Cybrids

• Somatic hybrids can be obtained where nucleus is derived from one parent and cytoplasm is derived from both the parents, thus resulting cytoplasmic hybrids, also called as cybrids. Whereas, sexual hybridization is an exact mixture of parental nuclear genes but the cytoplasm is derived from the maternal parent only.
• In cytoplasmic hybridization, nucleus from one protoplast is inactivated or segregated out in early stage such that one protoplast contributes the cytoplasm while the other contributes the nucleus alone or both nucleus and cytoplasm.
• There are different ways of inactivating the nucleus of one protoplast.
• In cybrids, there is fusion between protoplasts containing the full component of nucleus, mitochondria and chloroplasts with functional cytoplasmic component of second protoplast.

Ways of inactivating protoplast to obtain cytoplasmic hybrids:

i. By application of lethal dosages of X-rays or gamma rays to one parental protoplast population:

• Ionizing radiation treatment damages the nucleus, thus the protoplasts become inactivated and non-dividing but they function as an efficient donor of cytoplasmic genophores when fused with recipient protoplasts.
• Nicotiana protoplasts can be inactivated by 5-kr dose of X-rays. Other protoplasts may require different doses.

ii. By treatment with iodoacetate to metabolically inactivate the protoplasts:

• Pre-treatment with iodoacetate will cause the degeneration of non-fused and auto-fused protoplasts while fusion of iodoacetate pre-treated protoplasts with non-treated protoplasts will cause metabolic complementation and result in viable hybrids.
• In an experiment, iodoacetate- treated Nicotiana plumbaginifolia cell suspension was fused with X-rays irradiated N. tabacum mesophyll protoplasts.
• All regenerated cybrid plants had N. plumbaginifolia morphology but most of them contained N. tabacum chloroplasts.
• The iodoacetate treatment does not impair the nucleus of the treated protoplasts.
• Thus the latter can complement an X-rays irradiated protoplast.

iii. Fusion of normal protoplasts with enucleated protoplasts:

•  The high-speed centrifugation (20,000–40,000x g) for 45–90 minutes in an iso-osmotic density gradient with 5–50% percoll will yield enucleated protoplasts.
• Additional exposure of isolated protoplasts to cytochalsin B in combination with centrifugation has also been found beneficial for enucleation.

iv. Fusion of cytoplasts with protoplasts:

• Isolated protoplasts can be experimentally induced to fragment into types of sub-protoplasts called mini-protoplasts or cytoplasts.
•  The term mini-protoplast was coined by Wallin et al. (1978) for sub-protoplasts having nuclear material which can divide and may be able to regenerate into plants.
• The other similar terms for mini-protoplasts are karyoplast (evacuolated sub-protoplast) or nucleoprotoplast.
• The nuclear free subprotoplasts which donot divide but are important in the process of cybridization are termed as cytoplasts.
• Maliga et al. demonstrated that streptomycin resistance encoded by chloroplasts could be transferred by cytoplasts.
• Similarly, Tan (1987) claimed to have obtained cybrids by fusion of cytoplasts from Petunia hybrida and protoplasts of Lycopersicon peruvianum.
• Fusion of a normal protoplast with another in which nuclear division is inhibited.
• Cybridization thus opens an exciting avenue to achieve alloplasmic constitution in a single step without the need to perform a series of 8– 12 time-consuming backcrosses.
• Alloplasmic lines contain nucleus of one parent genome with the cytoplasmic constituents from other parent.
• Application of cybrids would be the directed transfer of cytoplasmic male sterility or herbicide resistance from a donor to a recipient crop plant species.
• Transfer of cytoplasmic male sterility (CMS) from N. tabacum to N. sylvestris by protoplast fusion was first reported by Zelcer et al. (1978).
• Resistance of plants to herbicide atrazine has been transferred from Brassica campestris to B. napus via fractionated protoplast fusions.
• CMS has been successfully transferred in various crop species of Oryza, Lycopersicon, Brassica, Nicotiana, etc.

Application of cytoplasmic hybridization:

1. Production of hybrid organisms:
   • Production of novel interspecific and intergeneric crosses between plants that are difficult or impossible to hybridize conventionally.
   • Both interspecific and intergeneric hybrids can be acquired by somatic hybridization.
2. Overcomes sexual incompatibility barriers:
   • Somatic hybridization overcome the sexual incompatibility barriers during breeding or cross fertilization.
   • For example, fusion between protoplasts of Lycopersicon esculentum (tomato) and Solanum tuberosum (potato) created the pomato first achieved by Melchers et al. (1978).
   • Asymmetric hybrids also develop when there is partial hybridization.
   • These asymmetric hybrids have abnormal or wide variation in chromosome number than the exact total of two species.
   • Efficient cell fusion can be achieved between sexually compatible and incompatible parents involving interspecific or intergeneric combination.
   • Attempts to overcome conventional breeding barriers by interspecific fusion of rice with four different wild species including Oryza brachyantha, O. eichingeri, O. officinalis and O. perrieri were more successful.
   • Mature plants with viable pollen could be obtained in all but the first Oryza combination.
   • The production of fertile interspecific diploid rice hybrid plants as well as cybrids demonstrates that the fusion technology can be extended to graminaceous crops.
3. Somatic hybridization for gene transfer:
   • i) for production of Disease resistance variety:
     • Many disease resistance genes viz. potato leaf roll virus, leaf blight, Verticillium, Phytophthora, etc. have been transferred to Solanum tuberosum from other species where normal crossings would not be possible due to taxonomic or other barriers.
     • Resistance to blackleg disease (Phoma lingam) has been found in Brassica nigra, B. juncea and B. carinata .And, resistant hybrids have been developed after production of symmetric as well as asymmetric somatic hybrids between these gene donors and B. napus,.
     • Resistance has been established in tomato against various diseases like TMV, spotted wilt virus, insect pests and also cold tolerance.
   • ii) for production of Abiotic stress resistance:
     • Work related to somatic hybridization for abiotic stress has been mainly done on families Fabaceaa, Brassicaceae, Poaceae, Solaneae and relates to cold and frost resistance.
     • Rokka et al. (1998) developed somatic hybrids between cultivated potato (Solanum tuberosum) and wild relative (Solanum acaule) possessing several disease and early frost resistance characters.
   • iii)  for production of Quality characters:
     • Somatic hybrids produced between Brassica napus and Eruca sativa were fertile and had low concentration of erucic acid content (Fahleson et al., 1993).
     • Likewise, nicotine content character has been transferred to N. tabacum.
4. Transfer of Cytoplasmic male sterility:
   • Various agriculturally functional traits are cytoplasmically encoded, including some types of male sterility and certain antibiotic and herbicide resistance factors.
   • Pelletier et al. (1988) reported Brassica raphanus cybrids that contain the nucleus of B. napus, chloroplasts of atrazine resistant B. campestris and mitochondria that confer male sterility from Raphanus sativus.
   • Cybridization has been successfully used to transfer cytoplasmic male sterility in rice (Kyozuka et al., 1989).
   • Sigareva and Earle (1997) produced cold tolerant cytoplasmically male sterile (cms) cabbage (Brassica oleracea ssp. capitata) by the fusion of cabbage protoplasts with cold tolerant ogura CMS broccoli lines.
5. Production of resistant variety:
   • Resistance to antibiotics, herbicide as well as CMS has been introduced in so many cultivated species.
6. Production of auto-tetraploids:
   • Somatic hybridization can be used as an alternative to obtain tetraploids and, if this is unsuccessful, colchicine treatment can be used.
   • Protoplasts of sexually sterile (haploid, triploid, aneuploid, etc.) plants can be fused to produce fertile diploids and polyploids.
7. Hybridization becomes possible between plants that are still in the juvenile phase.
8.  Production of heterozygous lines within a single species that normally could only be propagated by vegetative means, e.g. potato and other tuber and root crops.
9.  To study cytoplasmic genes:
   • Somatic cell fusion is useful in the study of cytoplasmic genes and their activities. This information can be employed in plant breeding experiments.
10. Production of unique nuclear-cytoplasmic combinations:
    • Mitochondrial and chloroplast recombination has also been reported to result in unique nuclear- cytoplasmic combinations.
    • These unique combinations using protoplasts will aid the development of novel germplasm not obtainable by conventional methods.

Limitation of cytoplasmic hybridization:

There are certain limitations to the use of these types of somatic hybridization:

1. Plants regenerated from some of the combinations in somatic hybridization are often sterile, deformed, and unstable and are thus not viable, particularly if the fusion partners are taxonomically far apart.
2.  Application of protoplast methodology requires efficient plant regeneration from protoplasts. Protoplasts from any two species can be fused. However, production of somatic hybrid plants has been limited to a few species.
3. Sometimes, the major problem is the lack of an efficient selection method for fused product.
4. The development of chimaeric calluses in place of hybrids. This is usually due to the nuclei not fusing after cell fusion and dividing separately. Plants that are regenerated from chimaeras usually lose their chimeric characteristics, since adventitious shoots or embryos usually develop from a single cell.
5. Somatic hybridization of two diploids leads to the formation of an amphidiploid which is generally unfavourable (except when tetraploids are formed intentionally). For this reason in most cases, the hybridization of two haploid protoplasts is normally recommended.
6. Regeneration products after somatic hybridization are usually variable because of the somaclonal variation, chromosome elimination, translocation, organelle segregation etc.
7. It is never certain that a particular characteristic will be expressed after somatic hybridization.
8. The genetic stability during protoplast culture is poor.
9. To achieve successful integration into a breeding programme, somatic hybrids must be capable of sexual reproduction.
10. In all cases reported, somatic hybrids containing a mixture of genes from two species must be backcrossed to the cultivated crop to develop new varieties.

Cytoplasmic hybrid (Cybrid)-applications and limitations