Applications of Haploids, Doubled Haploids and Gamete Biotechnology in Citrus Breeding

Production of triploids

The importance of triploids in Citrus improvement derives from the seedlessness of their fruits. This is a desirable trait of commercial importance and one of the main goals in Citrus breeding programmes. Triploids can be conventionally pro- duced by 2 x ´ 4 x and 4 x ´ 2 x crosses: two seedless triploid hybrids were produced from 2 x pummelo and 4 x grapefruit hybridization (Soost and Cameron, 1980,

1985).

Triploid plants can also be obtained through in vitro culture of endosperm, which, being the fusion of three haploid nuclei, is triploid. Triploid hybrid Citrus plants were recovered by in vitro embryoge- nesis from endosperm-derived calli (Gmitter et al., 1990).

One of the most interesting applica- tions of haploids in Citrus breeding is the

possibility of obtaining triploid somatic hybrids by fusion between haploid and diploid protoplasts.

Protoplasts isolated from nucellar calli of ‘Juman’ Satsuma mandarin, ‘Ohmishima’ navel orange or ‘Trovita’ sweet orange were electrically fused with protoplasts of two haploid strains of clementine (Kobayashi et al., 1997).

Triploid Citrus hybrids were obtained via somatic hybridization performed by electrofusion between a gynogenetic hap- loid cell line of clementine and nine diploid cultivars (Ollitrault et al., 2000). The diploid protoplasts were isolated from mesophyll of Marumi kumquat and from embryogenic calli obtained from ovule cul- ture of the following genotypes: Willow Leaf mandarin, Sunki mandarin, Murcott mandarin, Kinnow mandarin, Shamuti orange, Valencia late orange, Star Ruby grapefruit and Mexican lime. Triploid and tetraploid hybrids were obtained for each combination, along with a few pentaploid hybrids.

Ploidy analysis by fl ow cytometry of 94 regenerants from clementine anther culture showed that as many as 82% of them were tri-haploids, rather than haploids or dou- bled haploids as expected (Germanà et al., 2005b). Regeneration from anther culture

was therefore proposed as a rapid and attractive method of obtaining new triploid varieties in clementine.

Production of diploid somatic hybrids

Through haploid protoplast fusion, new interspecifi c and intergeneric hybrids can be obtained, by-passing the barriers that inhibit wide crosses and avoiding the tetraploid stage, which is usually characterized by undesirable traits such as thick rind.

Gametosomatic hybridization

The production of triploid interspecific hybrids in Nicotiana and intraspecific hybrids in Petunia by fusing tetrad proto- plasts with callus protoplasts are examples of successful gametosomatic hybridization (Pirrie and Power, 1986; Lee and Power, 1988). A preliminary study on gametoso- matic fusion between P. trifoliata tetrads and somatic protoplasts of C. sinensis cv. ‘Jincheng’ was reported by Z.A. Deng et al. (1992). One chimeric plantlet was regener- ated.

Androclonal variation and its utilization

Generally, a large amount of variation is generated throughout pollen embryogene- sis, including gene mutation and chromo- some variation (Hu and Huang, 1987).

Inbreeding depression, due to complete homozygosity, may be associated with a reduced vigour observed in doubled hap- loid plants (Veilleux, 1994).

A higher incidence of genetic changes in plants derived from male cells has been observed in comparison with plants derived from female cells. However, this mechanism is poorly understood. It seems probable that since paternal organelles are not usually transmitted to the progeny, their mutations in nature are less important and male gamete cytoplasm is, for this reason, less stable (Huang, 1996).

Gametoclonal variation can be of nuclear origin or it can concern organelles as in albino (chlorophyll-defi cient) plants, frequently found in cereals when microspores are induced to regenerate into haploid plants.

Haploid and albino embryoids have also been regenerated in Citrus from anther culture of ‘Mapo’ tangelo (Citrus deliciosa ´

C. paradisi) (Fig. 7.24) (Germanà and Reforgiato, 1997).

Male gametophytic selection (MGS)

The angiosperm pollen grains may easily be manipulated to improve the effi ciency, the rapidity and the precision of plant breeding methods. The production of doubled hap- loids by culturing previously selected pollen could avoid the stress-buffering abil- ity of the pistil during pollen germination and pollen tube growth which occurs when selective stress is applied to the whole plant. Gametophytic selection, based on the overlap (~70%) in genetic expression

Fig. 7.24. Haploid and albino embryoids from tan- gelo ‘Mapo’ anther culture.

between the gametophytic and the sporo- phytic generation (Mascarenhas, 1990) and on the similarity in their response in the presence of several external factors (Hormaza and Herrero, 1992), could have several advantages deriving from the char- acteristics of the gametic phase: haploidy and large population size. Plant breeding programmes are costly and time consum- ing: large population sizes are needed to obtain the required combination of charac- ters in the new genotypes. This is especially important in woody perennials due to the large size of the plants and their long juve- nile phase. MGS can already be included in plant breeding programmes when the aim is to screen genotypes or to increase the vigour of the next sporophytic generation and, when knowledge regarding the repro- ductive processes is more advanced, it will be useful to transmit selected traits into the progeny.

Angiosperm pollen also has great potential by culturing the isolated microspores and exerting stress during cul- ture. In this way, microspores, which result from recombination, are subjected to selec- tion, and it is possible to recover, via gametic embryogenesis, mutants for physio- logical and biochemical traits (Evans et al., 1990). For this aim, however, a well- defi ned procedure of regeneration through gametic embryogenesis is necessary.

Transformation

Microspores may also be considered attrac- tive candidates for gene transfer by co-culti- vation with Agrobacterium tumefaciens and eventual pollen culture, or by microin- jection of transfer DNA into pollen embryos (Heberle-Bors et al., 1990; Sangwan and Sangwan-Norrel, 1990). The delivery of DNA into embryogenic microspores further advances genetic improvement of crops by producing homozygous transgenic plants in a single step.

Embryogenic callus production in monoembryonic Citrus

Citrus clementina is characterized by monoembryony, and it is very diffi cult to obtain embryogenic callus from monoem- bryonic Citrus species. On the other hand, embryogenic calli are essential for the genetic improvement of crops through biotechnology.

In our experiments, embryogenic calli of different cultivars of C. clementina (Nules, SRA 63 and Monreal) have been obtained (Germanà et al., 1994, 2000a, 2005b; Germanà and Chiancone, 2003).

Haploid and doubled haploid propagation

Haploid micropropagation by in vitro tissue culture is particulary important in cases, such as woody species, where the fre- quency of haploid induction by anther cul- ture is low. It is possible to multiply haploid plants by culturing shoot meris- tems or axillary buds. Numerous haploid plants are easily obtained when highly embryogenic callus is produced by anther culture, as in C. clementina.

Protoplasts were isolated from the stem and leaf of a haploid golden delicious apple clone and protoplast-derived shoots were successfully in vitro propagated via organo- genesis (Patat-Ochatt et al., 1993). Similarly, haploid protoplasts could be used as a means of clonal propagation of rare haploids.