**5. Conclusions**

A proficient regeneration protocol was developed for elite sugarcane genotypes. Using the protocol, species-specific plastid transformation vector, cell-autonomous selection, and visual detection approaches, plastid transformation was achieved in sugarcane. Streptomycin-resistant clones remained heteroplasmic after selection and regeneration on streptomycin-containing medium, referring to the complex pattern of division and sorting of cells in the meristematic tissues as another important factor in the purification of transplastomes to homoplasmy in sugarcane.

**Author Contributions:** M.S.K. designed experiments and managed funds for the said research. G.M. performed experiments under the supervision of M.S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science & Technology (MoST), Islamabad, Pakistan to M.S.K.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank the Ministry of Science & Technology, Pakistan for providing funds; and Sugarcane Research Institute, AARI Faisalabad, Pakistan for providing the plant material.

**Conflicts of Interest:** The authors declare no conflict of interest relevant to the publication of this manuscript.
