Problems associated with wide crosses and their solution

The barriers to inter-specific hybridization can be broadly classified into pre- and post- fertilization barriers (Hovin, 1962b; Evans, 1962b; Chen and Gibson, 1972). For circumventing pre-fertilization barriers, various techniques have been used, including, (1) use of compatible pollen with inactivated nuclei mixed with the desired pollen (Taylor et al., 1980), (2) mixtures of compatible and incompatible pollen with subsequent identification of hybrids versus selfs in the progeny, (Brown and Adiwilaga, 1991), (3) cutting stylar tissue to remove the obstacles caused by the stigma and the inhibition factors present there (Ascher and Peloquin, 1968) and (4) application of chemicals like gibberellins or auxins and cytokinins to enhance pollen tube growth (Dionne, 1958; Alonso and Kimber, 1980; Sastri and Moss, 1982; Baker et al., (1975). Mujeeb-Kazi and Rodriguez (1980) also reported the use of immunosuppressants for enhancing hybridization in cereals and legumes. Post-fertilization barriers result from differences in ploidy levels, chromosome loss/rearrangement, genic incompatibilities (Stebbins, 1958) cytoplasmic incompatibilities, physiological abnormality, seed dormancy and hybrid breakdown resulting from lethal or low plant vigour in the first or subsequent generations (Taylor et al., 1980; Williams, 1987; Repkova et al., 2006). Like other species, T. repens improvement through hybridization is predominantly hampered by strong postzygotic barriers (Chen and Gibson, 1970).

Chou and Gibson (1968) reported that in crosses of 2x and 4x T. occidentale with T.

nigrescens pollen tubes penetrated the style and reached the ovule. So the failure of these crosses was due to postzygotic barriers caused by the lack of normal endosperm development. In the failed crosses, the endosperm first became abnormal and started degenerating and then the embryo collapsed. In T. repens, all ovules were fertilized within 24 hours of pollination when stigmas were pollinated with T. repens pollen (Chen and Gibson, 1972). But when T. repens was pollinated by other species, pollen germination took comparatively longer and the frequency of germination was lower. The pollen tubes in the inter-specific crosses were shorter than those in intra-specific pollination. However, pollen tubes of the species like T. occidentale and T. nigrescens grew more normally than T. uniflorum and T. ambiguum on the stigma of T. repens probably due to close genetic relationship. Pollen of autotetraploid T. occidentale appeared to germinate better on white clover stigmas than that of diploid T. occidentale. Pollen of the other species (T. hybridum, T. uniflorum and T. ambiguum) mostly swelled,

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burst or coiled in the style or ovary of T. repens and very few pollen tubes reached the ovules. Roy et al. (2004) developed a hybrid, T. alexandrinum x T.

constantinopolitanum and reported slow development of the embryo in the cross as compared to the intra-specific cross of T. alexandrinum.

After fertilization and gamete fusion in wide crosses, in vitro culture techniques are usually employed to recover young embryos before they abort due to endosperm degeneration (Brewbaker and Keim, 1953; Pandey, 1957; Evans, 1962; Williams, 1987c). Ovule culture is used in cases where embryos abort very early and, alternatively, sequential culturing of ovules and then embryos can be employed (Przywara et al., 1989). Embryo rescue/ovule culture has been used by several researchers for developing hybrids between different Trifolium species (Williams and Verry, 1981; Yamada and Fukuoka, 1986; Pandey et al., 1987; Ferguson et al., 1990).

When the genotypes used in the cross are at the same ploidy level and have common genomes, then genetic recombination is a straightforward event. An important aspect of wide hybridization is the complexity created by different ploidy levels of the species used in the crossing scheme. Many cultivated crops are polyploids while their wild relatives are diploids and there is often reproductive isolation between the polyploid species and its progenitor or related species. Many crop species that were, until recently, considered typical diploids, are in fact ancient polyploids but behave cytologically like diploids (Leitch and Bennett, 1997; Wolfe, 2001). The narrow genetic base of these polyploids due to their reproductive isolation can present problems to plant breeders trying to cope with evolving biotic and abiotic stresses. The genomic imbalances in the hybrids between species with different ploidy levels can be overcome through various approaches. First is direct crossing followed by chromosome doubling in the hybrid by chemical treatment to restore fertility in the F1. Later on, this plant is either backcrossed with the cultivated parent as a recurrent parent or selfed to generate spontaneous chromosome reduction to the ploidy level of the cultivated species. A second method is to first raise the ploidy level of the species having lower ploidy and then cross it with the other species which is usually the cultivated species. This approach is successful for crops which are autopolyploid, but for allopolyploid species, high sterility can occur in the F1. Using bridging species at a lower ploidy level and then chromosome doubling can help circumvent sterility problems in the desired hybrid (Simpson, 1991). Some species can be manipulated to lower the chromosome number of the higher ploidy

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species as reported by Voigt (1971), Burk et al. (1979) and Peloquin and Ortiz (1992). Ploidy manipulation with haploids, 2n gametes and use of wild species is an impressive and fascinating method which offers big opportunities for the improvement of crop germplasm. Another approach, re-synthesizing ployploids for creating diversity and gene introgression, has also been used and has proved successful in wheat (Fernandes et

al, 2000; del Blanco et al., 2001), and Brassicaceae (Lu et al., 2001; Summers et al., 2003; Pires et al., 2004).

2.5.3 Endosperm balance number (EBN) and inter-specific crosses in Trifolium