromosome doubling), so it is therefore an important tool for varietal development through conventional breeding (Charne and Beversdorf, 1991) and for genetic manipulation at the cellular and molecular level.

The dihaploid (DH) technique is a breeding tool for the rapid and cost effective production of homozygous inbreds to be used as parents in synthetic and/or hybrid varieties of Brassica.