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Cannabis sativa L. Due to the psychedelic effect of one of the compounds, tetrahydrocannabinol THC , many countries had regulations or bands on Cannabis growing, also as fiber or seed crop. Recently, as many of these regulations are getting less tight, the interest for the many uses of this crop is increasing. Cannabis is dioecious and highly heterogenic, making traditional breeding costly and time consuming. Further, it might be difficult to introduce new traits without changing the cannabinoid profile. Genome editing using new breeding techniques might solve these problems. The successful use of genome editing requires sequence information on suitable target genes, a genome editing tool to be introduced into plant tissue and the ability to regenerate plants from transformed cells. This review summarizes the current status of Cannabis breeding, uncovers potentials and challenges of Cannabis in an era of new breeding techniques and finally suggests future focus areas that may help to improve our overall understanding of Cannabis and realize the potentials of the plant. Cannabis Cannabis sativa L. It is wind pollinated and flowers under short day conditions. The plants have been used by man for at least years Li, Cannabis has traditionally been classified as a single species Small and Cronquist, This is supported by molecular studies Oh et al. However, Cannabis is often divided into subspecies or groups based on chemotype, ecotype, crop-type fiber or drug or leaflet morphology Grassi and McPartland, ; McPartland, ; Small, Grouping is often problematic, as the types readily inter-cross and a lot of hybrids exist and as classification based on crop-type is somewhat dependent on legislation. In this paper, we focus on Cannabis used for medical purposes, e. Cannabis produces a range of secondary metabolites, the best known are the phytocannabinoids. Although as many as different cannabinoids have been reported see Radwan et al. Acidic forms of cannabinoids are biosynthesized in the trichomes on the female flowers Sirikantaramas et al. The storage of the toxic cannabinoids in trichomes minimizes the risk of self-intoxication Sirikantaramas et al. THC is the main psychoactive compound in drug type Cannabis Pertwee, , but also beneficial effects are reported Grotenhermen, Moreover, other cannabinoids, especially CBD, have attracted interest for their pharmacological properties. CBD has been reported to act as an antidepressant, to relieve pain and anxiety and to reduce inflammation. The resin from the glandular trichomes also contain terpenes Flores-Sanchez and Verpoorte, Terpenes does not only add flavor to the product but also have medical properties, not least in synergic action entourage effect with cannabinoids Ferber et al. In planta , the secondary metabolites are believed to protect the plants against various pathogens and insects. Both cannabinoids and terpenes have been shown to possess antifungal activity McPartland, ; Wanas et al. Solving the many challenges in Cannabis sativa calls for the use of all available tools. In the current review, we discuss the challenges and possibilities for New Breeding Techniques, such as genome editing in medical Cannabis. To set the scene, we first present a brief status of Cannabis breeding and the level of genetic diversity. We then address the requirements for successful tissue culture and transformation with a focus on the status of micro propagation, regeneration and transient as well as stable transformation. Finally, we discuss the many opportunities for using genome editing in the improvement of medical Cannabis. Due to the high-value products, medical Cannabis is often produced in greenhouse or indoor facilities where the plants are propagated like a horticultural crop using stem cuttings Vassilevska-Ivanova, ; Monthony et al. The use of cuttings ensures that only female plants with a higher level of cannabinoids are used for production. The dioecy behavior of Cannabis, making it an obligately outbreeding species, the limited number of genetic markers and the anecdotal start of breeding of medical cultivars make breeding of drug type Cannabis challenging. Early breeding was selection, done by the illegal market with decades of interbreeding and hybridization without record of parentage Barcaccia et al. This means that the genetic identity of a medical Cannabis strains cannot be reliably inferred from its name, as studies have shown that some strains with different names were genetically similar, and some strains with identical names were genetically different Sawler et al. However, the fact that breeding has been illegal does not mean that it is inefficient, as the level of THC has increased Mehmedic et al. For a comprehensive review on medical Cannabis breeding, readers are referred to Barcaccia et al. The level and composition of cannabinoids and terpenoids as well as stability in production, flowering time and lower resource input are in focus in modern medical Cannabis breeding, with more focus on CBD and other non-psychoactive cannabinoids. Resistance against insects, pathogens and viruses is also in high demand. Healthy mother plants are essential. However, maintenance of mother plants in contained humid environments poses a challenge in relation to attacks by plant pathogens such as powdery mildew. This significant challenge can only be kept at tolerable levels by a strict growth control including air circulation, ventilation, and moisture control as the strict regulations for medical products do not permit any use of pesticides. The production of doubled haploids DH in Cannabis would be highly advantageous, as it would be possible to produce female pure lines in one generation. Later chromosome duplication in the haploid plants is performed, either spontaneously or by chemical treatment. Although DH production via microspore culture has been investigated in Cannabis, successful DH production has so far not been established Adhikary et al. The method used for successful doubled haploid production seems to be species dependent wherefor also the other methods should be investigated for their usefulness in Cannabis. As also suggested by others Hesami et al. Polyploidization is used as a tool in plant breeding to improve desirable plant characteristics such as larger organs and higher yield Sattler et al. This might give a changed chemical profile with lower or even missing production of secondary metabolites, making it difficult to predict the outcome of polyploidization in new species or even other genotypes. In Cannabis, the effect of polyploidization has been studied in hemp as well as drug-type Cannabis Bagheri and Mansouri, ; Mansouri and Bagheri, ; Parsons et al. In both types, the tetraploid plants had broader leaves with bigger and less dense stomata, both clear signs of polyploidization. Cuttings of the drug-type Cannabis had reduced rooting ability, a phenomenon also observed in hop Trojak-Goluch and Skomra, ; Parsons et al. When the level of cannabinoids was analyzed, only small changes were found. The terpene profile was not analyzed in the hemp-type Cannabis, but in the drug-type, the terpene profile changed after polyploidization, as mainly the contents of sesquiterpenes increased Parsons et al. A change in terpene profile after polyploidization was also found in hop. Although the general level was lower, there was an increase in terpenes desirable for the brewing industry Trojak-Goluch and Skomra, These experiments did not show very promising results as far as an increase in cannabinoid is concerned. It should be reminded, however, that only one genotype per experiment gave tetraploid plants that could be analyzed. As there is often a difference between genotypes, the effect of polyploidization on the level of cannabinoids, terpenes and other important traits might have a different and more positive outcome in other trials, not least after crossing of polyploids with different genetic backgrounds. The genetic differences between the groups are distributed across the genome and are not restricted to loci involved in cannabinoid production Dufresnes et al. Whether hemp or drug type Cannabis is having more heterozygosity seems to depend on the study, which probably reflects differences in the selected cultivars Sawler et al. The lower genetic diversity in drug type Cannabis compared to hemp found in some studies Sawler et al. However, studies including a broader set of genotypes are needed to create more knowledge about the heterozygosity of Cannabis from all regions Kovalchuk et al. Such studies also provide the widest genetic background for medical Cannabis breeding. The analyses not only showed diversity between but also within cultivars Punja et al. The high diversity within a cultivar means that hardly any reduction in the genetic variation was found after one round of selfing Punja and Holmes, Some of this high variation found after selfing might be due to accumulation of somatic mutations within plants been propagated as cuttings for a long time Adamek et al. The high genetic diversity found in Cannabis is very useful for breeding new varieties. For medical Cannabis, however, homogenous material is needed, and varieties must be multiplied via cuttings. Introduction of single gene traits like disease resistance genes in Cannabis by traditional cross breeding, without affecting the genetic background and thereby the cannabinoid and terpene profile is difficult. Genome editing might solve this issue, see below. The use of genetic markers in drug-type Cannabis has mainly focused on analysis of Cannabis samples and plants to discriminate between hemp type and drug type material, to evaluate genetic variance and to identify female plants. Some examples will be highlighted here, for more comprehensive information, especially on early work, readers are referred to Onofri and Mandolino ; Punja et al. Further, a panel of 41 robust SSRs, with an average of four markers per chromosome, is provided by Barcaccia et al. As drug type Cannabis is illegal in many countries, there is a great need to be able to detect the presence of this type of Cannabis in seized samples. An important issue to consider here is the balance between speed, simplicity of the analysis, affordability, and accuracy. Many different assays have been developed, not only to discriminate between hemp and drug types, but also to establish from which geographic location the sample might originate. As DNA extracted from the seized material is analyzed, there is no need to wait for plants to grow in case of seed material or to extract cannabinoids to analyze for the level of THC and CBD. The markers reported by Kojoma et al. Other types of markers have also been shown to be useful; autosomal microsatellite markers and markers based on mitochondrial and chloroplast DNA. It seems that a limited number of markers, from 6 to 13, is enough, not only to sort Cannabis from hop, but to individualize and differentiate between types drug versus hemp and to say something about geographic origin Houston et al. Often markers can be multiplexed Dufresnes et al. Thus, genetic markers are useful as forensic tool to give a confirmation whether a sample is Cannabis and to discriminate between hemp and drug type samples. This can otherwise be difficult, especially with seed samples where there is no obvious difference. Also, information about the geographical origin of samples can give valuable information about distribution routes of illegal products. A mentioned, the karyotype is composed of 9 pairs of autosomes and one pair of sexual chromosomes X and Y Divashuk et al. Monoecious hemp cultivars having both male and female flowers on the same plant, have two X chromosomes Faux et al. Different genetic markers have been developed to sort male and female plants Mandolino et al. It seems that the sex determination is somewhat leaky, as the environment, especially photoperiod, hormones and unknown genetic components other than the sex chromosomes also plays a role Schaffner, ; Faux et al. This ability of female plants to produce male flowers independent of the presence of the Y chromosome is used in the production of feminized seeds, as female plants can be treated with thiosulfate to produce male flowers Lubell and Brand, Due to the many uses of Cannabis, genome editing would be a very desirable tool. Not only would we get a deeper insight into the cannabinoid pathway, but also many other genes important for the many uses of Cannabis could be investigated. The successful use of genome editing requires a genome editing tool, sequence information of suitable target genes, introduction of the construct into plant tissue and the ability to regenerate shoots from explant tissue. New Breeding Techniques NBT have emerged as alternatives to classical plant breeding and conventional transgenesis. These new techniques facilitate development of novel varieties more precisely and faster than by classical breeding giving genome modifications indistinguishable from those introduced by conventional breeding and chemical or physical mutagenesis Lusser et al. By HDR it is thus possible to make specific nucleotide changes. The system is based on a Cas9 nuclease which can be targeted to a specific genomic sequence by an easily engineered single guide RNA sgRNA of 20 base pair bp. Originally, the purpose of the PAM sequence was to distinguish self from non-self in prokaryotes. Ease of multiplexing, i. Although many report the use of CRISPR techniques in the coding sequence of genes, they can be used in promoters and upstream open reading frames as well Holme et al. Recently DNA editing in plant plastics has also become a possibility Kang et al. The technology is quickly developing Zhu et al. Nucleases that only make a single-strand break nickases can be used in pairs, each requiring a sgRNA. By positioning the two nicks close to each other on opposite strands, a break is created. As two sgRNAs are required, this paired nicking dramatically increases the specificity and thus reduces off-targeting in unwanted places. A new technique for editing is the prime editing technique, where bases in the target site is edited based on the sequence of a prime editing guide RNA Lin et al. Further, multifunctional genome editing systems are now emerging, making it possible to do both base editing and knockout simultaneous using a single construct Li et al. Some new techniques are firstly developed for monocots and optimization might be needed to use them in dicots such as Cannabis. Online tools are available to guide the design of efficient sgRNAs. Five different sgRNA designing tools and their main characteristics have been reviewed by Hesami et al. The algorithms in such programs are developed based on the assessment of many thousands of gRNAs targeting genes. However, most tools are not developed based on plant data and the predicted efficiency is not always in accordance with the found results Pauwels et al. An updated tool based on plant data is needed to increase the efficiency of plant sgRNAs. On top of the reference genome, NCBI lists thirteen different Genbank accessions of genome assemblies, five of which are at chromosome level Supplementary Table S1. These thirteen assemblies mostly represent female plants but are made from different types of Cannabis TDC high, CBD high, mixed profile and hemp , making them a valuable tool for finding sequence data for potential candidate genes. Reference genomes and other assemblies for both the chloroplast and mitochondrial genomes are also available Supplementary Table S3. It must be stressed that for gene editing techniques, the precise genetic sequence of the cultivar at hand is needed in order to design guides. This means that both alleles of each candidate gene to be targeted must be sequenced. Further, the availability of genomic sequence information not only provide the candidate genes for targeting but also gives the opportunity to analyze for possible off-targets, not only in gene families with high sequence similarity but also unexpected off-targets in unrelated genome regions. However, a very accurate screening for off-targets might be difficult in the highly heterozygous Cannabis genome. The plant material for production of medical Cannabis is obtained from cuttings from mother plants. The maintenance of mother plants demands a lot of space and considerable effort to keep the material free from diseases. Further, there is a need to store valuable breeding material. Propagation and maintenance of material via in vitro culture would be a way to solve this issue and tissue culture has been a topic in several papers. Several papers have reported methods for in vitro propagation in Cannabis, methods that often are described as regeneration Table 1 Lata et al. However, most papers do not describe regeneration from a single non-differentiated cell but instead report shoot formation from pre-existing meristems, once the apical dominance is broken. Summaries of published micropropagation studies were recently published Hesami et al. Table 1 List of Cannabis sativa studies reporting micropropagation from pre-existing meristems. In general, finding the right growth medium seems to be very cultivar dependent Grulichova et al. This makes it difficult to compare studies using different combinations of cultivars and media. It remains to be uncovered if this is due to a very limited number of plants being very willing to respond in vitro , or whether the ideal medium composition still needs to be discovered. This cultivar dependence implies that the use of hemp as a proxy for medicinal type Cannabis may not be successful Page et al. Although there might not be a big difference in multiplication rate between fiber and drug types Table 1. The results might also be dependent on whether the starting material is taken directly from the greenhouse or from tissue that has been in tissue culture for some time. The position basal versus apical of the stem explant also seems to affect multiplication rates Hesami et al. There could be a lingering effect from plant growth regulators found in plants with roots that might influence the multiplication rate Page et al. A high number of shoot proliferation has been reported in some cases Lata et al. The use of floral reversion might be a way to move forward, as this method increased the multiplication rate up to eightfold Piunno et al. It should be noted that achieving the highest multiplication rate might give lower quality and lower rooting ability Stephen et al. Rooting is very important for successful micropropagation and has been a topic in several papers e. There is a general need for improvement in more cultivars, if micropropagation should be of general use. Scientific research can be successful, even though a very limited or only one number of plant cultivars can be used. However, in production, medical Cannabis growers need to be able to use the technique in all their material before they invest in tissue culture facilities. It is most probable that several companies already have developed successful protocols for micropropagation. Most of these are, however, kept as trade secrets, if not funded by public means Adhikary et al. Resent papers show that improvement of micropropagation is still an interest also in universities Borbas et al. Micropropagation using synthetic seeds has been investigated as an alternative solution for propagation and conservation of germplasm. The use of synthetic seeds in Cannabis was first reported by Lata et al. Cryopreservation has also been investigated as a means for long term storage on in vitro material Lata et al. Although these methods are not of immediate importance for genome editing, they represent valuable ways of storing high-value material such as modified cultivars. In vitro propagation from pre-existing meristems is very useful for multiplying material for medical Cannabis production. However, if regeneration from single cells is optimized, a much higher multiplication rate might be obtained. In Cannabis, such de novo regeneration seems very difficult to obtain from callus or tissue without preformed meristems. This recalcitrance to regeneration is the main obstacle to an efficient genome editing protocol in Cannabis Monthony et al. Recalcitrance is a common problem in tissue culture Altpeter et al. For years, many different combinations of explants and plant growth regulators have been used to try to solve the difficulty of regenerating plants from very many species, often without great success. The ability to regenerate seems to be not only species dependent but also cultivar dependent. This calls for further research into whether there is a general explanation for recalcitrance to regenerate across plant species. Testing many different combinations of explant type, explant age, type of gelling agent, type of carbohydrate source, type, and balance of PGRs and addition of other supplements such as Zn or polyamines is often tedious and time consuming, sometimes with a low success rate. Machine learning has already been used in Cannabis to optimize in vitro seed germination Hesami et al. Further, as many research projects are performed by PhD students and post docs, with a strong demand for an outcome of scientific papers, the focus is often turned away from comprehensive, long-term research aiming at optimizing regeneration protocols. There might also be a lack of reports showing negative results. All this might have slowed down the progress of regeneration. Although the lowest percentage of responding explants is reported in hemp Slusarkiewicz-Jarzina et al. The biggest difference can be explained by the type of explants used. Experiments using hypocotyl, stem, or stem nodes have a higher response rate in general Table 2 Wielgus et al. Table 2 List of Cannabis sativa studies reporting regeneration experiments without transformation. One experiment, using leaf explants, stands out, as almost all explants gave shoots, with an average of This study has been replicated using ten other drug type Cannabis genotypes Monthony et al. Here, the experiment failed to induce shoots in all the genotypes tested, making it clear that regeneration might not only be tissue specific but also very dependent on genotype. The importance of genotype for regeneration in Cannabis is stressed by the fact that Zhang et al. Further, the lack of reproducibility stresses the difficulty of transferring tissue culture methods to other laboratories. These results are very promising but remains to be seen whether this method can be transferred to medical Cannabis. Transformation in Cannabis has been a research topic for more than 20 years. Three main transformation techniques have been used, the transformation with Agrobacterium rhizogenes to get hairy roots, transient transformation, and stable transformation. The status of transformation in Cannabis has also been reviewed by others Feeney and Punja, ; Simiyu et al. For an in-depth discussion of Agrobacterium strains, promoters and selection markers, readers are referred to Hesami et al. Hairy root cultures often have an enhanced ability to synthesize secondary metabolites Srivastava and Srivastava, Hairy root cultures were established after transformation with A. Several types of media containing a range of different combinations of hormones were tried, but although callus developed from the hairy root cultures, no shoots were obtained Wahby et al. No cannabinoids were present in the hairy root cultures. Similarly, no cannabinoid production was found in cell suspension cultures Flores-Sanchez et al. Contrary, hairy root cultures developed from callus without the use of A. However, higher levels would probably be toxic to the cultures. The ability of callus cultures to form roots was also seen by Feeney and Punja Unfortunately, regeneration of plants from hairy roots, although possible in some species Crane et al. Transformation using Agrobacterium tumefaciens is often used in plants, both for transient and stable transformation Dunwell and Wetten, ; Krenek et al. Studies with wild-type A. However, there seems to be a cultivar difference in the susceptibility to A. As the infection with Agrobacterium might be considered as a pathogen attack by the plant and the secondary metabolites is known to protect Cannabis against pathogens McPartland, ; Wanas et al. This might partly explain why all stable transformations in Cannabis is done in hemp cultivars see below; Table 4. Most probably, the recalcitrance in Cannabis explants is also due to the developmental state of the explants. In many plants, the first choice of explant material for transformation would be very young tissue, like that obtained from young seedlings. Such material is easily obtained from hemp, where seeds are available in big amounts. In medical Cannabis, however, very specific combinations of cannabinoids and terpenes might be lost if seeds must be produced. A method where the meristems of medical plants could be the starting material, is therefore highly adventitious. Further, developmental regulators can be used to induce new meristems from somatic cells during the transformation process Maher et al. One should be aware that the plants obtained from these meristematic techniques might have a higher degree of mosaic tissue than plantlets obtained by regeneration via callus. A study from Wahby et al. A patent by Roscow JR. The treated plant parts were producing trichomes comprising secondary compounds on non-flowering parts of the plant. It is, however, unclear if this property was inherited to the next generation. Transient transformation using agroinfiltration is also reported by several others Schachtsiek et al. Results show that the recalcitrance to be transformed might be due to the plants ability to protect itself against pathogen attack, as the addition of ascorbic acid, scavenging excess ROS, had a positive effect of the transformation efficiency Deguchi et al. Other methods for transient transformation include vacuum infiltration of DNA coated gold nanoparticles Ahmed et al. Transient transformation, although not leading to stably transformed plants, is a very useful tool for overexpression and silencing studies of many genes. Valuable information about the interaction between Cannabis and Agrobacterium which might be used to improve stable transformation. It seems that transient transformation is possible in both hemp and drug-type material Deguchi et al. Stable transformation is likely needed for genome editing of Cannabis. The first successful stable transformation of hemp was reported by MacKinnon et al. The report gives very little information about the methods used and the transformation efficiency obtained. Hemp suspension culture cells were transformed by Feeney and Punja in Feeney and Punja, After these two year-old papers, stable transformation was not reported for almost 15 years, most probably reflecting the lack of success of regenerating transformants. A patent filed by Sirkowski describes a method for Agrobacterium -mediated transformation of medical Cannabis. The material used is sections of hypocotyl and plants are regenerated from the tissue, but the efficiency is not mentioned. It is not clear in which genotypes the regeneration was successful and if plants were regenerated directly via a callus phase. This transformation system is very fast as tissue could be analyzed for GUS already one month after transformation. Both genotypic and explant differences were observed, with the least genotype dependency and the highest transformation rate found using hypocotyls. This method seems very promising, but the use of hypocotyls might be hampering the transferability to medical Cannabis that is usually propagated using cuttings. However, there was a low transformation rate using meristems as one transformant from one genotype was obtained from this material. The first paper describing the use of genome editing in Cannabis was published in Zhang et al. Using immature embryo hypocotyls from hemp as a starting material, shoots were regenerated from callus. Zhang et al. During the last years there has been a lot of progress in the prerequisites for genome editing in medical Cannabis. There is now a reference genome for Cannabis and a lot of additional sequence data available. This is a very important basis for the development of sgRNAs for different genome editing techniques. Not only can sequence for candidate genes be found, but also analyses for potential off-targets can be made. The editing techniques are in rapid development with new techniques and improvements emerging every year. The biggest leap forward is the recent reports of regeneration and Agrobacterium -mediated transformation of Cannabis. There is, however, still challenges when it comes to using genome editing in medical Cannabis. The two main challenges are genotype dependency and the selection of transformable explant material from cuttings. There is without doubt genotype dependency, not only between chemotypes but also between hemp cultivars. It is our belief that the future will bring more improvements and refinements as recalcitrance to regeneration and genotype dependency is found in several plant species. Using hypocotyls from immature embryos for transformation in medical Cannabis is not the obvious choice. The plants are usually clonal propagated to maintain valuable cannabinoid and terpene profiles, which will be changed if plants are propagated by seeds. All these methods need to be thoroughly investigated in many genotypes of medical Cannabis. The possible outcome of the combination of floral reversion and transformation should also be pursued. Due to the multiple applications of Cannabis, many traits might be interesting to improve. Single gene traits are easier to work with than very complex traits. However, successful editing of several genes is reported in other species Gao et al. Clean cannabinoid products can be obtained by producing them in other systems like yeast Luo et al. However, the therapeutic response is often higher using plant products, probably due to a synergistic or entourage effect between various cannabinoids or between cannabinoids and terpenes Ferber et al. Genome editing tools would be very useful to study and manipulate the biosynthetic pathways of cannabinoids and terpenes, not only for pure scientific purposes, but also to improve the products in the medical Cannabis industry. Gene editing facilitates gene knockout studies as well as studies altering gene expression levels and tailoring of specific genes. The review by Hesami et al. The sequences of these synthases are very similar, and studies have shown that they are positioned at two closely linked loci in a very complex region Van Bakel et al. This complexity makes it challenging, although not impossible, to make changes using genome editing, as guides need to be designed to hit target genes without off-target effects. It is essential that drug formulations contain as low a THC content as possible as the THC contamination causes short- and long-term side effects Volkow et al. The use of extra purified CBD or synthetic CBD instead of the plant extract results in a lack of other naturally occurring cannabinoids and bioactive compounds such as terpenes involved in the entourage effect. This may have a decisive influence on the therapeutic effect of the product Russo, One amino acid change at AV increased the catalytic activity 3. Further studies are needed to uncover the potential of modulating the specificity of this enzyme in planta. Another need is to produce plants with a higher content of rare cannabinoids or to change the content of different terpenes independent of the cannabinoid profile. CBG is a compound having medical properties of its own, but it is often present in quite low levels. Genome editing approaches have been used to obtain resistance against plant diseases caused by viruses, fungi, and bacteria Borrelli et al. This is highly relevant in medical Cannabis production as the loss of production batches due to attack by fungal diseases is a serious problem. The plants cannot be treated with fungicides as traces of these compounds might be found in the final product. Controlling the humidity is highly energy demanding, but if not done, production batches might be lost. Amino acid changes in the Mlo gene s are known to give resistance against powdery mildew in a range of plants Kusch and Panstruga, In medical Cannabis, where the use of pesticides is not possible, the growth of resistant plants is highly desirable. When medical Cannabis is produced in greenhouses, there is a high humidity, which is the perfect environment for the development of the fungal disease powdery mildew. CRISPR-mediated changes in the Mlo gene would give resistance against powdery mildew without any site effects in the cannabinoid production. Grey mold due to the fungus Botrytis cinerea is a severe problem in indoor Cannabis production. Botrytis is a necrotrophic fungal pathogen, but it apparently starts its infection in a biotrophic manner Veloso and van Kan, Rather than indiscriminately killing its host, Botrytis gently guides the host plant towards committing suicide through apoptosis, making the fungus able to colonize and digest the plant tissue. If the spore density is low, the infected plants might be without symptoms. When these asymptomatic plants approach flowering, the fungus might switch to necrotrophic lifestyle causing the host plant to succumb Veloso and van Kan, This might be the reason why growers often see a very sudden, severe attack of grey mold. Although the molecular mechanisms are not fully understood, silencing of susceptibility genes has been shown to impede infection Sun et al. This and similar research might lead the way for some resistance against Botrytis obtained by genome editing. Varieties with less apical dominance might be easier to regenerate in tissue culture and would therefore be the obvious candidates for transformation. These more uniform plants make harvest of inflorescences easier. However, plants treated with phytohormones showed the same or reduced inflorescence dry weight, dependent on genotype, with no change in CBD content Burgel et al. Although drug type Cannabis, due to selection, has become shorter and more densely branched, there is a great demand for varieties with a standardized plant type, not least with same height and branching, as such plants are suitable for automation. Thus, genes involved in apical dominance, branching and plant height would be very interesting candidates for genome editing. This would be a very useful technique to use in Cannabis, where no successful doubled haploid technique is yet available. With a robust transformation platform in Cannabis, haploid inducer lines obtained with this technique is surely possible. Haploid-inducer lines with a hemp genetic background would be useful in medical Cannabis as well, as the haploids will not contain any parental DNA from the haploid-inducer parent. There are several other traits of general interest for Cannabis production, also in hemp cultivars. Flowering time, fiber quality, lower phytic acid content of seeds, and soil remediation properties are obvious candidates for investigation via genome editing Shiels et al. The very high genetic diversity in Cannabis sativa is a great advantage for conventional breeding. However, conventional breeding through crossing and selection is very time consuming. It requires several rounds of backcrossing and is complicated by the dioecious nature of the plants. Further, the introduction of new traits might compromise the cannabinoid and terpenoid profile of medical Cannabis. Targeted improvements at predetermined positions in the genome by gene editing might mitigate some of these challenges. Cannabis has traditionally been considered a recalcitrant species, in which techniques like genetic transformation and genome editing were very complicated. However, our review providing insight into recent progresses within tissue culture, genetic transformation, gene editing and necessary sequence information on Cannabis sativa indicates that this is not the case anymore. Currently, plant regeneration has been reported not only in hemp but also in a few medical Cannabis cultivars. Our study suggests that future efforts could be directed towards development of a robust regeneration protocol for differentiated or meristematic tissue from medical Cannabis and thereby form the basis for future targeted improvements of medical Cannabis. Genome editing is a great tool for scientific investigations and precision breeding for secondary metabolites such as cannabinoids and terpenes. Precision breeding further gives the possibilities to add new traits such as disease resistance or changed plant architecture without meddling with a known cannabinoid profile. Conventional methods adding new genetic variation will continue to be a corner stone in breeding but once transformation is a routine tool in Cannabis, there are almost unlimited possibilities to improve a wide range of plant traits. Furthermore, since medical cannabis is often grown under contained facilities, any GM regulatory requirements of genome edited plants will be easier to meet. HB-P designed the project. CI wrote the initial manuscript which was carefully revised by HB-P. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Keywords: Cannabis sativa , medical Cannabis, drug type Cannabis, genome editing, plant regeneration, transformation. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Top bar navigation. About us About us. Sections Sections. About journal About journal. Article types Author guidelines Editor guidelines Publishing fees Submission checklist Contact editorial office. Challenges and potentials of new breeding techniques in Cannabis sativa. Introduction to medical Cannabis Cannabis Cannabis sativa L. Breeding, genetic diversity and genetic markers Breeding Due to the high-value products, medical Cannabis is often produced in greenhouse or indoor facilities where the plants are propagated like a horticultural crop using stem cuttings Vassilevska-Ivanova, ; Monthony et al. Doubled haploids The production of doubled haploids DH in Cannabis would be highly advantageous, as it would be possible to produce female pure lines in one generation. Polyploidization Polyploidization is used as a tool in plant breeding to improve desirable plant characteristics such as larger organs and higher yield Sattler et al. Use of genetic markers The use of genetic markers in drug-type Cannabis has mainly focused on analysis of Cannabis samples and plants to discriminate between hemp type and drug type material, to evaluate genetic variance and to identify female plants. Markers for chemotype As drug type Cannabis is illegal in many countries, there is a great need to be able to detect the presence of this type of Cannabis in seized samples. Sex markers A mentioned, the karyotype is composed of 9 pairs of autosomes and one pair of sexual chromosomes X and Y Divashuk et al. The prerequisites for genome editing Due to the many uses of Cannabis, genome editing would be a very desirable tool. Gene editing techniques New Breeding Techniques NBT have emerged as alternatives to classical plant breeding and conventional transgenesis. Tissue culture and plant regeneration Micropropagation The plant material for production of medical Cannabis is obtained from cuttings from mother plants. Table 3 List of Cannabis sativa studies reporting transient transformation. Table 4 List of Cannabis sativa studies reporting stable transformation. Keywords: Cannabis sativa , medical Cannabis, drug type Cannabis, genome editing, plant regeneration, transformation Citation: Ingvardsen CR and Brinch-Pedersen H Challenges and potentials of new breeding techniques in Cannabis sativa.

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