# Releasing the Full Potential of Cannabis through Biotechnology
## 4 Overcoming Limitations in Cannabis Gene Editing
### Target Tissue Selection  
The development of a stable and robust regeneration system is critical to successful transformation. Since Cannabis plants are highly recalcitrant to _Agrobacteriumium_-mediated transformation, a variety of explants have been tested with the alteration of the tissue culture conditions for the purpose of regeneration. The totipotency of plant tissues is the foundation of most plant regeneration. DNA, dsRNA, or plasmid are delivered to isolated somatic tissue of Cannabis (be it hairy roots, leaves, flowers, stem, immature embryos) and selected for positive transgenic plants regenerated from the modified tissues [54]. Despite being developed over decades, the process is still genotype-dependent and far from routine in laboratories. Since Cannabis explants have been demonstrated to be susceptible to transient expression instead of stable transformation, the focus of researchers turned to the call tissue developed from embryos [54; 57]. Calli derived from immature embryos or mature plants were two main sources for _Agrobacteriumium_-mediated transformation in several plant species [58]. It has been demonstrated that both cotyledons and young true leaves are amenable to transient transformation, while notoriously recalcitrant to traditional genetic transformation methods [57]. To increase transformation rates, our focus turned to the calli developed from immature embryos. The embryogenic calli can provide competent cells, extremely susceptible to transformation by _Agrobacteriumium_[59]. To increase the regeneration rate and optimize the protocol, [PERSON] et al. (2021) performed a regeneration assay for one hundred Cannabis varieties [44]. These Cannabis varieties were obtained from the national germplasm bank of the Institute of Bast Fiber Crops. The immature grains were collected about 15 days after flowering because the hypocotyls collected at this time can produce more shoots. After sterilization, these hypocotyls were isolated and incubated in the mediums to estimate the regeneration ability. Among all the varieties evaluated, DMG278 has the highest shoot induction rate of 7.09%, suitable for the _Agrobacteriumium_-mediated transformation [46]. As one of the major limitations of regeneration and transformation, this barrier needs to be overcome before releasing the full potential of gene-editing technology in Cannabis.  
In addition, the genetic background of the _Agrobacteriumium_ strains influenced the efficiency of T-DNA transfer into cellular nuclei. Some _Agrobacteriumium_ strains were demonstrated to be more virulent than others for specific species [46]. Because of the limited transformation studies in this species, few reports mentioned about the appropriate _Agrobacteriumium_ strains for Cannabis. [PERSON] et al. tested the transformation efficiency of EHA105, LBA4404, and GV3101, which belonged to three types of _Agrobacteriumium_ strains, succinampine, octopine, and nopaline respectively [1]. The GV3101 strain is the optimal choice for Cannabis due to a significantly higher GUS expression than the other strains, which means the octopine-type strains might have better compatibility with Cannabis. Other _Agrobacteriumium_ strains in this type, such as GV3100, GV3850, C58C1, A136, and EHA 101, should be further tested for Cannabis transformation efficiency.