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In this article we will discuss about Hybrid Selection:- 1. Hybrid Identification of Plant Protoplast 2. Hybrid Selection of Plant Protoplast 3. Hybrid Isolation 4. Post-Fusion Events 5. Importance of Protoplast Fusion and Somatic Hybridisation.
Contents:
- Hybrid Identification of Plant Protoplast
- Hybrid Selection of Plant Protoplast
- Hybrid Isolation of Plant Protoplast
- Post-Fusion Events of Plant Protoplast
- Importance of Protoplast Fusion and Somatic Hybridisation
1. Hybrid Identification of Plant Protoplast:
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Following fusion of protoplasts, identification of protoplast fusion product is necessary to quantitate fusion frequency and to monitor the fusion products. The fusion frequency may vary due to either protoplast quantity or fusion conditions.
The preliminary identification of fusion product is done under microscope. The microscopic identification is based on differences between the parental cells with respect to pigmentation, presence of chloroplast, nuclear staining, cytoplasmic marker etc.
A system that has been used successfully consists of fusing protoplasts of leaf mesophyll cell containing chloroplasts with those from cell cultures lacking chloroplasts. At the initial stage, the fusion products at the light microscope level are seen to contain chloroplast in one half and colourless starch granules in other half.
As a result, the fused cell can easily be distinguished from un-fused parental protoplasts. Similarly, the protoplast of flower petal is usually vacuolated and pigmented. So, the protoplast fusion products between petal-mesophyll or petal-cell culture protoplast can readily be identified.
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If both types of parental protoplasts look alike, i.e., either colourless or pigmented, then the fusion products can be distinguished using nuclear staining technique. A hybrid cell contains two nuclei of two different parental protoplasts. Such dikaryotic cells can be identified using conventional aceto-orcein or aceto-carmine straining procedure.
But the presence of two different parental nuclei in the hybrid cell, i.e., hetero-dikaryotic condition, can more precisely be distinguished using carbol-fuschin staining technique because carbol-fuschin stains two parental nuclei differently.
Non-toxic fluorochromes are often used for the identification of heterokaryon or fusion products. For example, fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate (RITC) or rhodamine B are used as fluorochromes.
The advantage of using fluorochromes for the identification of fusion products is that it does not depend upon the types of protoplast being used. For example, in a fusion experiment, heterokaryons between FITC labelled suspension cell protoplast and un-labelled mesophyll protoplasts exhibit an apple-green fluorescence of FITC and a red fluorescence from chlorophyll of the mesophyll partner.
2. Hybrid Selection of Plant Protoplast:
In the mixture of both fused and un-fused protoplasts, the latter usually predominate. So, after plating these mixed protoplasts in the solid medium, it is very difficult to identify the hybrid cells microscopically. On the other hand, in most fusion experiments, the division rate of fused protoplast is relatively low.
At the same time, one or both un-fused parental protoplast may also divide and very shortly the hybrid protoplast can no longer be distinguished from parental cells. So some types of selection technique are required at the level of culture to recover hybrid cells and its callus tissue following fusion.
Since the cultural behaviour of protoplasts and their nutrient and hormone requirements may vary from plant to plant, several selection procedures have been developed.
3. Hybrid Isolation of Plant Protoplast:
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Several selection methods, as described above, are not applicable for the selection of all types of fusion products at the cultural level. Sometimes selection is so specific for a particular inter-generic somatic hybridisation. Various mutant cell lines are often used in some selection methods. But such methods are limited by the fact that mutant cell lines are not easy available in plants.
Again, it has been observed that, in fusion product, chromosome elimination may occur from the fused products. Therefore, use of mutant or genetic complementation may fail in attempts for the selection of hybrid produced from widely divergent sexually incompatible genera.
To overcome the limitation of selection methods, recently specifically fusion products following fusion are isolated physically before culturing them in either solid or liquid medium.
Hybrid isolation methods are given below:
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(i) Micropipette Technique:
Kao (1977) first developed this technique. By this technique, heterokaryons are isolated from the fusogen treated protoplast suspension, under a microscope using micropipette. But very few heterokaryons are obtained by a lot of time and efforts.
(ii) Density Gradient Fractionation of Protoplast Suspension after Fusion:
Harms and Potrykus (1978) used this technique to isolate heterokaryons from protoplasts on a large scale. Protoplast suspension after fusion is suspended in KMC solution (equal volume of 0.35M KC1, 0.245 M MgCl2, 0.254 MCaCl2 pH 6.0, 660 ± 20 mOs/kg H20) and is placed on the top of iso-osmotic KMC/sucrose-density gradients.
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Gradients are centrifuged at 20°C for 5 minutes at 50-100g. The fused protoplasts will form a band in intermediate density position. Heterokaryons are carefully pipetted off using Pasteur pipettes and are examined under the microscope to determine the number. Finally, the heterokaryons are washed once with liquid culture medium before plating.
4. Post-Fusion Events of Plant Protoplast:
Following membrane fusion, cytoplasm and its organelles of both parental protoplasts are intermixed with each other and such mixing forms a heteroplasmic cytoplasm. It offers an opportunity of obtaining heterozygosity of extra chromosomal material. This fusion differs from a zygote in that there is no strict maternal inheritance of cytoplasmic organelles.
In fused protoplast, normally, a dikaryotic condition is established. It means that the ratio of 1 : 1 nucleus of each species occurs most frequently in heteroplasmic cytoplasm. Two types of dikaryotic condition may be observed.
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Few fused protoplasts may be homokaryons which result from the fusion of similar parental protoplasts, but are of little significance in somatic hybridisation. Others are heterokaryons which are formed by the fusion of dissimilar parental protoplasts.
Thus, the protoplast population in culture is composed of a mixture of un-fused parental protoplasts and fused homokaryotic and heterokaryotic protoplasts. Sometimes more than two protoplasts are involved in fusion and produces multinucleated giant cells incapable of mitosis and subsequent development (Fig. 6.20).
Heterokaryon can produce either hybrid or cybrid cells. Only nuclear fusion takes place in case of hybrid cells. This event can be detected one day after fusion and requires several hours to complete. Nuclear fusion possibly occurs through the formation of nuclear membrane bridges. Nuclear fusion forms a synkaryon which contains a mixed chromatin.
Sometimes, nuclei of the hetero-dikaryotic condition do not fuse to form a synkaryon and one nuclei of any one parent may be eliminated in the subsequent developmental stages. Thus, a cybrid cell is produced with the nuclear genome of any one partner and the cytoplasm of both parents (Fig. 6.21).
The hybrid or cybrid protoplasts regenerate a wall around them and enter the mitotic cycle. Since diploid protoplasts are generally used for somatic hybridisation, tetraploid somatic hybrid should be expected. But, particularly at the wide cross level (inter-generic, interspecific), such tetraploid cell is considered as amphidiploid.
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In wide crosses, a few or several of chromosomes of one parent may be eliminated during segregation. It has been found that in hybrid cell between Glycine max and Nicotiana glauca where most of the larger chromosomes of N. glauca are eliminated.
In certain crosses some chromosomes of both parent are eliminated as in hybrid between Arabidopsis thaliana and Brassica campestris. Difference in mitotic cycle times in species which are not compatible sexually may result in such chromosome elimination.
A hybrid or cybrid cell undergoes mitotic divisions and, ultimately, forms callus tissue. Complete hybrid or cybrid plants can be regenerated from such callus tissue. But plant regeneration has, to date, been achieved successfully to only a small number of plant species and is mainly confined to some interspecific sexually compatible species.
In most of the other cases, particularly sexually incompatible species, reports of plant regeneration is very limited.
5. Importance of Protoplast Fusion and Somatic Hybridisation:
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Protoplast fusion and somatic hybridisation have opened up a new avenue in plant science. It is now a well-known fact that the somatic hybridisation in plants can be used in the improvement of plants. One of these is the production of hybrids which is not possible through normal sexual fusion or fertilisation process.
In other words, it includes the formation of somatic hybrids between two species which are sexually incompatible. Thus, protoplast fusion provides a method of combining the different genomes of different genera and species, with the potential of overcoming sexual incompatibility barrier between plants. A brief list of somatic hybrid plants raised through protoplast fusion is given below.
(a) Interspecific Hybridisation:
(i) Sexually Compatible Combination:
Daucus carota + D. capillifolius, Nicotiana glauca + N. langsdorffii, N. tabacum + N. alata, Petunia parodii + P. hybrida, Solanum tuberosum + S. chacoense.
(ii) Sexually Incompatible Combination:
Datura innoxia + D. Candida Nicotiana, sylvestris + N. knightiana, N. tabacum + N. nesophila, Petunia parodii + P. parviflora.
(b) Inter-Generic Hybridisation:
Arabidopsis thaliana + Brassica campestris, Daucus carota + Aegopodium podagaria, Solanum tuberosum + Lycopersicon esculentum.
The cytoplasmic mix obtained from protoplast fusions is novel with the opportunity for the production of cybrids coupled with the opportunity for the formation of mitochondrial recombinants. Mitochondria can segregate or recombine their DNAs to form a new type of mitochondria.
Chloroplasts segregate but do not appear to undergo recombination. Thus, with the production of hybrid or cybrid, the mixing of cytoplasm of both parental protoplast can improve the extra nuclear genetic elements. In sexual hybridisation, only maternal cytoplasm—i.e., the cytoplasm of egg cell take part in the formation of hybrid (Fig. 6.22).
Cybrids are generally produced due to elimination of total genome of one parent after the fusion of two protoplasts.
If one parental nucleus completely disappears, the cytoplasm of the two parental protoplasts are still hybridized and the fusion product is known as cybrid or cytoplasmic hybrid or heteroplast. But the use of a certain compound like cytochalasin has been found to completely extrude the nucleus from the protoplast—thus producing enucleate protoplasts.
The fusion of the enucleate protoplast with nucleate protoplast may lead to the production of male sterile somatic cybrid where male sterility is present in the cytoplasm.
In the experiment, cybrid may also arise by the following ways:
1. Fusion between a normal nucleate protoplast and a protoplast containing a nonviable nucleus;
2. Elimination of one of the nuclei after heterokaryon formation;
3. Selective elimination of chromosomes at the later stage.
The formation of cybrid has some application in plant improvement programme. The importance of cybridity has been confirmed by breeding experiments. The transfer of cytoplasmic male sterile cytoplasm by protoplast fusion to somatic hybrid should be of interest to the plant breeders. This may be of critical importance in male sterility based hybrid seed production.
Chromosome elimination in fusion product can be used for gene mapping as in fusion products of animal cells. Studies of fusion product can give information about compatibility or incompatibility of the nuclei or cytoplasm.
It has in recent years been repeatedly emphasised that plant tissue culture, per se, appears to be an unexpectedly rich source of genetic variation; this has stimulated effort to find out whether such genetic variation can be enhanced by protoplast cloning. This opportunity will undoubtedly lead to the production of new genetic variation.
By protoplast fusion, it is possible to transfer some useful genes such as disease resistance, nitrogen fixation, rapid growth rate, protein quality, frost hardiness, drought resistance etc. from one species to another and thereby widen the genetic base for plant breeding.
Hybrid vigour is well-known in sexual hybridisation and it has been suggested that somatic hybridisation may produce even greater vigour in hybrids. A critical evaluation of this suggestion is required since it could result in enhanced yields in many crops.
In case of vegetatively reproducing plants, the genetic variation can be induced through protoplast fusion of two species, varieties or two different genera. In case of sugarcane, which is vegetative, the production of somatic hybrids between different varieties followed by regeneration of whole plants can produce improved varieties which may be highly beneficial to the sugarcane industry.
Similar advantage may be obtained in potato and other horticultural plants for their improvement.
