Read this article to learn about the identification and selection of hybrid cells in plants.
For this purpose several methods are available which depends on (a) Physical properties of fused cells (b) Biological properties of fused cells and (c) Biological properties of colonies derived from fused cells.
Following fusion treatment, the protoplasts in liquid culture medium regenerate cell walls and undergo mitosis resulting in a mixed population of parental cells, homokaryotic fusion products and heterokaryotic fusion products or hybrids.
Hybrid cells must be distinguished from the other cells present. Heterokaryotic recognition techniques as well as colonies selection methods are thus highly desirable. For this purpose, several methods are available.
These are dependent on:
a. Physical properties of fused cells.
b. Biological properties of fused cells.
c. Biological properties of colonies derived from fused cells.
Following methods have been adopted for various types of protoplasts.
(I) Fusion partners such as non-green protoplasts (albino protoplasts from tissue culture) and leaf derived chloroplast protoplast allow microscopic visual detection of heterokaryotes.
A few hours or a few days after fusion products are pipetted and cultivated individually. Because visual techniques applied are tedious, other non- visual methods have been sought.
(i) By centrifugation in density gradient, enriched bands of fusion products can be recovered at an intermediate density.
(ii) Vital staining, specially using fluorescent dyes is another possibility to detect heterokaryotes providing a means of automatic separation with cell sorter.
(iii) Controlled fusion by electric field should provide a possible way to cultivate only true heterokaryotes.
(iv) A method adapted from animal cells to plant protoplasts allows the recovery of fusion products by complementation between inactivated protoplasts by different metabolic inhibitors. Cybrids as well as hybrids selection in vitro should be even more effective by use of complementary markers selectable in vitro.
(v) One of the principal features to characterize somatic hybrid is the chromosome complement. Chromosome number and morphology may be compared to the patterns displayed by the fusion parents to determine whether or not presumptive hybrids are true hybrids.
(vi) Supportive evidences for hybridity can be obtained from an electron microscopic morphometric determination of chromatic texture in hybrid nuclei. This technique was used for the verification of Arabidopsis-Brassica somatic hybrids and it may prove useful for identifying other inter-specific and inter-generic hybrids.
(vii) Morphological characters of the hybrid plants and parents are used to ascertain the hybrid nature of the regenerated hybrids.
(viii) Isoenzymes-proteins ultimately manifest the diversity laid down in the genetic programme of each individual. Isoenzymes (multiple molecular forms of an enzyme exhibiting identical and similar catalytic properties) are frequently used for this purpose. Isoenzymes used for hybrid identification include esterase, peroxidase, amylases, phosphatase, alcohol dehydrogenase, lactate dehydrogenase, aspartate and aminotransferase etc.
(ix) More recent techniques are – RFLP (Restriction Fragment Length Polymorphism), use of reporter gene and RAPD (Random Amplified Polymorphic DNA) for looking into genetic variability and transformations in the plants regenerated after hybridization or DNA uptake studies.