Let us make an in-depth study of the types of procedures that can be used for the identification, selection and isolation of hybrid cells.
Hybrid Identification:
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 protoplasts of flower petal are usually vacuolated and pigmented. So the protoplast fusion products between petal-mesophyll or petal-cell culture protoplast can readily by identified.
If both types of parental protoplasts look alike, i.e. either colourless or pigmented, then the fusion products can be distinguish 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. heterodikaryotic condition, can more precisely be distinguished using carbol-fuschin staining technique because carbol-fuschin stains differently two parental nuclei.
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 an 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.
Hybrid Selection:
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.
Some important selection procedures are discussed below:
Auxin Autotrophy:
The selection of the hybrids of Nicotiana glauca and N. langsdorffi is based on auxin autotrophy of the hybrid cells (Fig 13.3). The parental protoplast or cell requires an auxin compound in order to proliferate, whereas hybrid callus tissue needs no such requirement because the cells are auxin autotrophic. Therefore, somatic hybrid cells can be isolated selectively by growth on auxin free culture medium. Auxin autotrophy of the hybrid cell is expressed only as a result of the genetic combination of the two parental protoplasts.
Use of Genetic Complementation:
Melcher and Lalib (1974) first use genetic complementation to isolate green somatic hybrids following fusion of two distinct homozygous haploid recessive albino mutants of Nicotiana tabacum. A population of albino protoplasts are fused with either a population of protoplasts isolated from a second non-allelic albino mutant or with a population of normal green mesophyll protoplasts. In this process, the parental protoplasts forms the albino colony whereas the hybrid protoplast will produce either light green or green colony. This can be usually distinguished at the cultural level.
Sometimes a single recessive albino mutation as one parental line is not always sufficient to distinguish hybrid protoplasts. So morphological markers have also been used in combination with genetic complementation.
For an example, when albino, Daucus carota protoplasts are fused with wild type of D. capillifolius protoplasts, then D. capillifolius and hybrid protoplasts are both able to regenerate green shoots which are apparently looked alike. However, origin of shoots can be traced as the morphology of the leaves of the hybrid plant are more closely resembled with D. carota leaves.
Use of Uncommon Amino Acids:
Attempts have also been made to utilize uncommon amino acids as selective agents. Conavaline which is present in some legume, inhibits division of soya bean and pea cells but sweet clover and alfalfa are unaffected. Heterokaryon obtained by the fusion of protoplast from soya bean with those from any one of the resistant plant will divide in presence of the conavaline.
Use of Cells Resistance to Amino Acid Analog:
A number of cell line resistant to amino acid analogs have been isolated and are used routinely for the selection of hybrid cells following protoplast fusion. For an example, using cell lines resistant to 5-methyl-tryptophan (5-MT) and S-2 amino ethyl-cysteine (AEC), the interspecific hybrids of Nicotiana sylvestns are selected after protoplast fusion using medium containing both amino acid analogs.
In case of Daucus, two different cell lines have been raised for the selection of hybrid cells. A non-regenerating cell line of D. carota is resistant to 5-MT and azetidine 3-carboxylate (AZC), whereas a totipotent wild type line of D. capillifolius is sensitive to 5-MT. Hybrid colonies are selected by growth on 5-MT added medium and their ability to form plant through embryogenesis.
Use of Phytotoxin:
Some of the well-known fungal toxin may be used in selecting the fusion product. For an example, the protoplast of cultured soya bean cells resistant to HmT toxin produced by Helminthosporium maydis race T, whereas the leaf protoplasts of Zea mays are sensitive to this toxin. It has been observed that fusion products of soya bean and Zea mays survive on toxin containing medium. On this it is suggested that toxin may be a useful selective agent in fusion experiment.
Use of Antibiotics:
Cell lines or strains resistant to antibiotics are easy to obtain and their usefulness is being employed in hybrid selection. For instance, the drug actinomycin D has been used in the selection of somatic hybrids of two Petunia species. Cells from fusion products of protoplasts from P. parodii and P. hybrida can give rise to the complete plant via callus formation. The cells of P. hybrid falls to grow in the presence of actinomycin D. Adjustments in the medium results preferential growth of the hybrid cells and subsequent plant regeneration, whereas P. parodii fails to regenerate plants.
Similarly, a Kanamycin resistant cell line Nicotiana Sylvester’s has been used as a genetic marker to identify the fusion products between N. Sylvester’s and N. knightiana Streptomycin resistant mutant of N. tabacum are also used to recover interspecific hybrids with N. sylvestris. Cyclohexamide resistant cell line of Daucus carota can be used as marker for the fusion with albino cell line of D. carota.
Use of Auxotrophic Mutant:
Nutritional or auxotrophic could be the most attractive material because hybrid could be selected at the cellular level and plant regeneration would not be an essential part of this selection procedure. Auxotrophic mutants has been successfully used to isolate hybrid protoplast in Spherocarpus donnelii.
Hybrids obtained by fusion of protoplasts from nicotinic acid and glucose requiring mutants are selected on minimal media. The regenerated hybrid plants are identified on the basis of morphology and karyotype. Nutritional mutants also have been used in somatic hybridization with Physcomitrella petens.
Use of Metabolic Mutant:
A series of nitrate reductase deficient mutants have been obtained from mutagenized haploid cells of Nicotiana tabacum cultured on medium containing chlorate and with amino acids as the nitrogen source. Cells with nitrate reductase convert chlorate to chlorite which is cytotoxic. The isolated mutants are unable to grow on nitrate containing medium and lack nitrate reductase and other molybdenum-protein containing enzymes. Such mutants may be suitable for hybrid selection.
Chlorophyll deficient mutants have also been selected from haploid cells of Datura in- noxia after radiation treatment. Metabolic mutants of Arabidopsis and a pro-line requiring mutant of corn have been reported. Threonine deaminase and nitrate reductase deficient mutants have been obtained using haploid plants of Nicotiana plumbaginifolia.
Using Isoenzyme Analysis:
Isoenzymes are multiple molecular forms of an enzyme with similar or identical substrate specificity occurring within the same organism. Now-a-days, isoenzyme analysis has been extensively used to verify hybridity. Isoenzymes of different constitutive enzymes exhibit the unique banding pattern or zymograms in polyacrylamide gel electrophoresis.
The number of band and Rf value of isoenzyme are constant and specific for each parental plant species. The summation or intermediate banding pattern of isoenzyme may be found in the hybrid callus tissue. This analysis thus helps to select the hybrid cells.
Use of Herbicides:
Plants possess differences in their capacity to metabolize herbicides. This property can be utilized effectively for selection. For an example, rice plants are resistant to propanil (3, 4-dichloropropionanilide). This resistance is based on the ability of rice cells to metabolize propanil.
Chromosome Analysis of Hybrid Cell:
Chromosome preparation from actively growing small cell colonies derived from protoplasts and their karyotype assay clearly indicate the hybridity.
Hybrid Isolation:
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 hybridization. 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:
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 for a lot of time and efforts.
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 KCl, 0.245 M MgCl2, 0.254 M CaCl2 pH 6.0, 660 ± 20 mOs/kg H2O) and is placed on the top of iso-osmotic KMC/sucrose- density gradients.
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.
Post Fusion Events:
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. Few fused protoplasts may be homokaryons which result from the fusion of similar parental protoplasts, but are of little significance in somatic hybridization.
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 13.4).
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 heterodikaryotic 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 parent (Fig 13.5).
The hybrid or cybrid protoplasts regenerate a wall around them and enter the mitotic cycle. Since diploid protoplasts are generally used for somatic hybridization, tetraploid somatic hybrid should be expected. But particularly at the wide cross level (inter-generic, interspecific), such tetraploid cell is considered as amphidiploid.
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 parents 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.