Abstract
Wild crop relatives are important source of genetic diversity. Plant tissue culture provides proficienct means of conservation of endangered wild plants sustaining biodiversity, improving plant traits by virtue of induced somaclonal variation and trangensis. The objective of study was to establish direct in vitro regeneration and biolistic transformation protocol for wild tobacco N. plumbaginifolia using uidA gene. For direct in vitro regeneration, six combinations of IAA and kinetin were used. Then, biolistic transformation was carried out using BioRad PDS 1 000 (He) gun and 1 100-psi rupture discs. Transformation was confirmed through histochemical gus assay. A proficient and reproducible in vitro regeneration system was standardized for wild tobacco named as Nicotiana plumbaginifolia . In present study 1 mg/L kinetin in combination with 0.25 mg/L IAA proved best for direct in vitro shoot regeneration from mature leaf-based explants. Success in direct in vitro regeneration circumvented the callogensis step, accelerating the process of in vitro regeneration and increasing the chances of obtaining true to type plants. Individual shoots were placed on MS medium with/without IBA for rooting. Individual shoots were successfully rooted on MS medium without IBA. Then, explants were transformed with uidA gene using gene gun. Transient assay of bombarded calli showed gus activity on histochemical assay even after a week. Analysis of gus expression has been found as a principal method of plant transformation optimizations.
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