Genetic engineering is a method that has been widely used to improve traits and to obtain new cultivars with desirable characteristics. To achieve this, breeders can use conventional breeding, genetic modification or genome-editing methods [131]. Genetic modification entails the transfer of genetic materials from one species to another (transgenic) or within the same species (cisgenic), whereas in genome editing a specific part of a gene can be edited using sequence-specific nucleases such as zinc-finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs) and regularly interspaced short palindromic repeat-associated endonucleases (CRISPR/Cas) [131]. The use of CRISPR/Cas allows breeders to edit a gene sequence using an enzyme and a small guide RNA which can be removed afterwards, making the edited lines free of foreign genetic materials [10]. The molecular tools needed to achieve genome modification or editing can be delivered into the cell system by various methods, including Agrobacterium-, nanoparticle- and polyethylene glycol (PEG)-mediated transformation. In Cannabis, the aforementioned methods have been used in gene modification studies [48]; however, regeneration of the transgenic plants has remained a major challenge. For example, Cannabis callus was successfully transformed using Agrobacterium tumefaciens (strain EHA101) carrying a gene encoding phosphomannose isomerase, but regeneration of the transformed calli was not achieved [132]. In another study, Agrobacterium rhizogenes transformation of hemp hypocotyls resulted in transgenic hairy roots, with no report of regeneration of the transformed tissues [133]. A lack of replicable and reliable Cannabis regeneration protocols prompted more studies on transient gene transformation for studying gene functions. Virus-induced gene silencing (VIGS) was used for reverse genetic studies in hemp seedling and leaf to produce transient transformants [134]. An agroinfiltration protocol (using A. tumefaciens strain EHA105) coupled with vacuum infiltration resulted in successful transient transformation of cotyledons and true leaves of young Cannabis seedlings [135]. A more efficient transformation of mature leaves, flowers, stem and root tissues using similar methods of agroinfiltration concluded that A. tumefaciens strain GV3101 is 1.7 times more efficient than strains EHA105 and LBA4404 [136]. Additionally, the transient transformation of Cannabis protoplast using PEG-mediated transformation was also recently reported to be effective in studying gene functions [137]. In another study, a nanoparticle-mediated transformation was successful in delivering soybean genes into Cannabis leaf tissues using silicon-dioxide-coated gold nanoparticles [138]. Together, these studies provided important protocols for Cannabis transient transformation that do not require going through the indirect organogenesis procedures that have been a bottleneck in genetic engineering studies. Although transient transformation can be effectively applied to study gene function in Cannabis, establishment of stable transformation is still required for genetic analysis of the progeny. A recent study reported a protocol for stable Agrobacterium-mediated transformation of Cannabis hypocotyls and cotyledons [11]. The study concluded that the rate of transformation and regeneration was higher in hypocotyl explants, but it varied among different Cannabis varieties. However, transformed explants were directly regenerated without going through the callus phase. In another recent study, a successful stable transformation using immature embryos followed by indirect organogenesis of their calli was reported [12]. To enhance the indirect regeneration potential, Agrobacterium-mediated transformation was used to overexpress endogenous developmental regulator genes which are known to have positive influences on shoot organogenesis. Moreover, the same study used CRISPR/Cas9 for the first time in Cannabis to edit the phytoene desaturase gene, resulting in gene-edited seedlings. Development of homozygous inbred lines of a crop with a desired trait is a tedious process that can take up to 10 generations by traditional methods [139]. Shortening of this breeding time is crucial for both scientific and commercial ends in crops such as Cannabis which have high commercial value. Doubled haploid (DH) technology can be employed for this purpose, as it has been used successfully in other crops. However, DH technology in Cannabis is yet to be fully developed.

Doubled haploids (DH) are plants that carry genetic information from a single parent [140]. DH technology is used by plant breeders to rapidly make genetically homogeneous progenies in fewer generations than it takes in the traditional breeding. This technology has been used extensively in maize and other crops such as wheat, potato and sorghum [141]. To make haploid plants, breeders can use in vitro haploid gametophyte tissue culture, interspecific crossing or pollen irradiation procedures [140]. Once haploid plants are obtained, their chromosomes are doubled to make them fertile and robust by chemically blocking cell division without blocking chromosome duplication [140]. In Cannabis, interspecific crossing and pollen irradiation studies to make haploid plants have not been carried out, but studies to regenerate haploid plants directly by embryogenesis from microspores (androgenesis) have been conducted. In one study, a small percent (0.05-0.5%) of in vitro cultured hemp microspores formed embryo [10]. Recently, a study on two hemp varieties reported that cold-shock pretreatment of floral buds before in vitro culture increased the embryogenesis potential of the microspores [142]. The rate of embryo formation in the study (1.11 embryo formation for every 100 anthers), was also low and the microspore-derived embryo did not develop beyond 9 weeks. It has been reported that for successful embryogenesis, microspores have to be at, or just after, the first mitotic division when the cells are still in a uni-nucleate or early bi-nucleate stage [10]. Microspore culture at an appropriate developmental stage is therefore crucial but challenging. One other method to make haploid plants is the use of in planta gene-mediated haploid induction systems. This method entails crossing a line carrying the desired genetic information with a haploid inducer (HI) line [141]. In this crossing, the chromosomes from the HI will not be part of the embryo’s genome after fertilization. This haploid induction method has been used in plants such as Zea mays, where a HI carrying a MATRILINEAL (MTL)/Patatin-Like Phopholipase A1 (ZmPLA1)/NOT LIKE DAD (NLD) gene was used in a cross that resulted in haploid offspring [143]. Additionally, a mutation in the Centromere histone H3 (CENH3) gene has been reported to cause haploid induction in desired plants when these plants are crossed with an HI carrying this mutation [100,144]. Other genes that have been reported to have haploid induction ability are DOMAIN OF UNKNOWN FUNCTION 679 membrane protein (DMP) [145] and indeterminate gametophyte1 (ig1)/LATERAL ORGAN BOUNDARIES (LOB)-domain protein [146]. The molecular basis behind haploid-inducing ability of maize HI has been discovered [141,143]. This has allowed breeders to render haploid induction ability to plant lines through genetic engineering [144,145,147]. Although the use of HI has not yet been implemented in Cannabis, success in other plants indicate that this method can be used for haploid induction and DH studies. Once well established in Cannabis, DH technology can be useful in fixing desired characteristics and in reducing breeding generations required to obtain a desirable line. Being a plant with high variability in chemical and physical characteristics, as well as high commercial value, DH offers a promising way forward for Cannabis breeding. Moreover, researchers have made use of HI to deliver gene-editing tools into the egg cell of a plant through the methods called HI-Edit [148], and haploid-inducer-mediated genome editing (IMGE) [149]. These methods can also be used in gene editing procedures of Cannabis once well established. The fact that the HI genome, and the gene-editing machinery it carries, do not become part of the transformed embryo makes this technology appealing for commercial utilization of the engineered plants.