Distant Hybridization of Crop Plants (1992)
G. Kalloo et al. (eds.)

Chapter 4
Overcoming the Barriers in Hybridization
G. S. Khush and D. S. Brar1

4.1 Introduction

1 Principal Plant Breeder and Head, and Associate Plant Breeder, respectively. Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, Manila, Philippines

The wild relatives of crop plants are an important reservoir of genetic variability for various economic characteristics such as disease and insect resistance, tolerance for abiotic stresses, male sterility, increased biomass, grain yield, and improved quality characteristics. The role of wild relatives in crop improvement has been emphasized in several reviews (Harlan 1976; Knott and Dvorak 1976; Sanchez-Monge and Garcia-Olmedo 1978; Stalker 1980; Sears 1981; Frey 1983; Sharma and Gill 1983b; Swaminathan and Gupta 1983; Brar and Khush 1986; Khush and Brar 1988; Kalloo Chap. 9, this Vol).

Several barriers are encountered while transferring genes from wild into cultivated species. Some barriers prevent fertilization in distant crosses and hamper the development of the hybrid embryos in others. Blakeslee (1945) and Stebbins (1958) reviewed these barriers. Pre-fertilization barriers include (1) failure of pollen germination, (2) slow pollen tube growth and poor penetration through the stigma wall, and (3) arresting of pollen tubes in the style, ovaries and ovules, resulting in incomplete fertilization. The post-fertilization barriers include (1) hybrid inviability and weakness, (2) hybrid sterility, and (3) hybrid breakdown. Hybrid inviability may be due to abnormal endosperm development resulting in embryo abortion. The hybrid seedlings are sometimes lethal or sublethal. Hybrid sterility may be due to chromosomal or genic differences. Hybrid breakdown occurs in F2 or later generations. Techniques that have been developed for overcoming these barriers (Table 4.1) are reviewed in this chapter.

4.2 Techniques for Overcoming Pre-Fertilization Barriers

4.2.1 Manipulation of the Chromosome Number

Many difficulties arise when hybridization is attempted between species with different ploidy levels. Several crop plants such as wheat, cotton, potato, tobacco, peanut, oat, sugarcane, Brassica napus, B. juncea, and several forage crops are polyploids. Many of the wild relatives of these crops with useful genes are either diploid or have lower chromosome number than the cultivated species. On the other hand, some of the wild relatives of diploid crop plants such as rice, maize, barley, pearl millet, and pulses have higher chromosome number. Crosses between cultivated and wild species with different ploidy levels are difficult to achieve.

Table 4.1. Techniques for overcoming barriers to wide hybridization

Barrier Technique for overcoming the barrier
1. Failure of pollen germination Mechanical removal of pistil followed by pollination of the exposed end of the style
Use of recognition pollen
2. Slow pollen tube growth Use of recognition pollen
In vitro fertilization
Use of growth hormones and immunosuppressants
3. Pollen tube unable to reach the style Shortening the style
4. Arresting of pollen tube in the style, ovary and ovule In vitro fertilization
5. Failure to obtain sexual hybrids Protoplast fusion
6. Differences in ploidy level Chromosome doubting of species or species hybrid before hybridization with the recipient species
Bridging species technique
Reducing the chromosome number of cultivated polyploid species before hybridization
7. Hybrid inviability and weakness  
  Embryo abortion Embryo rescue
In vivo/vitro embryo rescue/embryo implantation
  Embryo abortion at very young stages Ovule culture
In vitro fertilization
  Lethality of F1 hybrids Reciprocal crosses
Grafting of hybrids
Regenerating plants from callus
  Chromosome elimination Altering genomic ratios of two species
Inducing chromosomal exchanges before onset of elimination
8. Hybrid sterility Chromosome doubling (amphiploid production)
9. Hybrid breakdown Growing larger F2 populations
10. Lack of recombination Inducing chromosomal exchanges through tissue culture
Inducing chromosomal exchanges through irradiation
Inducing homoelogous recombination through genetic manipulation of chromosome pairing system

Chromosome doubling in either of the putative parents helps overcome such hybridization barriers. In potato, doubling the chromosome number of the wild species Solanum chacoense greatly increased its crossability with S. tuberosum (Livermore and Johnstone 1940). In other studies, the chromosome number of several diploid (2n = 24) potato species was doubled before they could successfully be hybridized with the tetraploid S. tuberosum (2n = 48). These crosses were made to transfer frost resistance into the potato cultivars. Amphiploids of Nicotiana species hybrids were crossed with Nicotiana tabacum to transfer disease and nematode resistance (Stavely et al. 1973).

Beasley (1940c) doubled the chromosome number of the F1 hybrid (Gossypium thurberi x Gossypium arboreum) and crossed this synthetic amphiploid with natural amphiploid G. hirsutum (cultivated American cotton). Subsequent backcrosses with G. hirsutum resulted in lines with superior lint strength. Knight (1953, 1954) transferred genes for blackarm resistance from Gossypium arboreum (AA) to G. barbadense (AADD) by first doubling the chromosome number of G. arboreum and then crossing it with G. barbadense. Wernesman et al. (1965) obtained no hybrid seed from the cross of Lotus tenius (2n = 12) with L. corniculatus (2n = 24). However, crosses were successful between an advanced generation tetraploid of L. tenius (2n = 24) and L. corniculatus. Sanchez-Monge and Martin (1982) could produce barley hybrids by crossing autotetraploid Hordeum chilense (2n = 28) with cultivated barley H. vulgare (2n = 14).

4.2.2 Bridging Species Technique

When direct crosses between two species with the same or different ploidy levels are difficult or impossible to accomplish, a third species (bridging species) is used to produce such crosses. This technique has been used to make wide crosses in wheat, tobacco, potato, lettuce, and sugarbeet. Many diploid species of wheat and its related genera with desirable genes do not cross with the hexaploid wheat Triticum aestivum. In this case, a tetraploid species serves as a bridge for transferring genes from a diploid wild species to the cultivated hexaploid wheat. Sears (1956a) could not obtain viable seeds when Aegilops umbellulata was used either as a male or as a female parent in crosses with hexaploid wheat. An amphiploid of T. dicoccoides and Ae. umbellulata was produced and this was crossed with T. aestivum. Crosses between Aegilops ventricosa and hexaploid wheat are difficult to obtain. Doussinault et al. (1983) used T. turgidum as a bridge species and transferred resistance to eyespot from Ae. ventricosa to hexaploid wheat. Eenink et al. (1982) transferred resistance to lettuce leaf aphid from Lactuca virosa to cultivated lettuce (L. sativa) by using L. serriola as a bridging species.

In potato, Solanum acaule has been used as a bridging species for producing hybrids of cultivated S. tuberosum with S. bulbocastanum, S. cardiophyllum, and S. pinnatisectum (Dionne 1963). Chavez et al. (1988a, b) could not cross wild species S. commersonii with S. tuberosum; however, hybridization was successful when S. lignicaule and S. capsicibaccatum were used as bridging species. Crosses of Nicotiana repanda (2n = 48) with N. tabacum (2n = 48) are difficult to make. Burk (1967) used N. sylvestris (2n = 24) as a bridging species to transfer nematode resistance from N. repanda to N. tabacum.

4.2.3 Shortening the Style

Several species that differ widely in stylar length are difficult to hybridize. The style of one species may be too long for the pollen tubes of the other species to reach the ovary. This kind of barrier is encountered in crosses of corn (Zea mays) with Tripsacum dactyloides. The styles of corn are generally more than 30 cm long, whereas the styles of Tripsacum are usually less than 2 cm in length. Hence, the distance to be traversed by the Tripsacum pollen tubes to reach the ovaries of maize is 15 times greater than that which they need to reach Tripsacum ovaries.

To overcome this barrier, Mangelsdorf shortened the styles of corn to less than 2 cm and applied Tripsacum pollen. Each of the five ears so treated set some seeds and, thus, a hybrid between corn and Tripsacum was produced (Mangelsdorf and Reeves 1939). In another such experiment, 382 corn ears with an estimated 184925 styles were pollinated with Tripsacum pollen. On the average, 4.54 seeds per 10000 pollinated styles were obtained. The mature seeds were small and shriveled. They were cultured on sterile agar in Petri dishes. From 84 seeds, 45 seedlings were recovered, 29 of which survived transplanting to the field. The hybrids had traits of both parents, but resembled the Tripsacum parent much more closely (Mangelsdorf 1974). Dhaliwal and King (1978) also shortened corn styles to 1 to 2 cm before attempting in vitro pollination with corn or sorghum pollen.

4.2.4 Use of Recognition Mentor Pollen

*Incompatibility and the Pollen-Stigma Interaction. Annual
Review of Plant Physiology 26:403-425 (June 1975)

Sometimes the pollen grains of one species do not germinate on the stigmas of another. If these incompatible pollen grains are mixed with the killed maternal pollen grains, germination of the incompatible pollen grains is obtained. Pollen grain walls are known to contain extracellular proteins, which are released on the stigma after pollination. These cell wall proteins play an important role in pollen-stigma interactions (Heslop-Harrison et al. 1975)*. When the compatible pollen is killed with ethanol and mixed with incompatible pollen for use in pollination, the proteinaceous recognition factors released from the walls of the killed compatible pollen grains mask the rejection reaction of the recipient stigma, thus allowing the alien pollen grains to germinate. The killed maternal pollen is called recognition or mentor pollen.

This method was used to overcome interspecific incompatibility in Populus (Knox et al. 1972a, b). Stettler (1968) produced interspecific hybrids in Populus by mixing live incompatible pollen with gamma irradiated (killed) compatible pollen. Sastri and Shivanna (1976) also used recognition pollen to overcome incompatibility in Sesamum indicum x S. mulayanum cross. The fresh pollen of S. indicum was killed with anhydrous methanol treatment for 2-3 min. Pollinations were made by applying the recognition pollen sparsely on the stigma followed by pollination with fresh incompatible pollen of S. mulayanum. The pollen grains of wild species germinated and the pollen tubes entered the stigma. However, only a few pollen tubes reached about half the length of the style, and no pollen tubes were observed at the base of the style. Although effective in overcoming pollen grain germination, the recognition pollen technique in Sesamum was ineffective in overcoming incompatibility in the style. The use of this technique in overcoming stylar incompatibility in wide crosses has not been well investigated.

4.2.5 Use of Growth Hormones and Immunosuppressants

Various growth hormones have been found to stimulate pollen tube growth and embryo development. They also prolong the receptivity of stigma and prevent early abscission of pollinated flowers. The use of gibberellic acid (GA3) and other growth hormones improves the chances for producing wide hybrids. Application of 75 ppm GA3 to the maternal plant 1 or 2 days before and after pollination has become a routine procedure for producing interspecific and intergeneric hybrids in Triticum and Hordeum. In general, GA3 increases both seed set and embryo yield. Application of GA3 and indole acetic acid (IAA) to the maternal parent promoted the growth of pollen tubes and markedly increased the frequency of zygote formation in barley-rye crosses although the zygotes failed to proceed beyond the proembryo stage (Larter and Chaubey 1965). Crane and Marks (1952) reported that the cross pear x apple succeeded following 40 mg/l naphthalene acetic acid (NAA) application to the ovary and base of the style of the female parent. Brock (1954) reported that the application of 40 mg/l NAA to the stigmas of pear stimulated the growth rate of apple pollen tubes and prevented early abscission of flowers and fruits, and thus allowed fertilization to occur between pear and apple. The F1 seedlings were intermediate between the two parents. Sastri and Mallikarjuna (1985) reported that GA3 application to pollinated flowers in interspecific crosses of peanut stimulated the initiation and geotropic elongation of the pegs. Subsequent application of IAA, NAA, or kinetin further increased the number of pods formed. Park and Walton (1990) sprayed GA3 (0.2%) on the f;orets of the female parent Elymus canadensis 2 days before and after anthesis to promote embryo development in crosses of E. canadensis x Psathyrostachys juncea.

Bates and Deyoe (1973) suggested that the crossability barriers in wide crosses are mediated through stereo-specific inhibition reaction (SIR) analogous to immunochemical mechanism in animals. The SIR affects both fertilization and subsequent embryo development. Several immunosuppressants such as E-amino caproic acid (EACA), chloramphenicol, acriflavin, and salicylic acid have been used with varying degrees of success in plants also. Taira and Larter (1977) used EACA in Triticum turgidum x Secale cereale crosses. The injection of either EACA or L-lysine into the T. turgidum parent, as early as 1 day after pollination with rye pollen, significantly enhanced the embryo development in vitro. Baker et al. (1975) applied EACA to flower buds of Vigna radiata before pollination to produce its hybrids with V. umbellata. This technique appears to be useful to partially overcome the physiological barrier to hybrid embryo development. However, results on the application of immunosuppressants are not very conclusive.

4.2.6 In Vitro Fertilization

Any manipulation of excised maternal and paternal tissue to accomplish pollen tube penetration to the embryo sac to accomplish fertilization is referred to as in vitro fertilization. It is an important technique for overcoming the barriers in stigma and style that inhibit pollen tube growth and embryo abortion in early stages of development. In vitro fertilization followed by culturing of fertilized ovules to maturity is a promising approach and may be a viable alternative to parasexual or somatic cell hybridization. Stewart (1981) and Tilton and Russell (1984) have reviewed the usefulness of the in vitro fertilization technique for producing wide hybrids.

Various methods are used for in vitro fertilization. The whole gynoecia are excised and placed on medium for 24-48 h followed by dusting of pollen on the stigma. Kanta et al. (1962) were the first to try in vitro pollination technique in Papaver somniferum. Since then, a number of reports on this technique have been published (Stewart 1981). However, the technique has been used to a very limited extent in wide crosses. Zenkteler (1980) reported the production of intergeneric hybrid plants through in vitro pollination of Melandrium album x Silene schafta and M. album x Viscaria vulgaris.

Dhaliwal and King (1978) tried hybridization between corn x corn and corn x teosinte (Zea mexicana). The styles of corn were removed and the exposed nucellus of caryopsis on cultured sections of scape were dusted with pollen of Z. mexicana. Seed set was about 5%. No seed developed after application of sorghum pollen to corn styles. Application of sorghum pollen directly to corn ovules resulted in a rapid and massive growth of nucellar tissue without any indication of embryo and endosperm development.

Slusarkiewicz-Jarsina and Zenkteler (1983) obtained hybrid plants from in vitro pollination of ovules of Nicotiana tabacum var samsum with N. knightiana pollen. Embryos produced callus, which later differentiated into shoots and formed plants. Refaat et al. (1984) produced hybrid plants through in vitro pollination of Gossypium hirsutum with G. barbadense pollen.

4.2.7 Protoplast Fusion

The barriers to hybridization among some species are so strong that sexual hybridization is impossible. To overcome such barriers, protoplast fusion can be attempted followed by regeneration of somatic hybrids. Since the production of the first somatic hybrid between Nicotiana glauca and N. langsdorffii by Carlson et al. (1972), a number of somatic hybrids have been produced. Notable examples are tomato+potato (Melchers et al. 1978), Arabidopsis+Brassica (Gleba and Hoffmann 1980), Moricandia arvensis+Brassica oleracea (Toriyama et al. 1987a), Eruca sativa+Brassica napus (Fahleson et al. 1988), Eruca sativa+Brassica juncea (Sikdar et al. 1990), O. sativa+several wild species of Oryza (Hayashi et al. 1988).

4.3 Techniques for Overcoming Post-Fertilization Barriers

4.3.1 Hybrid Inviability and Weakness Embryo Rescue
Among the post-fertilization barriers, abortion of hybrid embryos is the most common. Embryo abortion can occur at different stages of development, depending upon the genomic relationships of two parental species. Such abortive embryos can be dissected from the developing seed, cultured on nutrient medium in test tubes, and grown into mature hybrid plants. Since the first demonstration by Laibach (1925, 1929) that embryos from nonviable seeds of Linum perenne x L. austriacum could be cultured on nutrient medium and raised to maturity, the technique has been routinely used for producing hybrids between distantly related species. Using embryo rescue, a large number of interspecific and intergeneric hybrids have been produced in wheat, rice, barley, cotton, Brassica, forages, legumes, and various horticultural and ornamental plant species (Collins and Grosser 1984; Raghavan 1986a; Williams et al. 1987; Bhojawani et al. 1988).

The embryo rescue technique consists of excising immature embryos at different stages of development depending upon the species, and culturing them in a simple nutrient medium. In cereals, such as wheat and rice, hybrid embryos are excised and cultured usually 10-14 days after pollination. Murashige and Skoog's (MS) medium is commonly used (Murashige and Skoog 1962) either with or without growth hormones such as IAA and kinetin, and supplemented with casein hydrolysate, yeast extract, and coconut milk. In the initial stages, embryos are allowed to germinate in dark but are later put under diffused light. In many cases, particularly in cereals, gibberellic acid (GA3 75-150 ppm) is applied to the florets 1-2 days before and after pollination. Young seedlings arising from hybrid embryos are transferred to the nutrient solution and are later transferred to pots.

Brink et al. (1944) made a cross between Hordeum jubatum and Secale cereale cv. Imperial rye. Gametic union occurred 4 h after pollination, as in selfed H. jubatum. The hybrid embryos started to degenerate within 6 days but some survived as long as 13 days. Histological observations showed that hybrid embryos do not remain viable beyond this period. The incompletely developed hybrid embryos (9-12 days old) were dissected and cultured on nutrient medium consisting of White's major mineral elements (White 1954) supplemented with yeast extract. Of the 81 embryos cultured, 34 remained free from contamination. The growth of most of the embryos was of an undifferentiated nature. However, one embryo developed into a seedling with roots. The hybrid plant flowered normally but was completely sterile.

Gill et al. (1981) followed embryo rescue to produce viable hybrids between Aegilops squarrosa and Triticum boeticum. Fassuliotis and Nelson (1988) cultured embryos 34-99 days after pollination from a cross of Cucumis metuliferus x C. anguria and obtained hybrid plants. Embryo rescue has been used to produce intergeneric hybrids between Elymus canadensis and Psathyrostachys juncea (Park and Walton 1990) and between Triticum aestivum and Psathyrostachys juncea (Plourde et al. 1990). Several incompatibility barriers are encountered in crosses of rice with other Oryza species (Sitch 1990). Embryo rescue has been used successfully to produce several interspecific hybrids in rice (Jena and Khush 1984).

Younger embryos require media containing not only the standard macro and micro salts and an energy source such as carbon, but also an array of organic and hormonal substances. In Vivo/ Vitro Embryo Culture (Embryo Implantation)
In vivo/vitro embryo culture is a modification of the embryo rescue technique and is used when embryo abortion starts at very young stages of development. In this technique the endosperm of the female parent serves as a nurse tissue for the hybrid embryo. Normal endosperm from the female parent is placed on the surface of the nutrient medium but not in direct contact with the hybrid embryos. Sometimes the endosperm extract is added to the nutrient medium. Immature endosperm is known to provide critical nutrients to the developing embryo and helps induce the germination of the immature embryo. Ziebur and Brink (1951) used barley endosperm and endosperm extract for growing young (0.3-1.1 mm) barley embryos in vitro. Kruse (1974) used this technique to produce Hordeum x Triticum hybrids. The success rate in Hordeum x Secale crosses increased to 30 to 40% through nurse tissue technique as compared with 1% through the traditional embryo rescue technique.

A slightly modified technique was used by Williams and de Lautour (1980). This technique involves the insertion of a hybrid embryo into a healthy endosperm dissected from a normally developing ovule and their transfer to the nutrient medium. The normal embryo is pressed out of the sac of endosperm, and the hybrid embryo is inserted into the exit hole of this endosperm. These implanted embryos are cultured on nutrient medium to produce hybrid seedlings. This technique has been used to obtain interspecific hybrids in Trifolium, Lotus, and Ornithopus (Williams and de Lautour 1980). Ovary Culture
In many wide crosses, embryo abortion starts at very early stages of development. Such small embryos are difficult to excise and culture. To overcome this problem the ovaries are cultured. The technique consists of excising ovaries 2-15 days after pollination depending upon the cross combination and culturing them on nutrient medium. The calyx, corolla, and stamen are removed. Before culturing, the tip of the distal part of the pedicel is cut off and the ovary is implanted with the cut end inserted into the medium. The nutrient medium is generally simple, consisting of inorganic salts, auxins, cytokinin, yeast extract, and casein hydrolysate. Both semi-solid and liquid media can be used. After the embryos become visible, they are excised aseptically and cultured on nutrient medium following all the steps of embryo rescue described in the preceding section.

Ovary culture has been successfully used to produce interspecific hybrids in Brassica. Inomata (1978) excised ovaries 4 days after pollination of Brassica campestris with B. oleracea pollen and cultured them on White's medium containing mineral salts, vitamins, and casein hydrolysate (300 mg/l). The ovaries were cultured in a room at 20'C in natural diffused light (300-500 lx) for about 12 h/day. After 36 days of culturing, the embryos from these ovaries were excised and cultured on the same medium under similar conditions. The plantlets arising from cultured embryos were transferred to the soil and about 70 hybrid plants were obtained.

Delourme et al. (1989) produced intergeneric hybrids between Diplotaxis erucoides and Brassica napus through the ovary culture technique. Takahata (1990) produced hybrids between Moricandia arvensis and Brassica oleracea through ovary culture. The ovaries were excised 3-8 days after pollination and cultured on MS medium free of hormones. The embryos from these ovaries were excised after 3-4 weeks of ovary culture and transferred to the same medium. The embryos failed to develop into plantlets. Therefore, shoots were induced from hypocotyl segments. The plants were regenerated from such shoots. The hybrid plants were morphologically intermediate between the parents. Ovule Culture
Like ovary culture, ovule culture is sometimes helpful in overcoming the barriers affecting growth of the zygote in earlier stages of development. In this procedure, ovaries are harvested l-12 days after pollination depending upon the species, surface-sterilized, and cut open with a sterilized scalpel. The fertilized ovules are scooped out and placed as evenly as possible on the nutrient medium. The nutrient medium generally consists of either Nitsch's (1951) or White's (1954) inorganic salts, auxins, and cytokinins, and other complex growth substances such as coconut milk, casein hydrolysate, and yeast extract. With ovule culture, the hybrid embryos can be cultured at earlier stages than excised embryos.

Cotton is the classical example where ovule culture has been successfully used to raise interspecific hybrids. Stewart and Hsu (1978) cultured ovules 2 to 4 days after anthesis. After 8 to 9 weeks of culture, all ungerminated ovules were dissected and the embryos were transferred to the same medium used for germinating seedlings. This technique, commonly referred to as in ovulo embryo culture overcomes the incompatibility between the A genome diploid cottons and the AD genome tetraploid cottons. Hybrid plants have also been produced through ovule culture from crosses of Gossypium hirsutum x G. australe, G. barbadense x G. australe, and G. arboreum x G. australe. Ovule culture has also been used in obtaining hybrids between cultivated and wild species of Nicotiana, Glycine, and Lens. Reed and Collins (1978) used ovule culture to produce hybrids of cultivated tobacco Nicotiana tabacum with three wild species: N. nesophila, N. stocktonii, and N. repanda. Arisumi (1980) found that in order to produce interspecific hybrids in Impatiens, embryo rescue was successful only when well-developed embryos, I to 2 weeks beyond the late heart stage, were cultured. In contrast, rescue by ovule culture was possible with much younger, globular stage embryos. The reasons for the recovery of hybrids in some wide crosses by ovary or ovule culture rather than embryo culture are not well understood. The embryos developing within the ovule may have more favorable chemical and physical environment for growth and development than embryos cultured outside the ovule. It is also possible that tissues of the ovary and the ovule serve various functions such as nutrition and protection of the enclosed embryos. Grafting Hybrids
Wide cross progenies sometime show seedling lethality and are inviable. For example, when Melilotus alba is crossed with M. dentata, fairly plump and readily germinable seeds are obtained but the hybrid seedlings are albinistic and die in early stages. Smith (1943) showed that such lethal hybrid seedlings may be grown to maturity by grafting them upon normal Melilotus plants. The hybrids between cultivated sugarbeet Beta vulgaris and wild species are inviable: the F1 seedlings fail to develop roots. Johnson (1956) grafted F1 seedlings onto sugarbeet plants to enable them to survive. Savitsky (1975) grafted F1 seedlings of B. vulgaris x B. procumbens on to sugarbeet plants and these F1 plants were used for transferring nematode resistance from B. procumbens to B. vulgaris. Reciprocal Crosses
In some interspecific crosses, nuclear cytoplasmic interactions result in sterility or degeneration of F1 plants. Reciprocal crosses have no such problem. The hybrid Nicotiana debneyi x N. tabacum was male sterile, but the reciprocal cross was male fertile (Clayton 1958). Barley-wheat hybrids having barley cytoplasm are associated with pistilloidy. Similarly, the wheats having the cytoplasm of Triticum timopheevi and the nucleus of T. aestivum are male sterile (Wilson and Ross 1962). Reciprocal crosses, on the other hand, are fertile. Regenerating Plants from Callus of Hybrid Embryos
In most distant crosses, hybrid embryos are manipulated to produce F1 plants directly. In some crosses, however, the hybrid embryos fail to differentiate into plants. To overcome this barrier, the undifferentiated embryos are induced to proliferate as callus on a culture medium, and hybrid plants are regenerated from the callus. Thomas and Pratt (1981) employed this technique to increase the chances of recovering F1 plants from the cross Lycopersicon esculentum x L. peruvianum. In the cross Nicotiana suaveolans x N. tabacum, viable seeds are produced, but the F1 seedlings die soon after emergence at the first- or second-leaf stage. Lloyd (1975) cultured the young cotyledons on a callus-inducing medium and from the callus regenerated several shoots which were grown to maturity. Pandey et al. (1987) also obtained normal hybrid plants from the partially necrotic F1 seedlings of Trifolium repens x T. uniflorum via a callus phase. Similarly Fiola and Swartz (1985) obtained hybrid plants from interspecific crosses of Rubus by culturing embryonic axes on proliferation and shoot induction media. Poysa (1990) could not obtain hybrid plants from the cross of Lycopersicon esculentum x L. peruvianum following embryo rescue, ovule culture, and through the use of immunosuppressants and hormonal treatments. The barrier was overcome through embryo-callus culture technique. Altering Genomic Ratio
Chromosome elimination sometimes occurs in wide crosses. A typical example is the elimination of Hordeum bulbosum chromosomes after fertilization in H. vulgare x H. bulbosum crosses (Kasha and Kao 1970). Such chromosome elimination also occurs in crosses of wheat with H. bulbosum (Barclay 1975). In wheat x maize crosses, maize chromosomes become eliminated from the hybrid zygote in very early stages (Laurie and Bennett 1986). Thus, chromosome elimination is a barrier to gene transfer in such wide crosses. Chromosomal elimination may be avoided by altering the ratios of parental genomes in wide crosses. For example, chromosome elimination in H. vulgare x H. bulbosum crosses could be avoided by changing the genomic ratio. When diploid H. vulgare was hybridized with tetraploid H. bulbosum, there was no chromosome elimination in the zygote (Kasha 1974). Mutagenic treatment of fertilized egg cell could be used to induce chromosomal exchanges before the onset of chromosome elimination.

4.3.2 Hybrid Sterility Chromosome Doubling
Distant hybrids often show high levels of sterility. This sterility is attributed to genie or chromosomal differences between the parents. Sterility is overcome by doubling the chromosome number of the hybrid to produce amphiploids. During the last 45 years, amphiploids have been produced through colchicine treatment from numerous sterile hybrids. Triticale is the classical example of a manmade cereal produced through chromosome doubling of the sterile F1 hybrids of Triticum x Secale species. Tritordeum is another amphiploid produced from a cross of Hordeum chilense and Triticum turgidum (Martin and Sanchez-Monge 1982). The amphiploids can be backcrossed to one of the parents several times to isolate the desirable stable progenies with genes from the other parent. Backcrossing
Most of the wide cross F1s are sterile. The fertility can be restored either by doubling the chromosome number or through backcrossing. The amphiploids per se are not of much agricultural value because the agronomic characteristics of the component wild species are poor. Backcrossing of the sterile hybrids with the cultivated species as a recurrent parent leads to restoration of fertility and incorporation of a few desirable genes from the wild parent. This procedure has been used successfully to transfer genes from distantly related species into crop plants. As an example, a dominant gene for resistance to grassy stunt virus of rice was transferred from wild Oryza nivara to cultivated rice O. sativa through backcrossing (Khush 1977). Similarly, genes for resistance to brown planthopper and whitebacked planthopper from O. officinalis (CC genome) were transferred to O. sativa (AA genome) through the backcrossing of their sterile hybrid with the cultivated parent (Jena and Khush 1990).

4.3.3 Hybrid Breakdown

Hybrid breakdown has been a major unsolved problem in wide hybridization. In some interspecific crosses, the F1 is fertile but the recombinants in the F2 or later generations are either lethal or weaker and are gradually eliminated, resulting in only parental types. Many a time, the F2s or later generations look like backcross populations. The probable causes of hybrid breakdown include centromeric affinity, cryptic structural hybridity, gene substitution, and unfavorable nuclear-cytoplasmic interactions. Lethality may be due to differences in gene content between parents. The F1 hybrids tolerate chromosome duplications or deficiencies because they are in heterozygous condition. However, F2 progenies homozygous for these abnormalities are lethal (Stebbins 1958). Tetraploid Gossypium barbadense and G. tomentosum cross readily with G. hirsutum. The F1 plants are vigorous and fertile and show normal chromosome pairing. However, F2 progenies contain many weak, dwarf, chlorotic, and inviable plants. Stephens (1949) reported a strong selection in favor of parental types and against recombinant progenies. In backcrosses, genes of the donor parent were selectively eliminated. Sano and Kita (1978) reported similar observations in Melilotus hybrids. The reasons for hybrid breakdown are not well understood. It may be possible to isolate a few desirable recombinants by growing large F2 and later generation populations.

4.3.4 Limited Recombination

Success in the utilization of alien genetic material depends not only upon the production of wide crosses but also on the subsequent intergenomic recombination. Lack of chromosome pairing and recombination between genomes of alien and cultivated species are the major barriers in the utilization of wild species for crop improvement. These barriers are sometimes overcome through (1) tissue culture of wide hybrids, (2) irradiation-induced chromosomal translocation, and (3) manipulation of chromosome pairing system. Tissue Culture of Wide Hybrids
Chromosomal rearrangements sometimes occur during the tissue culture of distant hybrids and the regenerated progenies show intergenomic gene transfer. Larkin et al. (1989) have reviewed the potentials of cell culture for alien gene introgression. Orton (1980a) reported that the plants regenerated from the tissue culture of a sterile Hordeum vulgare x H. jubatum hybrid had enhanced bivalent formation as compared to the original hybrid, which was asynaptic. Two of the five haploids examined showed a few H. jubatum isozyme bands, indicating that some intergenomic exchanges occurred prior to chromosome elimination (Orton l980b). Lapitan et al. (1984) observed many chromosomal structural changes in amphiploids of wheat x rye regenerated from tissue culture. The analysis of karyotype by C banding of ten amphiploids showed three wheat-rye and one wheat-wheat chromosome translocations, seven deletions, and five amplifications of heterochromatin bands of rye chromosomes. This technique seems promising for obtaining gene transfers between nonhomologous genomes and may be applied to F1 hybrids or alien addition or substitution lines. Irradiation-Induced Chromosomal Translocations

Barriers to recombination may also be overcome through irradiation of wide cross derivatives. The method consists of irradiating the pollen or seed of the monosomic alien addition lines followed by recovery of the translocation of alien chromosome segments in the successive generation progenies either genetically or cytologically, or by both. Sears (1956a) used this technique for transferring a small chromosome segment carrying leaf rust resistance from Aegilops umbellulata to the 6B chromosome of common wheat. Sharma and Knott (1966) transferred stem rust resistance from Agropyron elongatum to common wheat. Driscoll and Jensen (1963) transferred rust resistance from rye to wheat by irradiation of the ditelosomic addition line. A number of strains, viz. Transfer, T4, Agatha, and Transec, possessing resistance to various diseases of wheat, have been developed using this technique. The most successful transfer involves translocation of the Ag. elongatum chromosome, carrying resistance to wheat stem rust, to chromosome 6A of wheat (Knott 1961). Radiation-induced translocations have been used in oats to transfer alien chromosome segments. Aung and Thomas (1976) successfully transferred the gene for mildew resistance from tetraploid Avena barbata into the cultivated oat by means of an induced translocation. The translocation involved the long arm of the shortest chromosome of A. sativa and the short arm of an A. barbata chromosome, which carries the gene for mildew resistance. Manipulation of the Chromosome Pairing System
Some of the cultivated crop plants such as wheat (2n = 42, AABBDD), oats (2n = 42), and cotton (2n = 52) are naturally allopolyploid. The suppression of homologous pairing resulting in the diploid-like meiotic behavior is genetically controlled in polyploid species such as wheat (Riley and Chapman 1958), oats (Rajhathy and Thomas 1972), and tall fescue Festuca arundinacea (Jauhar 1975). A similar mechanism is postulated to control pairing in cultivated tetraploid cotton (Kimber 1961).

Wheat is the classical example where there is no intergenomic pairing between A, B, and D genomes. Such pairing activity is regulated by a genetic mechanism controlled by ph (pairing homoeologous) gene (Wall et al. 1971; Sears 1976). Manipulation of the pairing system could induce homoeologous recombination between chromosomes of different species. Methods to introgress alien genes through induced homoeologous recombination have been discussed by Khush and Brar (1988). For example, three methods have been used in wheat to manipulate chromosome pairing and achieve homoeologous recombination: (a) crossing alien addition lines with wild species, (b) crossing nullisomic 5B stocks of wheat with the alien addition lines, and (c) crossing a ph mutant of wheat with alien addition lines. These are followed by isolation of intergenomic recombinants in the derived progenies.

Wild species such as Aegilops speltoides (2n = 14) and Avena longiglumis (2n = 14 accession CW 57) are known to induce homoeologous pairing in wheat and oats, respectively (Riley and Chapman 1958; Rajhathy and Thomas 1972). Riley et al. (l968a) made crosses between an alien addition line of wheat and a diploid relative of wheat (Aegilops speltoides). The rust resistance gene from the addition line carrying the 2M chromosome of Ae. comosa was transferred into wheat by induced pairing. The homoeologous pairing occurred because the dominant allele of Ae. speltoides neutralized the effect of ph. H. Thomas et al. (1980a) used Avena longiglumis to transfer mildew resistance from A. barbata to the cultivated oat through induced pairing. Similarly, Griffiths and Thomas (1983) used A. longiglumis to induce homoeologous pairing and transferred the gene for mildew resistance from A. prostrata to the cultivated A. sativa. The suppression of genetic control of bivalent pairing in hexaploid oat by Avena longiglumis (CW57) is comparable to role of Ae. speltoides in inducing homeologous pairing in interspecific crosses of wheat.

When nullisomic 5B wheat is crossed with the alien species, the F1 hybrids lack chromosome 5B and the chromosomes of the alien species pair with the homoeologous chromosomes of wheat. Using wheat nullisomic 5B, Sears (1973b) produced wheat lines carrying leaf rust resistance from Agropyron elongatum. As a result of induced homoeologous pairing, several wheat-Agropyron transfers have been obtained (Sears 1972, 1978).

The ph mutants for induced homoeologous pairing were isolated by Wall et al. (1971) and Sears (1977a). Wang et al. (1977) used the ph mutant and induced recombination between chromosome 4B of wheat and the homoeologous Agropyron intermedium chromosome carrying genes for streak mosaic virus resistance. Giorgi and Barbera (1981) crossed the ph mutant of durum wheat (Triticum durum) with four tetraploid Aegilops species and observed higher chromosome pairing than in the control hybrids.

4.4 Conclusions

Wild species are an important reservoir of useful genetic variability for traits of economic importance such as resistance to diseases and insects, tolerance for abiotic stresses, improved quality characteristics, and new sources of male sterility. There are several pre- and post-fertilization barriers that hinder the transfer of useful alien genes into crop plants (Table 4.1). The techniques of embryo rescue, ovary and ovule culture, in vitro fertilization, protoplast fusion, use of growth hormones, chromosome doubling, induced chromosomal exchanges through tissue culture, and irradiation and genetic manipulation of the chromosome pairing system have been refined to overcome the barriers encountered in wide hybridization. This has widened the scope for utilization of wild species in enlarging the gene pools of crop plants.