🧬 Products of Somatic Hybridization and Cybridization

After protoplast fusion, the main question is what type of product is actually obtained. This lesson helps distinguish somatic hybrids from cybrids and explains why chromosome behavior becomes a major selection issue after fusion.

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Products of somatic hybrids and cybrids

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Chromosome status of somatic hybrids

The chromosome numbers of the somatic hybrids successfully obtained through protoplast

fusion indicate that only few have exact number expected in an amphiploid. Hence selection will

also need to be applied at the cytological level, if true amphiploids need to be obtained. The

variability in chromosome number in hybrids could be due to any one of the following reasons:

(i) Multiple fusions give a higher chromosome number. In PEG-induced and electro

induced fusions between more than two protoplasts.

(ii) Asymmetric hybrids result from fusion of protoplasts isolated from actively dividing tissue

of one parent and quiescent tissue of the other parent.

(iii) Unequal rates of DNA replication in two fusing partners may also give asymmetric

hybrids.

(iv) Somaclonal variation in cultured cells used for protoplast isolation may also lead to

variation in chromosome number.

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Practical applications of somatic hybridisation and cybridisation

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Means of genetic recombination in asexual or sterile plants

Somatic cell fusion appears to be the only approach through which two different parental

genomes can be recombined among plants that cannot reproduce sexually. Further, protoplasts

of sexually sterile (haploid, triploid and aneuploid) plants can be fused to produce fertile diploids

and polyploids. There are several reports describing the amphidiploid and hexaploid plants

produced from fusion of haploid protoplasts of tobacco. Protoplasts isolated from dihaploid

potato clones have been fused with isolated protoplasts of Solanum brevidens to produce

hybrids of practical breeding value. Haploid protoplasts from an anther-derived callus of rice

cultivars, upon fusion also produce fertile diploid and triploid hybrids.

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Overcoming barriers of sexual incompatibility

In plant breeding programmes, sexual crossings at interspecific or intergeneric levels often fail

to produce hybrids due to incompatibility barriers. The bottlenecks in sexual hybridisation may

therefore, be overcome by somatic cell fusion. In some cases somatic hybrids between two

incompatible plants have also found application in industry or agriculture.

Schieder (1978) obtained amphidiploid Datura innoxia (+) D. discolor and D. innoxia (+) D.

stramonium, by fusing their diploid mesophyll protoplasts. These hybrids did not exist in nature

as conventional breeding procedures proved unsuccessful. Somatically produced amphidiploids

of these combinations of Datura species are propagated for industrial uses as they demonstrate

heterosis and higher (20-25%) scopolamine content than in the parental forms.

Nicotiana repanda, N. nesophila and N. stockonii are resistant to a number of diseases but are

not sexually crossable with tobacco ( N. tabacum ). However, fertile hybrids have been reported

in combination N. tabacum (+) N. nesophila and N. tabacum (+) N. stocktonii by protoplast

fusion. Somatic hybridisation of dihaploid and tetraploid potato protoplasts with isolated

protoplasts of Solanum brevidens, S. phureja and S. pennelii resulted in the synthesis of fertile,

partially amphieuploid plants possessing important agricultural traits, e.g., resistance to potato

leaf virus, potato virus Y and Erwinia soft rot. Using this approach, tomato ( Lycopersicon

esculentum ) hybridised somatically with a number of wild species has resulted in the synthesis

of hybrids which are fertile and used in breeding programmes. Interspecific somatic

hybridisation involving species that are sexually incompatible with egg-plant ( Solanum

melongena ) has also resulted in the production of amphidiploids with traits resistant to

verticillium wilt.

Rapeseed ( Brassica napus ) is a natural amphidiploid of B. o l eracea and B. campestris . Schenk

(1982) was the first to resynthesise rapeseed in vitro using protoplast fusion. Somatic

hybridisation between B. napus and B. nigra cultivar, possessing the gene for resistance to

Phoma lingam, yielded amphidiploid plants carrying this gene. These hybrids possess all the

three Brassica genomes (A, B and C) and are now incorporated in breeding programmes.

Recently, hybrids have been produced parasexually by protoplast fusion, between Brassica

juncea (a major oilseed crop of the tropical world) and the sexually incompatible species

Diplotaxis muralis and Erica sativa .

The potential of somatic hybridisation in perennial tree breeding is best illustrated by

interspecific and intergeneric somatic hybridisation among citrus species. Somatic hybrids

produced through these experiments are amphidiploids featuring characteristics for scion

improvement and increased rootstock potential.

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Somatic hybrids for cytoplasmic male sterility

Methods were also developed to substitute the nucleus of one species into the cytoplasm of

another species, whose mitochondria were inactivated. This type of substitution in some cases,

led to generation of cytoplasmic male sterility.

For this purpose, the two types of protoplasts, used for the production of somatic hybrids, were

treated differently, as follows:

(i) mesophyll protoplasts of tomato ( Lycopersicon esculentum ) were treated with

iodoacetamide (IOA) to inactivate mitochondria and

(ii) (ii) mesophyll protoplast of Solanum acaule (or S. tuberosum ) were irradiated with g or x

rays to inactivate nuclei.

The protoplasts were mixed in 1:1 ratio and induced to fuse using Ca [2+] and PEG, leading to the

production of heterologous or alloplasmic hybrids. Among the fusion products, some hybrid

tomato plants were indistinguishable from the original cultivars, with respect to morphology,

physiology and chromosome number (2n = 24), but exhibited various degrees of male sterility.

In five tomato cultivars, male sterility induced in this manner was inherited maternally over

several generations. Therefore, it was obviously cytoplasmic male sterility. The mitochondrial

DNA of these CMS hybrids did not resemble mtDNA of either parent, and was instead

recombinant type, representing a hybrid mitochondrial genome. Therefore, protoplast fusion can

be effectively used for production of CMS lines and has the following advantages:

(i) Only one step is required;

(ii) The nuclear genotype of the cultivar remains unaffected,

(iii) There are prospects that 100% of the progenies of somatic hybrids will be CMS. The

restorer lines for these CMS lines have also been shown to be available in tomato,

so that hybrid seed can be produced without manual emasculation.

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Generation of cytoplasmic male sterility by fusion

1. IOA (damages mitochondria), 2. γ rays or x-rays (inactivate nuclei), 3. Mitochondria, 4.

chloroplast (tomato protoplasts), 5.Ca [++] + PEG, 6. Protoplast fusion, 7. chloroplast (potato or

S. acaule protoplasts), 8. Nucleus, 9. tomato nucleus, 10. recombinant mitochondira, 11.

chloroplasts (mixture), 12. fused protoplasts, 13. somatic hybrid plants (CMS)-resemble tomato

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Cytoplasm transfer

Power et al . (1975) fused mesophyll protoplasts of Petunia with cultured cell protoplasts of the

crown gall of Parthenocissus and selected a line which contained the chromosomes of only

Parthenocissus but exhibited some of the cytoplasmic properties of Petunia for some time. This

was followed by direct application of cybridisation in agricultural biotechnology by transfer of

cytoplasmic male sterility from Nicotiana techne to N. tabacum, N. tabacum to N. sylvestris and

Petunia hybrida to P. axillaris . Besides cytoplasmic male sterility, the genophore of the

cytoplasm codes for a number of practically important traits, such as the rate of photosynthesis,

low or high temperature tolerance and resistance to diseases or herbicides. Recent experiments

on cybridisation have resulted in plants with reconstructed cytoplasm combining mitochondrial

DNA (mt DNA) and cp DNA encoded traits from both parents.

The best example illustrating the potential for protoplast fusion in reconstructing cytoplasm for

practical purposes is the genus Brassica . Two desirable traits coded by cytoplasmic genes have

been genetically manipulated through interspecific cybridisation between different species of

Brassica . These traits include cytoplasmic male sterility (cms) and resistance to atrazine

herbicides. The cms gene in Brassica plants, Diplotaxis muralis and Raphanus sativus is of

alloplasmic (the nucleus of one species into a foreign cytoplasm) origin. Raphanus sativus is of

interest because it leads to complete male sterility. Cms restorer genes have been introduced

into rapeseed ( Brassica napus ) from this plant. Mutants resistant to atrazine herbicide have also

been discovered both in Brassica napus and B. campesteris . Protoplast fusion experiments

(conducted in various laboratories) have resulted in the synthesis of cybrid plants with recon

structed cytoplasm combining both cms (coded by Raphanus mt DNA) and low temperature

tolerance or atrazine resistance (coded by Brassica cp DNA). Similarly, cytoplasmic genes

coding for atrazine resistance and cms have been transferred into cabbage, rice and potato.

In somatic hybridisation and cybridisation, the essential pre requisite is that parental protoplasts

and their fusion products regenerate to whole plants. Research in the past decade has shown

that plants can be raised in vitro from isolated protoplasts of species belonging to a range of

angiosperm families. Somatic hybrids have been produced between sexually compatible as

well as incompatible species. It could be possible to overcome prezygotic embryo/endosperm

( Petunia parodii (+) P. inflate ) and postzygotic ( Datura innoxia (+) D. stramonium ; Petunia

parodii (+) P. parviflora ), incompatibility barriers by protoplast fusion. Experiments on

intergeneric somatic hybridisation have also been successful in some cases such as potato (+)

tomato somatic hybrids and synthesis of ‘Arabidobrassica’. With these initial successes, and

subsequent advancements in protoplast technology it is desirable that efforts be concentrated

on important plant species which have potential in industry or for food production. Crops which

have not yielded satisfactory results through conventional methods of genetic manipulation

need to be aided by non-conventional in vitro techniques such as somatic

hybridisation/cybridisation, embryo culture, etc. to manifest their full potential.

Interspecific hybrids produced through protoplast fusion	Col2
Parent species and their chromosome numbers	Chromosome number of hybrid
Brassica oleracea (2n = 18) + B. Campestris (2n = 20) B. napus (2n = 38) + B. oleracea (2a = 18) B. napus (2a = 38) + B. nigra (2n = 16) B. napus (2n = 38) + B. carinata (2n = 34) B. napus (2n = 38) + B juncea (2n = 36)	Wide variation
Nicotiana glauca (2n = 24) + N. longsdorfii (2n = 18)	56-64
N. tabacum (2n = 48) + N. alata (2n = 18)	66-71
N. tabacum (2n = 48) + N. glauca (2n = 24)	72
N. tabacum (2n = 48) + N. glutinosa (2n = 24)	50-88
N. tabacum (2n = 48) + N. Knightiana (2n = 24)	44-137
N. tabacum (2n = 48) + N. mesophile (2n = 48)	96
N. tabacum (2n = 48) + N. octophora (2n = 24)	48
N. tabacum (2n = 48) + N. rustica (2n = 48)	60-91
N. tabacum (2n = 48) + N. stocktonii (2n = 48)	96
N. tabacum (2n = 48) + N. sylvestris (2n = 24)	72
N. tabacum (2n = 48) + N. phumbaginifolia (2n = 20)	-

Petunia parodii (2n = -48) + P. hybrida (2n = 20)	44-48
P. parodii (2n = 14) + P. hybrida (2n = 14)	46
P. parodii (2n = 48) + P. parviflora (2n = 18)	31-40
Solnum tuberosun (2n = 24, 48) + S. chapcoense (2n = 14)	60
S. tuberosum (2n = 24, 48) + S. brevidens (2n = 24)	-
Lycopersicon esculentum (2n = 24) + L. Peruvianum (2n = 14)	72
Daucua carota (2n = 18) + D. capillifolius (2n = 18)	36, 38
Datura innoxia (2n = 24) + D. capillifolius (2n = 24)	46, 48, 72
D. innoxia (2n = 24) + d. sanguinea (2n = 24)	46, 72, 96
D. innoxia (2n = 24) + D. candida (2n = 24)	-

Intergeneric hybrids produced through protoplast fusion	Col2
Plant species and their chromosome numbers	New genus
Raphanus sativus (2n = 18) + B. oberacea (2n = 18)	Raphanobrassica
Moricandia arvensis (2n = 24, 28) + B. oleracea (2n = 18)	Moricandiobrassica
Eruca sativa (2n = 22) + B. napus (2n = 38)	Erucobrassica
E. sativa (2n = 22) + B. juncea (2n = 36)	Erussica
Diplotaxis muralis (2n = 42) + B. napus (2n = 38)	Diplotaxobrassica
D. muralis (2n = 42) + B. juncea (2n = 36)	Diplotaxojuncea
Sinapis alba (2n = 24) + B. napus (2n = 38)	Sinapobrassica
S. alba (2n = 24) + B. oleracea (2n = 18)	Sinapo-oleracea
Nicotiana tabacum (2n = 24) + Lycopersicon esculentum (2n = 24)	Nicotiopersicon
N. tabacum (2n = 24) + Petunia inflorata (2n = 14)	Nicotiopetunia
Solanum tuberosum (2n = 24) + Lycopersicon esculentum (2n = 24)	Solanopersicon
Daucus carota (2n = 18) + Petroselinum hortense (2n = 22)	Daucoselenium
Datura innoxia (2n = 48) + Atropa belladonna (2n = 24)	Daturotropa
Oryza sativa (2n = 24) + Echinochloa oryzicola (2n = 24)	Oryzochloa

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Intertribal somatic hybrids produced within the family Brassicaceae

Arabidopsis thaliana (Tribe Sisymbrieae)	(2n = 10) +	B. Campestris (2n = 20) (Tribe Brassiceae)	Arabidobrassica
Thlaspi perfoliatum (Tribe Lepideae)	(2n = 14) +	B. napus (2n = 38) (Tribe Brassiceae)	Thlaspobrassica
Barbarea vulgaris (Tribe Arabideae)	(2n = 16) +	B. napus (2n = 38) (Tribe Brassiceae)	Barbareobrassica

Even somatic hybrids of sexually compatible plants may exhibit new variations as a result of

interactions between plastomes donated by parental species during protoplast fusion. The

technique of cybridisation, besides transfer of male sterility, can be adopted for the introduction

of genes for resistance into the new species. The modification of plants with respect to nitrogen

fixation can also be contemplated through transformation of protoplasts by uptake of exogenous

DNA, or organelles, carrying this trait. Further, genetically heterogenous clones can be derived

from protoplast culture and fusion which display a high frequency of variations for several

agronomic traits.

The above developments suggest an immense potential for somatic cell genetics in crop

improvement. However, the genetic diversity that can be generated via somatic cell fusion is still

poorly understood. This is because only a very limited number of the synthesised somatic

hybrids or cybrids have been fertile or amphiploids. Induction and control over the degree of

species-specific chromosome elimination in wide or distant somatic hybridisation requires to be

mastered in order to understand the mechanism of producing desirable asymmetric nuclear

hybrids.

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Questions

1. The variability in chromosome number in somatic hybrids could be due to …………

a). Multiple fusions give a higher chromosome number

c). Somaclonal variation in cultured cells

2. The somatic hybridisation is used …………

b). Asymmetric hybrids

d). All the above

a). Genetic recombination in asexual or b). Overcoming barriers of sexual sterile plants incompatibility

c). Both a and b d). None of the above

3. The first somatic hybrids are produced in …………

a). Datura innoxia b). Nicotiana repanda c). N. nesophila d). N. stockonii

4. The first somatic hybrids are produced by …………

a). Schieder b). Cocking c). Schenk d). None of the above

5. …………….. was the first to resynthesise rapeseed in vitro using protoplast fusion.

a). Schieder b). Cocking c). Schenk d). None of the above

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Summary Cheat Sheet

Quick Recall Points

• Define key terms in one line and revise their use in plant biotechnology.
• Memorize major steps, methods, and applications covered in this lesson.
• Practice exam-style distinctions between related concepts and techniques.

Exam Traps

• Do not confuse similar terms without checking context and biological level.
• Revise process order carefully; sequence-based questions are common.
• Link each method with its most likely application question.

References

1 source • [1]

[1]

Standard BSc Agriculture Plant Biotechnology notes

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