Efficient Regeneration of Tobacco (Nicotiana tabacum L.) Plantlets from Cotyledon, Hypocotyl and Leaf Explants: An Excellent Model Plant for Gene Function Analysis

Abstract

Tobacco has been widely used as a model plant for stable and non-stable gene function analysis. Successful Agrobacterium-mediated transformation mainly depends on in vitro regeneration of tobacco plant. However, a reliable and standard regeneration protocol of tobacco using multiple explants is limited. In this study, we established a reliable and reproducible regeneration protocol of tobacco using three different explants i.e. cotyledon, hypocotyl and leaf. Preliminary, surface sterilized tobacco seeds were germinated on growth regulator free MS medium. Thereafter, in vitro germinated explants were inoculated into Murashige and Skoog [1] media supplemented with different combination and types of growth regulators for callus induction and subsequent regeneration of plantlets. It was revealed that, regeneration ability of explants is greatly influenced by type and nature of the explant. Among the three explants, higher callus induction (95%) was obtained in MS medium supplemented with 2.0 mg l-1 kinetin + 2.0 mg l-1 IAA from leaf explant. Also, leaf explant exhibited much higher regeneration ability (95%) than hypocotyl (60%) and cotyledon (45%) explants. Significantly highest number of shoots (8.0) were regenerated from leaf explants cultured on MS medium supplemented with 3.0 mg l-1 Kinetin+1.0 mg l-1 IAA compared to the other hormone combinations. Regenerated mature shoots were showed normal root after transferred onto ½ MS medium containing 0.3 mg l-1 IBA. This study will provide valuable information related to in vitro regeneration of tobacco plantlets using cotyledon, hypocotyl and leaf explants and will be used as a standard protocol for Agrobacterium-mediated transformation for gene function analysis.