*Article* **Transmission of Engineered Plastids in Sugarcane, a C4 Monocotyledonous Plant, Reveals that Sorting of Preprogrammed Progenitor Cells Produce Heteroplasmy**

**Ghulam Mustafa and Muhammad Sarwar Khan \***

Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, University Road, Faisalabad, P.O. Box 38040, Pakistan; ghulam.mustafa@uaf.edu.pk

**\*** Correspondence: sarwarkhan\_40@hotmail.com; Tel.: +92-41-9201087

**Abstract:** We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARE-S (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.

**Keywords:** plastid transformation; sugarcane; unfurled leaves; streptomycin; heteroplasmy; mesophyll and bundle sheath cells
