Selection of Hybrid Cells

Selection of Hybrid Cells. In the past few blogs we had gone through an introduction to what is protoplast in detail, and also studied how the isolation of a protoplast takes place with all the methods. We also understood how to check the viability of protoplast along with their culturing in Protoplast Culture: Isolation. Then we discussed one of the applications of protoplast isolation which is somatic hybridization. In that blog, we saw how somatic hybrids are made or we can also say how the protoplast of two different species is fused together to form a heterokaryon. 

In this blog we are going to resume our journey where we left in the previous blogs ie. we are going to cover up the topic of-

a) Selection of Hybrid cells - In this, we are going to distinguish between a homokaryon and heterokaryon (somatic hybrid) cell in order to further grow that hybrid cell in to a hybrid plant.

b) Selection of Hybrid plant - In here we are going to learn how to distinguish a hybrid plant from a normal conventional plant ie. we are going to learn how a hybrid plant has so many different features to it which we do not see in a naturally grown plant.

Selection of hybrid cells

Selection of hybrid cells

Selection of Hybrid Cells is the process of selection of hybrid cells after the protoplast fusion step, cause after the fusion step, the protoplast hybrids that we get might consist of heterokaryons or, homokaryons.

Therefore, it is necessary to select the hybrid cells. The most used methods employed for the selection of hybrid cells are 

1) Biochemical Methods.

2) Visual Methods.

3) Cytometric Methods.

1) Biochemical Methods= The selection of hybrid cells by biochemical methods are based on the use of biochemical compounds in the medium (selection medium). These compounds help to distinguish the hybrid cells from parental cells.

   a) Drug sensitivity =This method is helpful in the selection of hybrids of two plant species if one of them is sensitive to a drug. Protoplasts of Petunia hybrida (species A) can form macroscopic callus on MS(Murashige and Skoog) medium but are sensitive to (inhibited by) actinomycin D. Petunia parodii protoplasts (species B) form small colonies, but are resistant to drug actinomycin D. When these two species are fused, the fused protoplasts derived, shows both the characters - formation of macroscopic colonies and resistance to actinomycin D on MS medium. This helps in the selection of hybrids, the parental protoplasts of both species fail to grow as protoplasts of Petunia parodii form very small colonies while that of P. hybrida is inhibited by actinomycin D.

   b) Auxotrophic mutants = Auxotrophs are those mutants that cannot grow on a minimal medium and therefore require specific compounds to be added to the medium. Nitrate reductase deficient mutants of tobacco (N. tabacum) is known. The parental protoplasts of such species can not grow with nitrate as the sole source of nitrogen while the hybrids can grow. Two species of nitrate reductase deficiency one due to lack of apoenzyme [nia-type mutant] and the other due to lack of molybdenum cofactor [cnx- type mutant] were taken. The parental protoplasts can't grow on a nitrate medium while the hybrid protoplasts can grow. The auxotrophic mutants selection is possible only if the hybrid cells can grow on a minimal medium

 

2) Visual methods= The selection of hybrid cells via the visual method, although tedious is very efficient. In some of the somatic hybridization experiments, chloroplast lacking [albino or non-green] protoplasts of one parent are fused with green protoplasts of another parent. This helps in the visual identification of heterokaryons under a light microscope. The heterokaryons are bigger and green in colour while the parental protoplasts are either small or colorless.

3) Cytometric methods =Some workers use flow cytometric technique and fluorescent-activated cell sorting techniques for the analysis of plant protoplast while their viability is maintained. The same techniques can also be applied to the sorting of heterokaryons. The hybrid cells derived from such selections have proved useful for the development of somatic hybrid plants.

 Identification of hybrid plants

The development of hybrid cells followed by the development of hybrid plants requires clear proof of genetic contribution from both the parental protoplasts. The hybridity must be established only from euploid and not from aneuploid hybrid plants. Some of the commonly used methods for the identification of hybrid plants are:-

a) Morphology of hybrid plants = Morphological features of hybrid plants usually are intermediate between two parents can be identified. For this, the vegetative and floral characters are considered. These includes leaf shape, leaf area, root morphology, flower shape, its structure, size and color, and seed capsule morphology. Some somatic hybrids such as pomatoes and topatoes which are the fused products of potato and tomato show abnormal morphology, and thus can be identified.

b) Chromosomal constitution= The number of chromosomes present in the hybrid cells can be directly counted as this provides information on the ploidy state of the cells. The somatic hybrids are expected to have chromosomes that are equal to the total number of chromosomes originally present in the parental protoplasts. Sometimes, the hybrids are found to have more chromosomes than the total of both parents. The occurrence of chromosomal markers is greatly useful for the genetic analysis of hybrid cells.

c) Molecular techniques= Many recent developments in the molecular biology field have improved the understanding of the genetic constitution of somatic plant hybrids. Some of them are listed down.

 l. Differences in the restriction patterns of chloroplast and mitochondrial DNAs.

 2. Molecular markers such as RFLP ( Restriction Fragment Length Polymorphism ), AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA ), and microsatellites are used.

 3. PCR technology.

Conclusion

Based on your prior knowledge we have completed somatic hybridization in the previous blog, if you have studied or read that blog you will know that we studied protoplast fusion in it with different fusion techniques. This blog is the continuation of that blog, cause after we developed hybrids we need to differentiate between homokaryons and heterokaryons (hybrids), so the first thing, we studied in this blog was the selection of the hybrid cells from a whole jungle of homokaryons and heterokaryons with the help of biochemical method, visual method, and auxotrophic methods, we differentiated with the help of callus forming ability of a cell in all these methods, then after we got our hybrid callus we did its tissue culture with the micropropagation steps and made it to grow in to a hybrid plant, then we studied the selection of hybrid plant. In this we discussed how a hybrid plant is distinguished from a normally naturally grown plant with different techniques. 

Reference

Biotechnology by U Satyanarayana