8. Use of Metabolic Mutants:
A series of nitrate reductase deficient mutants have been obtained from mutegenised haploid cells of Nicotiana tabacum cultured on medium containing chlorate and with amino acids as a 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. Chlorophyll deficient mutants have also been selected from haploid cells of Datura innoxia after radiation treatment.
Similarly, metabolic mutant of Arabidopsis and proline requiring mutant of corn have been reported.
9. Using Isoenzyme Analysis:
Isoenzymes are multiple molecular forms of an enzyme with a 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 Rr value of isoenzyme are constant and specific for each parental plant species. The summation or intermediate banding pattern of Isoenzymes may be found in the hybrid callus tissue. This analysis thus help to select hybrid cells.
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. This resistant is based on the ability of rice cells to metabolize propanil.
11. Chromosome Analysis of Hybrid Cell:
Chromosome preparation from actively growing small cell colonies derived from protoplasts and their karyotype assay clearly indicate the hybridity.
Cybrids or cytoplasmic hybrids are cells or plants containing nucleus of one species but cytoplasm from both the parental species. They are produced in variable frequencies in normal protoplast fusion experiments due to one of the following:
(i) fusion of a normal protoplast of one species with an enucleate protoplast or a protoplast having an inactivated nucleus of the other species,
(ii) elimination of the nucleus of one species from a normal heterokaryon, or
(iii) gradual elimination of the chromosomes of one species from a hybrid cell during the subsequent mitotic divisions.
Various approaches to produce cybrids
- By application of lethal dosages of X or gamma radiation to one parental population.
This treatment renders the protoplasts inactive and non dividing but they serve as an efficient donor of cytoplasmic genophores when fused with recipient protoplasts.
- By treatment with iodoacetate to metabolically inactivate the protoplasts.
Fusion of iodoacetate pretreated protoplasts with non treated protoplasts will cause metabolic complementation and result in viable hybrids.
- Fusion of normal protoplasts with enucleated protoplasts.
Enucleated protoplasts can be obtained by high speed centrifugation (20,000-40,000 x g ) for 45-90 minutes in an iso-osmotic density gradient with 5-50% percoll.
- Fusion of a normal protoplast with another in which nuclear division is suppressed.