NaNO3 Treatment, Successfully Utilized for Fusion, Protoplasts from Root tips, Maize Seedlings, Frequency of Fusion, Inverted Microscrope

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NaNO3 treatment

This method was successfully utilized fro fusion of protoplasts from root tips of oat and maize seedlings but is not preferred due to low frequency of fusion, particularly when highly vacuolated mesophyll protoplasts are used.

The method involves the following steps.

Procedure of NaNO3 treatment for production of interspecific somatic hybrids in the genus Nicotiana

1. Nicotiana Longsdorfii	2. Nicotiana Glauca
3. Leaf meosphyll cells treated with enzymes to digest away the cell walls	4. Protoplasts are mixed and suspended in NaNO3 to cause cell fusion
5. Protoplast suspensions plated onagar medium with no auxin or cytokinin; only fused hybrid cells grow	6.Hybrid cells differentiate to form shoots
7. Shoots grafted onto a parent plant; the hybrid scion matures to produce fertile flowers and seeds	8. Seeds germinate to produce seedings similar to sexually produced amphidiploid

(i) Isolated protoplasts are suspended in an aggregation mixture (5.5% sodium nitrate in 10% sucrose solution). This mixture works as a fusion inducing mixture and causes fusion on incubation (water bath maintained at 35oC).

In order to obtain a higher frequency of fused protoplasts, the mixture may be centrifuged and the pellet resuspended and incubated for one or more additional cycles.

(ii) The fusion inducing mixture is replaced by a liquid culture medium and the protoplasts in this mixture are incubated again; the cycle may be repeated once or twice before plating the protoplasts on a solid medium. The fusion of protoplasts may be monitored at different steps through examination under an inverted microscope.

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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