Selection of Fused Protoplasts, Protoplasts Population Consists, Homokaryons and Heterokaryons, Hybrids of Petunia Parodii, Hybrida

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Protoplast Culture Regeneration and Somatic Hybridization Protoplast Culture Regeneration and Somatic Hybridization - Introduction Isolation of Proplasts Mechanical Method Enzymatic Method Direct One Step Method Sequential Two Step Method Purification of Isolated Protoplasts Sedimentation and Washing Flotation Viability and Plating Density of Protoplasts Testing Protoplast Viability Minimum Plating Density MPD and Culturing Individual Protaplasts Protaplast Culture and Regeneration of Plants Isolation of Subprotoplasts Protoplast Fusion and Somatic Hybridization Techniques of Protoplast Fusion

Home >> Plant Biotechnology and Genomics >> Protoplast Culture Regeneration and Somatic Hybridization >> Selection of Fused Protoplasts

Selection of fused protoplasts

After fusion treatment as above, the protoplast population consists of a mixture of parental types, homokaryons and heterokaryons, of which heterokaryons (potential source of future hybrids) often make only 0.5%-10%.

Different Possible Products obtained due to fusing of two parental types (A and B) of protoplasts

Different Possible Products

Different Possible Products

Efforts have been made to select the heterokaryons or the calli derived from them. Following methods have been used for selection:
(i) The hybrids Nicotiana glauca ´ N. longsdorfii could grow on a medium, on which none of the parents could grow.

Auxin Independent Growth of Hybrids of Nicotiana Glauca and N. Longsdorffii

Auxin Independent Grwoth of hybrids of Nicotiana Glauca and N. Longsdorffii the two parental lines can not produce auxin and thus do not grow on auxin-free medium; the hybrid cells produce auxin and are thus able to grow and forms callus

(ii) Hybrids of Petunia parodii (green) with P. hybrid, P. inflorata or P. parviflora (albino), could be selected in the form of green callus, which represented only hybrid cells; similar approaches were also followed in hybrids of Datura innoxia (albino) with other species of Datura or with Atropa belladonna (wild type = green).

(iii) The protoplasts of two parents may be labelled by different fluorescent agents, which will then enable the selection of hybrids, as done in the genus Nicotiana.

Simplifeid diagram of the principle of a fluorescently activated cell sorter

1. Dropiet charging signal	6. Flow Cell	11. Aperture	16. Filter
2. Sample Introduction Port	7.Droplet deflection assembly	12. Dichroic Mirror	17. Laser
3. Sheat fluid line	8. Waste	13. Photomultiplier (i)	18. Light
4. laser light	9. microtiter plate (automatically indexed)	14. filter	19. Flow cell
5. Lens	10. lens	15. Photomultiplier (ii)	20.Droplet Formation

(iv) The selection of somatic hybrids can also be exercised using magnetic microbeads. In this technique, the protoplasts of one fusion partner (say, A), carrying no resistance to antibiotic, kanamycin Km-) are labelled with selected biotinylated lectins.

The protoplasts of other parent (say, B) are derived from antibiotic resistant source (e.g. Km+ = resistant to kanamycin), due to the presence of selectable marker neomycin phosphotransferase (NPTII).

After electrofusion, the mixture is labelled with magnetic microbeads coupled to Streptavidin, which facilitates labelling of only those protoplasts which were already labelled with biotin, so that AB, A and AA types will be labelled and B and BB will remain unlabelled.

The mixture thus labelled with microbeads is sorted by magnetic ell sorter (MACS), so that the magnetic fraction will contain only those protoplasts, which carry magnetic microbeads, i.e A (Km-), AA (Km-) and AB (Km+).

This magnetic fraction is then incubated on kanamycin containing medium, where only AB (Km+) will grow into calli, which will give hybrid plants on regeneration.

The frequency of hybrid plants, obtained by this method, could be as high a 82 per cent as against 28 per cent by other methods.

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Induced Fusion NaNo₃-treatment Treatment with Calcium Ions ca⁺⁺ at High pH Polyethylene Glycol PEG Treatment Electrical Fusion Selection of Fused Protaplasts Chromosome Status of Somatic Hybrids Interspecific Hybrids Produced Through Protoplast Fusion Intergeneric Hybrids Produced Through Protaplast Fusion Use of Protoplast Fusion and Somatic Hybrids Somatic Hybrids for Gene Transfer Asymmetry in Somatic Hybrids for Gene Transfer Somatic Hybrids for Cytoplasmic Male Sterility Cytoplasmic Hybrids or Cybrids Genetic Modification Transformation of Protoplasts

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