Hybrid Cells: Identification, Selection and Isolation| Plant Tissue

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 R_f 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.