CS-9-6 - Modifying the AEG1 Promoter from Cotton to Make It Tapetum-specific: A Step Towards Developing Male Sterile and Restorer Lines for Hybrid Seed Production in Cotton

Tapetum specific promoter is a key component for development of male sterile and restorer lines using barnase/barstar gene, for the production of hybrid seeds. In an earlier work, a gene, AEG1, expressing in the anther tissue was identified in cotton. The 1.5kb URM (Upstream Regulatory Module) of the AEG1 gene was observed to be active mainly in the tapetum tissue of anther of cotton with weak activity in roots. The present works report the development of a modified AEG1 URM which specifically express in the tapetum tissue. Two promoters were developed (i) AEG1ΔB wherein the -230bp to -500bp of the promoter (that carries several root specific cis-elements, ROOTMOTIFAPOX1) was deleted and (ii) AEG1ΔBMut where the root motifs in proximal region was mutated in the background of AEG1ΔB. Activity of the modified URMs was compared with that of wild type AEG1 URM in tobacco transgenics using β-glucuronidase as a reporter gene. Unlike cotton, in transgenic tobacco, wild type AEG1 URM was observed to be active in pollen instead of tapetum. Weak activity was also observed in roots. In case of modified URMs, AEG1ΔB and AEG1ΔBMut, activity was reduced and abolished respectively while retaining their activity in pollen. Further, transgenics in tobacco expressing the barnase gene under the wild type AEG1 URM and AEG1ΔBMut URM were developed. While no viable transgenics could be developed with barnase gene expressing under the control of wild type AEG1 URM, male sterile lines were produced when barnase gene was under the control of AEG1ΔBMut URM. The male sterile lines set seed on cross pollination with untransformed tobacco plants. Thus AEG1ΔBMut URM can possibly be used to develop male sterile lines in case of cotton.

Coauthors: Amita mehrotra – University of Delhi South Campus;PRADEEP BURMA – University of Delhi South Campus