The present investigation was carried out with the aim of development plant transformation vectors suitable for indication of male sterility in crop plants. This research intended to clone a mitochondrial, male sterility inducing gene orfH522 from PET1 CMS line of sunflower under a tapetum specific promoter, TA29 promoter from tobacco in frame with a mitochondrial target peptide coxIV pre-sequence from yeast, so that ORGH522 would be targeted into mitochondria in the target plants, evethough the protein is synthesized as a nuclear encoded protein in the transgenic plants. nos terminator from Agrobacterium was proposed to be used as termination signal in the gene cassette. As appropriate controls gene cassettes were developed where orfH522 was cloned under a constitutive promoter, CaMV 35S in frame with or without mitochondrial target sequence so that the effect of expressing ORFH522 constitutively could be determined in comparison to tapetum specific expression.

The development of the proposed gene cassettes was carried out using a three step strategy. In the first step, the component sequences TA29 promoter from tobacco, coxIV pre-sequence from yeast, orfH522 from sunflower and terminator sequence from Agrobacterium were amplified from their respective sources using appropriate PCR techniques and cloned in T/A cloning vector. The authenticity of the protein encoding sequences (coxIV pre-sequence and orfH522) was confirmed by sequencing.

In the second step, these sequences were cloned in plasmid vector pUC18 in appropriate/correct orientation by following serial directional cloning using the restriction enzymes sites incorporated in forward and reverse primers employed for amplifying each for the component sequences in the first step. The orientation of cloning of the component sequences in pUC18 was confirmed by appropriate restriction digestion analysis using cloning enzymes and PCR analysis using component sequence specific primers. Also, these cassettes were completely sequenced to obtain the ultimate level of confirmation of orientation of cloning and genetic information of the gene cassettes.

In the third step, these cassettes were excided out from pUC18 vectors and were cloned in binary vector pCAMBIA 1305.2. The cloning was confirmed by restriction digestions.

orfH522 with or without coxIV pre-sequence was also cloned under CaMV 35S promoter. For this, orfH522, ‘coxIV presequence-orfH522’ were amplified and cloned in T/A cloning vector and later cloned in pRT100 (which has CaMV 35S promoter and CaMV poly A signal flanking the MCS) with appropriate restriction enzymes. The two gene cassettes were excised out from pRT100 with HindIII and cloned in pCAMBIA 1305.2

Thus, in the present investigation four gene cassettes were developed which when successfully introduced into crop plants, could induce male sterility. These constructs could unequivocally establish the role of orfH522 in causing male sterility in PET1 CMS line of sunflower.