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Plant regeneration through callus-derived protoplasts of Phellodendron amurense Rupr.

Mustafa Abul KalamAzad

A method for the isolation, culture of the callus-derived protoplasts, and plantlet regeneration in Phellodendron amurense is described. In this study, the highest 92.0%viabilitywith the yield of 2.4  105 protoplast / g fresh weightwas obtained fromcallus, when the materials were treated for 6 hrs with Cellulase Onozuka R-10 plus Driselase. Sustained cell division and colony formation fromthe protoplasts were best supported by a 0.2% Gellan gumsolidified or liquid medium containing 2.0 MBAP with 4.0 M NAA, IBA or 2,4-D, where the plating density was 4  105 to 6  105 protoplasts/ml. The highest frequency (0.33  0.02%) of colony formation was observed, when the protoplasts were cultured on MS medium supplemented with 2.0 MBAP and 4.0 MNAA. The protoplast-derived colonies formed green compact calli on 0.2% Gellan gum solidified MS medium containing 2.0 MBAPwith 4.0 MNAAor IBA.Adventitious shoots from protoplast-derived callus were induced on MS medium supplemented with 2.0 MBAP and 1.0 MNAA or 2.5 MIBA. Shoot multiplication and elongation occurred onMSmediumcontaining 1.0 M BAP. The highest 90.0% of in vitro grown shoots were rooted on MS mediumcontaining 2.0 MIBA. Plantlets were transferred to the Kanuma soil and successfully established under greenhouse condition.