Perlecano como marcador de síntesis de una matriz reparadora del daño 111

Trifolium L. is one of the largest genera of the family Fabaceae (subfamily

Papilionoideae), comprising of around 250 herbaceous species which are commonly called clovers. This is the most widely spread, distinct and evolved genus of its tribe

and Heller, 1984). Section Lotoideais the oldest, largest and widest in its distribution. It includes the important perennials T. repens (white clover), T. hybridum and T.

ambiguum. The second largest section is Trifolium having important species like T.

pratense (red clover), T. incarnatum, T. alexandrinum, etc. Ellison et al. (2006)

proposed a new infrageneric classification of the genus based on results of phylogenetic study. They placed white clover in a new section Trifoliastrum of subgenus Trifolium. Their DNA sequences analysis showed that similarities among species are influenced by their geographic distributions. White clover was grouped with 14 species of European and Eurasian distribution in a clade. The genus Trifolium is widely distributed in temperate and sub-tropical parts of the world. The Mediterranean and Californian regions are its main distribution centers. Especially Mediterranean region is considered to be the centre of the origin of the genus and is the source of origin of cultivars by domestication and breeding.

Badr et al. (2002) analysed isozyme polymorphism in seven Trifolium species to investigate their ancestral relationships. Their analysis revealed that white clover was close to T. uniflorum and T. nigrescens. However, shared alleles were also detected between white clover and both T. occidentaleand T. isthmocarpum. Pairwise distances indicated that there were strong relationships between white clover,T. nigrescensand T.

uniflorum. They also suggested that T. occidentale might have contributed to the

genome of white clover but via T. nigrescens and T. uniflorum. Ellison et al. (2006) comprehensively studied the phylogeny of the Trifolium genus. They analysed 218 species representing a wide geographic and taxonomic range. Internal transcribed spacers from nuclear DNA and chloroplast (trnL intron) DNA were employed to construct a phylogenetic tree. Their results confirmed a monophyletic and Mediterranean origin of the genus during the Early Miocene period. They found that 16 is the ancestral diploid chromosome number. In the phylogenetic tree of section

Trifoliastrum, T. occidentale and T. uniflorum appeared as close perennial relatives to

white clover. This study showed that T. pallescens(in chloroplast DNA analysis) and T.

occidentale(in nuclear DNA analysis) are the likely diploid progenitors of white clover.

However, combined analysis did not pool these two species in one group.

Williams et al. (2006) explored the section Trifoliastrumand extracted a group of eight species which appeared to be closely related to white clover on experimental phylogenetic basis. They named this group as the “white clover complex”. It consists of

T. ambiguum, T. montanum, T. nigrescens, T. isthmocarpum, T. uniflorum, T.

occidentale, white clover, T. pallescens and T. thalii. The white clover complex has

significant diversity in terms of geographic and environmental adaptation, plant morphology, life cycle and tolerance to various biotic and abiotic stresses. The researchers developed a comprehensive interspecific hybridization programme to exploit this valuable genetic diversity.

The study of Williams et al. (2008) showed that 2x T. nigrescens (both subspecies

nigrescens and petrisavii) and 2x T. occidentale can be hybridized without much

difficulty. These hybrids produced normal F2, F3 and BC1F1 progeny (with T.

nigrescens as recurrent parent). Plants of the F2 and F3 generations lacked the

stoloniferous, perennial nature of T. occidentale. There was complete bivalent chromosomal pairing and evidence of introgression from T. occidentale into T.

nigrescens genome in the backcrossed hybrids. This showed incomplete and relatively

recent reproductive isolation of these species. Taxonomic closeness, success of hybridization and chromosomal homology showed that T. nigrescensand T. occidentale

were close relatives of white clover. However, based on the nonappearance of stoloniferous perennial plants among hybrids of T. nigrescens and T. occidentale and the results of DNA analysis by Ellison et al. (2006), the researchers concluded that T.

occidentale could be a parent of white clover but T. nigrescens was probably not.

However, this species clearly was a close relative of T. occidentale.

Abberton and Thomas (2010) highlighted the importance of conservation of Trifolium

germplasm from diverse origins. They emphasized the importance of conservation of wild Trifolium species for agriculturally important species like T. pallescens and T.

occidentale for white clover, and T. diffusum for T. pratense breeding programs. The

gene banks were found to have largest collections of accessions from wild populations. This could be highly valuable source for future efforts to transfer desired genes from wild types into cultivated species. Williams and Nichols (2011) have also comprehensively reviewed the distribution, diversity and usage of Trifoliumspecies.

2.5.2 Wide hybridization

Pandey (1957) suggested that speciation in Trifolium has resulted from mutation and chromosomal changes leading to cytological incompatibility among species.

Consequently hybridization among Trifolium species is difficult. This general absence of interspecific hybridization has evolutionary value. Zohary and Heller (1984) suggested that a predominance of selfing is the cause of the near-absence of interspecific hybridization. Ellison et al. (2006) found only 5–6 cases of interspecific hybridization among a large sample of 218 Trifoliumspecies. This extremely low rate of natural hybridization shows strong genetic barriers for interspecific hybridization in this genus. Attempts at artificial wide hybridization among Trifolium species have been done with two wide perspectives, i.e. to understand evolutionary relationships within the genus and to introgress desirable traits into agriculturally important species, mainly red clover and white clover. The success of these efforts, especially for intersectional hybridization, has been low.

Williams (1987b) reviewed interspecific hybridization in pasture legumes with special emphasis to Trifolium, Lotus and Ornithopus. The reviewer indicates that prefertilization and postfertilization barriers similar to other plant families prevent interspecific hybridization in these legumes. The genotypes of individual plants involved in interspecific hybridization affect the intensity of incompatibility. Normal pollen tube growth and pod enlargement is taken as an indication of absence of prefertilization barriers. Endospermic inviability can be an active postfertilization barrier for interspecific hybridization in these legumes. During its early development endosperm form a haustorium at the chalazal end of the embryo sac. Failure in formation or attachment of the haustorium with maternal tissues results in failure in development of endosperm. Embryo abortion results due to starvation in absence of endosperm and disturbance in normal water potential gradients of the seed. Embryo rescue and in vitro development can overcome these problems. Normally developing hybrid embryos, usually at early heart stage, are excised from the enlarged pods and placed in normal endosperm (nurse culture technique) or in carefully composed growth media for further growth. Embryos of white clover can be normally grown on EC6 medium without the aid of nurse endosperm.

Red clover (T. pratense L.) is a perennial diploid (2n=14) species widely cultivated in most temperate regions. It has high seedling vigor, rapid growth, salinity tolerance and high nutritive value. It is much shorter lived and less grazing tolerant than white clover. Improvement in persistence is one of the major areas of active research for T. pratense. Travin (1930) reported success of crosses between T. pratenseand T. medium. Taylor et

al. (1963) used T. pratenseas the female parent and crossed it with 47 Trifoliumspecies to explore transferability of longevity of growth period into T. pratense. They successfully generated an amphidiploid hybrid (2n=30) by a cross between colchicine- induced tetraploid T. diffusum (2n=32) and tetraploid T. pratense (2n=28). This hybrid was backcross compatible with both parents. It was intermediate between its parents in most respects but was annual in growth habit. This hybrid was projected as a valuable bridging source for future interspecific hybridization. Taylor and Collins (1989) reported a hybrid between strongly perennial T. sarosienseand the weakly perennial T.

pratense ('Kenstar'). This hybrid had 31 chromosomes, 24 from T. sarosiense and 7

from T. pratense and expressed complete absence of meiosis and fertility. The hybrid was perennial in nature and could be a useful source of improvement for T. pratense if its fertility could have been restored. Phillips et al. (1992) could recover only one F1

hybrid from crosses between T. alpestre and T. pratense. That hybrid had completely dysfunctional gametogenesis. A successful attempt to transfer the rhizomatous growth habit of T. medium (2n=80) into T. pratense(2n=28) was made by Sawai et al. (1995). Chromosomes were doubled by colchicine treatment of the hybrid embryo. The third backcross progeny, using T. pratense as recurrent parent, had a range of chromosome numbers (2n= 32 – 42), acceptable fertility and rhizomes. Isobe et al. (2002) observed seed production and vigor of four backcrossed generations (having T. pratense as recurrent parent) of these hybrids under field conditions. Fertility and survival improved with advancement in backcrossing. The BC4 generation had survival rates similar to T.

pratense. However, the rhizomatous trait did not appear in hybrids after the BC1

generation. Most of the BC4 plants had more than 28 chromosomes, and were different

from T. pratense in leaf size and shape. This indicated the presence of T. medium

genome in these hybrids. The genomic diversity, acceptable longevity and considerable seed production made these BC4 hybrids a potentially valuable breeding resource.

Kouame et al. (1997) screened T. pratense and seven related species against four root- knot nematode species i.e., Meloidogyne arenaria, M. hapla, M. incognita and M.

javanica. The various host and pest species combinations contributed to the appearance

of a wide range of resistance responses. The host response seemed to be associated with plant species life form and root habit. The majority (98%) of T. pratense were intermediately or highly susceptible to all four nematode types. More than 50% of T.

alpestreaccessions expressed high resistance to all nematodes species. About 33% of T.

provided the greatest variation, from highly resistant to highly susceptible. This analysis demonstrated that transfer of root-knot nematode resistance from wild relatives into T.

pratenseby interspecific hybridization might be possible.

Evans (1962) showed the existence of a high degree of incompatibility between

Trifolium species. Grafting as well as crossing techniques were used to screen for

compatibility among nine agronomically important species and eight taxonomically related species of Trifolium. Hybridization was significantly greater for graft compatible genotypes than for graft incompatible genotypes. Twenty eight hybridizations between naturally occurring diploids and polyploids resulted in seed production for five combinations. Induced polyploids of white clover, T. nigrescens, T. hybridum, T.

pratense,T. incarnatumand T. alexandrinumwere tested in 10 reciprocal combinations.

Five hybridizations produced seeds but only one hybrid i.e., white clover (8x) x T.

nigrescens(4x) matured into plants.

Chen and Gibson (1972) studied pollen germination, pollen tube growth and fertilization in Trifolium. They used white clover as the maternal parent in intra- and interspecific crosses with T. nigrescens (2x), T. uniflorum (4x), T. occidentale (2x), T.

uniflorum x T. occidentale (4x), T. hybridum (2x) and T. ambiguum (2x). Intraspecific

crosses performed clearly much better than interspecific crosses for all studied traits. Pollen germination during interspecific crosses was very slow, compared to intraspecific crosses. In interspecific crosses, pollen of T. occidentale (4x) germinated faster than other species. However, little difference was observed among other species for pollen growth. The most different pattern from white clover was shown by T.

uniflorum, whileT. occidentaleshowed the least deviated pattern of pollen tube growth.

A direct relationship was found between pollen tube growth and fertilization. Species like T. nigrescens and T. occidentale (2x) having normal pollen tube growth similar to that of white clover, fertilized more ovules than species like T. uniflorum and T.

ambiguum, which had abnormal and slow pollen tube growth. The researchers proposed

that as pollen tubes contacted only stigmatoid tissues of the pistil during their growth, the compatibility of these tissues with pollen is a crucial factor for pollen tube growth. Once the pollen tube enters into the embryo sac, there is no barrier to prevent fertilization. All interspecific crosses involving different species remained successful in fertilization after 24 hours from pollination. Post-fertilization barriers appeared to be responsible for incompatibility of some interspecific crosses. In an earlier study (Chen

and Gibson, 1971a), they observed that starvation of embryos was caused by abnormal development and disintegration of endosperm. This starvation eventually led to embryo abortion.

Ferguson et al. (1990) studied the potential of in vitro embryo and ovule rescue for

Trifolium hybrids. The cross between T. isthmocarpum (2x) and white clover (4x)

produced highly infertile triploid (3x) hybrids. A T. ambiguum x T. occidentale hybrid was produced, but it did not flower. A hybrid of white clover withT. occidentalefailed to produce viable embryos when crossed with T. ambiguum. Similarly, cross between white clover and T. ambiguum (2x, 4x and 6x) failed either to produce embryos or to regenerate plantlets. An important successful plant regeneration obtained was for the products of crosses between a trihybrid (RUO) of white clover, T. uniflorum and T.

occidentale, backcrossed to T. occidentale (as maternal parent) and hexapoloid T.

ambiguum (as paternal parent) i.e., {[(white clover x T. uniflorum) x T. occidentale

(2n=32)] x T. occidentale(2n = 32)} x T. ambiguum(2n = 48).

2.6

INTERSPECIFIC HYBRIDIZATION OF WHITE CLOVER