Hybridization

PROTOPLAST FUSION TECHNOLOGY IN VITRO HYBRIDIZATION

PROTOPLASTS * They are plant cells without cell wall. * They are functioning, living plant cells with only plasma membrane as outermost layer.

SOMATIC HYBRIDIZATION USING PROTOPLAST FUSION TECHNIQUE

[1] SELECTION OF EX-PLANT

[2] ISOLATION OF PLANT PROTOPLASTS A) Mechanical slicing; Yield is low

B) Enzymatic digestion By cellulase, hemicellulase and pectinase [pH 5.5.-5.8], Yield and purity of cells are high

[3] PURIFICATION OF PROTOPLASTS 1] Floatation technique 2] Centrifugation 3] Sieving * Removal of enzymes * mixe with 21% Sucrose in salt solution

4[A] VISUALISATION OF PROTOPLASTS 4[B] PROTOPLASTS VIABILITY CHECKING [A] [B]

[5] COUNTING OF PROTOPLASTS by tripple lined square counting chamber

[6] PROTOPLASTS FUSION At alkaline pH and high Ca++ content

1] Chemical agents; Salts NaCl, KCl, NaNO3, KNO3,

2] Fusogenic agents; Dextran sulphate, polyvinyl alcohol, lysilethicin, poly ethylene glycol (PEG)

3] Electrical fusion; Dielectrophoresis [1 MHz frequency]

4] Mechanical fusion Perfusion micropiptte

[7] PROTOPLAST MEDIA [+] Mannitol, Sorbitol : as a carbon source [+] Sucrose or Glucose : as a carbon source & osmotic balance [+] Maltose : to increase cell regeneration [-] Ammonium ions : reduces protoplasts survival

[8] CULTURING TRANFERED / NON TRANSFERRED PROTOPLASTS ON MEDIA

INTERPRITATION OF HYBRID PROTOPLASTS USING GEL ELECTROPHORESIS Fused protoplasts may be; 1] cytoplasmic hybrids / Cybrids (interspecific transformation) Hybrids (intergeneric transformation) 2] homokaryons or heterokaryons Colonies of transformed and non transformed protoplasts can be identified by its morphology

INTERPRITATION OF HYBRID PROTOPLASTS USING GEL ELECTROPHORESIS

TOTAL TIME CONSUMPTION FOR GETTING SOMATIC HYBRID PLANT BY PROTOPLAST FUSION TECHNIQUE

ADVANTAGES 1] Hybridization between * sexually incompatible plants * vegetative propagation favoring plants * sterile or subfertile species of plants * plants having very long life cycle

ADVANTAGES

2] To produce; * herbicide resistant plants * antibiotic resistant plants * insect resistant plants * plants with high yield of amino acids

APPLICATION Tomato

1] Hybrid plants with increased yield Brassica compestris × B. oleraceae Datura innoxia × A. belladona Nicotiana tabacum × Petunia hybrida Nicotiana tabacum × N. sylvestris Medicago sativa × M. falcata

Pomato Potato

APPLICATION 2] GENEIC TRANSFORMATION using bacterial vector; Agrobacterium tumefaciens for insect and disease resistant plants