The following points highlight the ten important procedures of hybrid selection. The procedures are: 1. Auxin Autotrophy 2. Use of Genetic Complementation 3. Use of Uncommon Amino Acids 4. Use of Cells Resistance to Amino Analog 5. Use of Phytotoxin 6. Use of Antibiotics 7. Use of Auxotrophic Mutant 8. Use of Metabolic Mutant 9. Using Isoenzyme Analysis 10. Use of Herbicides.
Procedure # 1.
Auxin Autotrophy:
The selection of the hybrids of Nicotiana glauca and N. langsdorffi is based on auxin autotrophy of the hybrid cells (Fig. 6.19). 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.
Procedure # 2.
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 form the albino colony whereas the hybrid protoplasts 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 apparently look 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.
Procedure # 3.
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.
Procedure # 4.
Use of Cells Resistance to Amino 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 example, using cell lines resistant to 5-methyl-tryptophan (5-MT) and S-2 amino ethyl-cysteine (AEC), the interspecific hybrids of Nicotiana sylvestris 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 Azatidine 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.
Procedure # 5.
Use of Phytotoxin:
Some of the well-known fungal toxins may be used in selecting the fusion product. For example, the protoplast of cultured soya bean cells resistant to HmT toxin are 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 a toxin may be a useful selective agent in fusion experiment.
Procedure # 6.
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. hybrida fails 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 sylvestris has been used as a genetic marker to identify the fusion products between N. sylvestris 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.
Procedure # 7.
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 hybridisation with Physcomitrella petens.
Procedure # 8.
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 innoxia after radiation treatment.
Metabolic mutants of Arabidopsis and a proline requiring mutant of corn have been reported. Threonine deaminase and nitrate reductase deficient mutants have been obtained using haploid plants of Nicotiana plumbaginifolia.
Procedure # 9.
Using Isoenzyme Analysis:
Isoenzymes are multiple molecular forms of an enzyme with similar or identical substrate specificity occurring within the same organism. Nowadays, isoenzyme analysis has been extensively used to verify hybridity.
Isoenzymes of different constitutive enzymes exhibit the unique banding pattern or zymograms in poly-acrylamide 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.
Procedure # 10.
Use of Herbicides:
Plants possess differences in their capacity to metabolize herbicides. This property can be utilised effectively for selection. For example, rice plants are resistant to propanil (3, 4-dichloropropionanilide). This resistance is based on the ability of rice cells to metabolize propanil.
Chromosome preparation from actively growing small cell colonies derived from protoplasts and their karyotype assay clearly indicate the hybridity.