Rice is a major staple food worldwide and can be genetically modified using various methods like Agrobacterium-mediated transformation, biolistic transformation, and chloroplast transformation. Agrobacterium transformation has advantages like simplicity but is limited by host specificity. Biolistic transformation has the disadvantage of random integration and tissue damage. Chloroplast transformation involves constructing vectors by cloning homologous fragments and promoters before transforming rice chloroplasts. Co-cultivation on filter paper with cysteine gave non-browning calli for Agrobacterium transformation selection. Transgenic plants were confirmed using Southern blotting for all three methods.

4. Seed sterilization Preinduction callus Preculture of callus Callus induction inoculation Culture A. Tumefaciens harboring pCAMBIA1301 in AB medium Preparation of inoculum Co-cultivation selection Shoot regeneration X-Gluc solution Examination of expression of GUS rooting potting Material and Methods – Agro bacterium transformation at 28 o C +16 h light +8 h dark cycle 3 weeks at 28 o C In dark 3 days Co-cultivation 2 mins 3 days 3 days

5. Continue… Material and Methods – Biolistic transformation

8. Chloroplast from leaves & DNA Isolated Two homologous fragments are amplified and purified Cloned into pBluescript SK (MB) - pSKE & pSKF sequencing vectors are constructed Double digested with Sac II & Bam H I & fragment inserted in pSKF Vector Rice homologous fragment - pREF Pu16S was digested by Sac I Cut was blunted with klenow fragments and again cut with Hind III Modified 16s promoter (150BP) Digested product was cloned into PT393 between xbal site & hind III site Expression vector p16ST PAZ – Digested by Xba I Cut was blunted and again cut with Hind III Product contain Bar sequence & it was purified Inserted into p16ST B/W Sac I – Hind III site Intermediate vector p16STB was formed Cut with Bam H I – DNA fragments Cloned into pREF – b/w Bam H I site Rice chloroplast pRB

9. Construction of chloroplast transformation vector Source : LIYi-nü etal

13. transplastomic rice lines - Biolistic transformation Molecular identification of the bar gene in transplastomic rice plants - Chloroplast transformation bar gene was identified in transplatomic rice plants by using Southern blotting - Chloroplast transformation

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