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In vitro Direct Regeneration and Agrobacterium tumefaciens Mediated in planta Transformation of Ocimum sanctum L.


Affiliations
1 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, Uttar Pradesh, India
2 CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow 226 015, Uttar Pradesh, India
 

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II (npt-II) & β-glucoronidase (uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Keywords

Gus-A, Kanamycin, MAPs, Npt-II, O. tenuiflorum, Petiole.
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  • In vitro Direct Regeneration and Agrobacterium tumefaciens Mediated in planta Transformation of Ocimum sanctum L.

Abstract Views: 173  |  PDF Views: 92

Authors

Sana Khan
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, Uttar Pradesh, India
Zakir Husain
CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow 226 015, Uttar Pradesh, India
Laiq ur Rahman
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, Uttar Pradesh, India

Abstract


An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II (npt-II) & β-glucoronidase (uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Keywords


Gus-A, Kanamycin, MAPs, Npt-II, O. tenuiflorum, Petiole.

References