The Importance of Haploids and Doubled Haploids

The life of higher plants comprises a sporo- phytic (2 n) and a gametophytic (n) genera- tion, where the gametes are products of meiosis, and the sporophytic generation, usually diploid, develops from the zygote produced by fertilization. In the course of evolution, the gametophytic generation has been progressively reduced in terms of both size and lifespan, in comparison with the

© CAB International 2007. Citrus Genetics, Breeding and Biotechnology (ed. I.A. Khan) 167

sporophytic generation. For this reason, the infl uence of the gametophytic phase on the sporophyte is usually underestimated and gametes are only considered as vectors for transmission of the genome to the next sporophytic generation; Heslop-Harrison (1979) termed this step of angiosperm life the ‘forgotten generation’.

More recently, both basic and applied studies have improved the knowledge of pollen biology and pollen biotechnology, making the manipulation of pollen devel- opment and function a reliable tool for crop improvement (Mulcahy, 1986). The most important application of pollen biotechnol- ogy in breeding and genetic studies is the ability to obtain haploids and doubled hap- loids.

Haploids are sporophytic plants with the gametophytic chromosome number because they originate from a single gamete. The importance of haploids in plant breeding and genetic research was recog- nized with the discovery of the fi rst natural haploid in Datura stramonium and Nicotiana (Blakeslee et al., 1922, 1924; Kostoff, 1929), but long before techniques for producing haploids by in vitro gametic

embryogenesis became available.

The discovery made by Guha and Maheshwari (1964, 1966) that, by in vitro culture of immature anthers of Datura innoxia, a change in the normal gameto- phytic development into sporophytic development can be induced and embry- oids with a haploid chromosome number can be obtained, led to further and exten- sive research on anther culture.

The interest of breeders in haploids or, by doubling the chromosome numbers, doubled haploids, lies in the possibility of shortening the time needed to produce completely homozygous lines compared with conventional breeding. In fact, hap- loidiploidization through gametic embryo- genesis allows the single-step development of complete homozygous lines from het- erozygous parents. In a conventional breed- ing programme, pure lines are developed after several generations of selfi ng and still may not be 100% homozygous. In the case

of woody plants, such as Citrus, generally characterized by a long reproductive cycle, a high degree of heterozygosity, large size and, sometimes, self-incompatibility, there is no other chance to obtain hap- loidization through conventional methods. Actually, the absence of pure lines in woody plants makes genetic studies rather diffi cult.

Haploid plants, with a gametophytic set of chromosomes in the sporophyte, and homozygous doubled haploid plants aroused interest in the fi elds of genetic and developmental studies, as well as for plant breeding. In fact, they have a potential use in mutation research, selection, genetic analysis, the production of inbred lines required to utilize hybrid vigour (heterosis), and genetic transformation.

New superior cultivars produced via gametic embryogenesis (above all through anther culture) have been reported for rice, wheat, tobacco, maize and pepper (Evans, 1989), and doubled haploids are being rou- tinely used in breeding programmes for new cultivar development in many crops such as aubergine, pepper, barley and rape (Veilleux, 1994).

Often, in vitro regenerated plants show differences in their morphological and- biochemical characteristics, as well in chromosome number and structures. ‘Gametoclonal variation’, the variation observed among plants regenerated from cultured gametic cells (Evans et al., 1984; Morrison and Evans, 1987), is another opportunity to use haploids in crop improvement. Unlike ‘somaclonal varia- tion’ which is related to the variation among plants regenerated from cultured cells or tissue (Larkin and Scowcroft, 1981), gametoclonal variation results from both meiotic and mitotic division. Moreover, because of their homozygosity, in the game- toclones it is possible to observe the direct expression of both dominant and recessive mutations. Several different sources of vari- ation have to be considered in order to explain gametoclonal variation, such as new genetic variation induced by the cell culture procedures, new variation resulting

Table 7.1. Haploids and doubled haploids obtained in Citrus by gynogenesis.

from segregation and independent assort- ment, new variation induced by the chro- mosome doubling procedure and new variation induced at diploid level, resulting in heterozygousity (Morrison and Evans, 1987; Huang, 1996).

Doubled haploids can also increase the effi ciency of crop breeding programmes because they are important in genome map- ping, providing excellent material to obtain reliable information on the location of major genes and qualitative trait loci (QTLs) for economically important traits (Khush and Virmani, 1996).