**1. Introduction**

Plastids have become attractive targets for genetic engineering since their genome offers several potential advantages, including accumulation of transporters to high levels with bona fide structures, biological containment of transgenes, gene stacking in operons, and absence of position effects [1,2]. Chloroplast genome has been engineered successfully to develop valuable traits, such as herbicide tolerance, insect and disease resistance, drought or salt tolerance, production of therapeutic proteins, antibodies, antibiotics, vaccine antigens, industrial enzymes, and other biosimilars in different plants [3–5]. Hence, more than 100 transgenes have been stably integrated and expressed in the chloroplast genome [6–8]. More recently, chloroplast genomes of major crops, including cotton, soybean, vegetables (carrot, lettuce, cabbage, eggplant, sugar beet), fruits (tomato), and trees (poplar and citrus), have been transformed [9,10].

Generally, monocots are recalcitrant to regeneration and there is no standard protocol available for efficient regeneration of plants from dedifferentiated cells, which could be used to transform plastids and to recover transgenic plastid-carrying cells. For each crop, different combinations of nutrients and auxins are used for callogenesis as well as

**Citation:** Mustafa, G.; Khan, M.S. Transmission of Engineered Plastids in Sugarcane, a C4 Monocotyledonous Plant, Reveals that Sorting of Preprogrammed Progenitor Cells Produce Heteroplasmy. *Plants* **2021**, *10*, 26. https://doi.org/10.3390/ plants10010026

Received: 18 September 2020 Accepted: 14 November 2020 Published: 24 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

organogenesis. To date, plastid transformation has been reported in rice, yet the regenerated shoots remained heteroplasmic at plastome, organelle, and cell levels [11,12]. One of the major bottlenecks in developing stable plastid transformation in monocots has been their regeneration from non-green embryonic cells, containing undifferentiated plastids. Other barriers in developing homoplasmic transplastomic plants, particularly of rice, might be the low level of marker gene expression in non-green plastids in embryogenic cells because of a low genome copy number and low rates of protein synthesis [13].

While addressing bottlenecks, we attempted to transform the sugarcane plastid genome where we developed an efficient regeneration protocol and species-specific chloroplast transformation vector carrying the dominant visual selectable marker. The development of transplastomic technology in sugarcane will open up exciting possibilities for novel gene introduction and expression for agricultural or pharmaceutical traits.
