IN VITRO DIRECT REGENERATION OF POLYPLOID CANNABIS PLANTS
20230044740 · 2023-02-09
Assignee
Inventors
Cpc classification
A01H4/005
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a new in vitro method for direct regeneration of a Cannabis sativa L. plant as well as the use of such method for micropropagation of selected elite clones belonging to Cannabis sativa L., development of polyploid Cannabis plants with enhanced levels of secondary metabolites, promotion of spontaneous rooting of in vitro regenerants in a shorter period of time than conventionally used methods, and production of mutagenized and transgenic or gene-edited Cannabis plants. The method advantageously comprises culturing selected explants lacking already developed meristems such as cotyledons, hypocotyls and/or epicotyls from Cannabis seedlings of short and neutral-day varieties in an appropriate culture medium without plant growth regulators nor chemical microtubule disruptors with a high toxicity grade.
Claims
1. A method for in vitro direct regeneration and induction of polyploidization in Cannabis plants by means of in vitro tissue culture of cotyledons, hypocotyls or epicotyls from a Cannabis seedling, said method comprising the steps of: a) Collecting seeds of a donor Cannabis plant; b) Surface sterilization of the seeds with ethanol, bleach, mercuric chloride or other chemical or physical disinfectant agent. c) Germination of the seeds d) Dissection of cotyledons, hypocotyls and/or epicotyls from a Cannabis plant in a phenological growth stage coded from 00 to 99 according to BBCH-scale of Mishchenko et al. (2017). e) Culturing of the explants in a culture media under controlled conditions of temperature at 22° C.±1° C. and 60%±1% relative humidity with a photoperiod of 16 hours of light per day during 2-3 weeks f) Selection of embryos, shoots and/or shoots with roots; g) Sub-culturing the specimens of f) individually to glass-tubes or other containers of different volumes with the culture media used in step e) until spontaneously rooted plants are generated or until spontaneously rooted plants develop enough; h) Transplanting spontaneously rooted plants in pots with fertilized substrate and acclimatizing as needed; wherein the culture media used in steps c), e) and g) is free of hormones and chemical microtubule disruptors in the case of hypocotyls and epicotyls culture, and free of chemical microtubule disruptors in the case of cotyledons culture.
2. A method for in vitro direct regeneration and induction of polyploidization in Cannabis plants according to claim 1, characterized in that the cotyledon, hypocotyl and/or epicotyl explants are dissected from a Cannabis donor plant in a phenological growth stage coded from 05 to 19 according to BBCH-scale of Mishchenko et al., (2017).
3. A method according to claim 1, characterized in that the culture medium used in steps c), e) and g) comprises macronutrients, micronutrients, vitamins, and carbon sources, with or without gelling agents.
4. A method according to claim 1 characterized in that the donor Cannabis plant belongs to Cannabis sativa L.
5. A method for micropropagation of selected elite clones belonging to Cannabis sativa L. which comprises in vitro direct regeneration of Cannabis plants according to claim 1.
6. A method for obtaining transgenic or gene-edited Cannabis plants which comprises direct in vitro regeneration of a Cannabis plant according to claim 1.
7. A method for inducing mutagenesis in order to generate variability in Cannabis sativa L. and produce new Cannabis plant genotypes which comprises direct in vitro regeneration of a Cannabis plant according to claim 1.
Description
DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0088] Plant regeneration involves the in vitro culture and aseptically growth of cells, tissues, and organs under defined physical and chemical conditions. Regeneration has long been known to occur in plants. In plants, non-differentiated cells are able to regenerate into the full array of organs and/or tissues under appropriate culture conditions. Regeneration can involve direct or indirect organogenesis. In direct regeneration, in vitro organs are directly induced from explant tissues; in indirect regeneration, a de novo organ is typically formed from an intermediate tissue, the callus. Plant calli are undifferentiated structures that can give rise to new tissues. Plant leaves, shoots, roots, and embryos can variously be elicited from a growing callus by treating it with different ratios of hormones. However, in vitro regeneration of a plant from a callus is often associated with loss of genetic fidelity of the regenerants with respect to the donor plant (Evans and Bravo, 1986; Ramirez-Mosqueda and Iglesias-Andreu, 2015).
[0089] It has been known from the prior art a method of in vitro regeneration of Cannabis sativa L. from explant hypocotyl (Movahedi et al., 2016a). However, such method is not a direct regeneration method, but an indirect in vitro regeneration method which probably due to the addition of plant growth regulators in the culture medium, comprises a callus formation phase taking place prior to shoot organogenesis. The formation of tissue or a new plant from the intermediate tissue (callus), seriously compromises the genetic fidelity of regenerants with respect to the donor plant (Evans and Bravo, 1986; Ramirez-Mosqueda and Iglesias-Andreu, 2015).
[0090] To the contrary, the present invention concerns a direct in vitro regeneration method of Cannabis plants which avoids formation of a callus phase and by avoiding this phase, does not compromise the genetic fidelity of the regenerants obtained with respect to the donor plant.
[0091] For that purpose, the method advantageously uses hypocotyls and/or epicotyls from a plant with a phenological growth stage coded from 05 to 19 according to BBCH-scale of Mishchenko et al. (2017), more preferably seven-days-old seedlings are used as donor Cannabis plants. By the method of the present invention, Cannabis plants are in vitro regenerated from said specific explants at a high rate of shoot organogenesis and/or embryogenesis. In other words, a high rate of shoot organogenesis and/or embryogenesis means that at least 43% of the explants give rise to Cannabis sativa L. in vitro plant regeneration. Preferably, regeneration occurs in at least 62% of cultured explants, even more preferably, regeneration occurs in at least 71% of cultured hypocotyl explants.
[0092] In a preferred embodiment, when hypocotyl and/or epicotyl explants are cultured according to the present invention's method, it is not even required addition of hormones, growth factors, plant growth regulators or any other substance or composition which induces or improves the formation or development of any multicellular structure or organ in the plant (e.g. embryo, shoot and/or root) to the culture medium.
[0093] As has been reported previously, one of the most important factors in adventitious organ formation is the endogenous auxin:cytokinin balance and not the amount of auxin or cytokinin added in the culture medium (Tanimoto and Harada, 1984).
[0094] Cytokinins are produced predominantly in the root meristem and auxins are synthetized in the shoot meristem, and both types of phytohormones can migrate from roots and shoots to their action site through phloem and xylem (Beck, 1996). It is believed that segmentation of both shoot and root meristems as a result of hypocotyl or epicotyl dissection could modify the endogenous hormonal interaction between auxins and cytokinins, leading to an appropriate environment for shoot and/or root organogenesis development in hypocotyls and epicotyls.
[0095] The present invention is also based in the findings that hypocotyl and epicotyl derived plants can root spontaneously in a hormone-free culture medium, which comprises no hormones, no growth factors and/or any plant growth regulator and/or any other substance or composition eliciting a plant growth regulator effect, thus being able to completely be acclimatized in only six weeks.
[0096] It is also believed that the same reasoning described above explains the fact that after shoot development in the top of the hypocotyl or epicotyl, auxins produced endogenously in the shoot meristem could promote the spontaneous rooting of in vitro regenerants, representing an added advantage of the method according to the present invention, since a separate auxin containing medium is not required for root induction.
[0097] Advantageously, the author of the present invention has found that hypocotyls cultured in a medium free of plant growth regulators reached the third highest shoot induction rate of the evaluated media without presenting significant differences with the other two media with better percentages of shoot organogenesis (see table 5 below). In the case of Cannabis epicotyl in vitro culture, hormone-free medium reached the highest shoot induction rate of the evaluated media (see table 3 below). In addition, thereto, when hypocotyls and/or epicotyls were used as explants in a hormone-free medium according to the method described in the present invention, plants spontaneously rooted and thereafter were completely acclimatized in just six weeks.
[0098] It is also believed that pericycle cells adjacent to xylem poles could be the origin of in vitro direct regenerated plants of Cannabis according to the present invention.
[0099] In order to infer the possible origin of in vitro regenerants from cotyledons, hypocotyls, epicotyls and true leaves coming from seven-days-old seedlings, the author of the present invention have examined transversal sections of hypocotyls and epicotyls and identified pith, cortex and epidermis. These observations are consistent with those documented in several prior art references. As presented by Behr et al. (2016), cross-sections of cannabis hypocotyls six and nine days after sowing are coincident with the results obtained by the author of the present invention, since epidermis, cortex and pith can be easily differentiated and their respective anatomy is also concurrent with the present findings.
[0100] The fact that the two primordia emerged from the top of hypocotyls and in the lower section of epicotyls were always distributed in the periphery of the organ, and aligned one in front of the other, led the author of the present invention to hypothesize that regenerated plants originated always from the same type of cells. In a former work by Miller (1959), hop hypocotyl cross-sections drawings detailed the connection between root and cotyledons of the seedling, describing not only the same regions than in the hypocotyl transversal section of the present invention's findings, but also two protoxylem poles situated in a peripheral position and distributed in opposite sides, whose location strongly resembles the regeneration area of hypocotyl and epicotyl derived meristems in the experiments of the present invention. Furthermore, this prior state of the art also describes how only one protoxylem pole is located in the median strand of the base of each cotyledon. The fact that in the present invention's study plant regeneration from cotyledons always was located in the central region of the basal zone of the explant, supports the hypothesis that cotyledon, hypocotyl and epicotyl derived plant regeneration in C. sativa originates from pericycle cells adjacent to xylem poles. It should be noted that hop (Humus lupulus L.) is the only species together with C. sativa belonging to Cannabaceae family, so it should be considered as an ideal candidate to compare with Cannabis sativa L. With respect to the present invention's observations concerning true leaf derived plant regeneration, although in this study only five plants were regenerated from leaves, it is remarkably how all of them were originated from leaf-petiole transition zone. The fact that vascularization also takes place in petioles, as it does also on stems, and that leaf regenerated plants always emerged from petioles, could fit with the present invention's hypothesis concerning pericycle-derived in vitro shoot organogenesis in this species. This extends the scope of the present invention's protocol towards micropropagation purposes, adding the possibility to produce multiple clones genetically identical to the mature elite plants already selected from which they could be derived. In this respect, it is important to emphasize how cotyledon, hypocotyl, true leaf and epicotyl derived regenerants, while were subcultured in vessels, continued producing multiple shoots even six months after culture initiation.
[0101] It has been described in the state of the art how pericycle cells encircling the xylem pole retain the capacity to undergo asymmetric cell division even when other cells have differentiated, and that some pericycle cells surrounded by differentiated cells can still become programmed to begin to proliferate, thus leading to the initiation of a new organ (Beeckman and De Smet, 2014). These findings explain how in the present invention, direct in vitro plant regeneration always developed directly with no need of cell dedifferentiation. It is believed that the implication of vascular traces on the regenerative capacity of the evaluated explants could explain the different shoot organogenesis processes according to the present invention.
[0102] The present invention is also based on the findings that polysomaty is present in cotyledons, epicotyls and hypocotyls of C. sativa seedlings.
[0103] The term polysomaty refers to the condition of those cells in the somatic tissues of a plant which contain multiples of the typical chromosome number (Ervin, 1941). Its role as one of the most crucial pathways in introducing speciation and broadening biodiversity, especially in the plant kingdom, has been highlighted by the authors skilled in the art (Van Hieu, 2019).
[0104] Endomitosis or endoreduplication are described as possible causes that may lead to polysomaty (D'Amato, 1964; Bubner et al., 2006), whose occurrence is related with growth and differentiation of tissues. It has also been described how plant tissues frequently contain a proportion of endopolyploid cells (Ramsay and Kumar, 1990, and references therein) and how portions of the plant such as storage organs and vessels often contain polyploid cells (Adelberg et al., 1993). Concerning polysomaty in C. sativa, it should be noted how it was first described in root meristems of the species by Litardiére (1925) and how it is known that the doubled number of chromosomes in root meristems coming from C. sativa resulted from two successive cleavages of each chromosome during the prophases (Langlet, 1927). Other authors proposed nuclear fusion as the cause of the polysomatic condition described in C. sativa roots (Breslavetz, 1926, 1932). The author of the present invention has analyzed the ploidy level of cotyledons, hypocotyls, epicotyls and true leaves coming from seven-days-old seedlings of C. sativa by means of flow cytometry and have described for the first time polysomaty in cotyledons, epicotyls and hypocotyls of C. sativa. In light of these results, while leaves preserved the diploid pattern typical of the species, cotyledons, epicotyls and hypocotyls displayed a polysomatic pattern containing diploid and tetraploid cells, and therefore should be considered as mixoploid organs. These findings concerning polysomaty found in cotyledons, epicotyls and hypocotyls of C. sativa, open new applications of the method of the invention, such as the development of polyploids through in vitro direct plant regeneration from these organs.
[0105] The present invention is also based on the findings that mixoploid plants can be regenerated after in vitro culture of hypocotyls, epicotyls and cotyledons coming from seedlings of Cannabis.
[0106] Polyploids are associated with enlarged organ sizes, increased biomass yield, phytochemical content and metabolic products, enhanced ability to adapt to biotic and abiotic stresses, and with changes on gene regulation (Van Hieu, 2019). Additionally, development of polyploid plants, in particular tetraploids, could be useful in plant breeding for development of triploid varieties with seedless fruits. Since polyploid nuclei may sometimes be the progenitors of a cell generation, giving rise to a patch of polyploid tissues (D'Amato, 1952) and after being aware of polysomaty in cotyledons, epicotyls and hypocotyls of C. sativa, the author of the present invention evaluated the ploidy level of in vitro regenerants. In this respect, no significant differences were detected between explants in terms of polyploidization of regenerated plants and it has been described how cotyledon, epicotyl and hypocotyl are the only explants capable to generate mixoploid plants. It should be noted how polyploidization uses to be associated with enhanced levels of secondary metabolites in a large number of species (Iannicelli et al., 2019).
[0107] Polyploidization in C. sativa has always been induced by treating apical meristems with chemical microtubule disruptors with a high toxicity grade (Mansouri and Bagheri, 2017; Parsons et al., 2019), for which polyploid plants often revert back to the diploid condition (Clarke, 1981), forcing to test the ploidy level of polyploid plants throughout generations.
[0108] In conclusion, due the high regenerative capacity of hypocotyls and epicotyls and that only hypocotyl and epicotyl-derived in vitro regenerants have been able to spontaneously rooting, together with the absence of significant differences between media with the best shoot induction rates with respect to number of shoots per responding explant and between cotyledon, hypocotyl and epicotyl derived plants in terms of polyploidization, the present invention contemplates as a preferred embodiment of the invention the culture of hypocotyls and epicotyls in hormone-free medium without chemical microtubule disruptors as the most suitable of the treatments evaluated in this study in order to obtain polyploid Cannabis plants. The present invention's method for in vitro direct regeneration of Cannabis plants has important connotations in exploitation of contemporary plant breeding techniques like genome editing (e.g., by using CRISPR/Cas gene edition) or mutagenesis, being also useful for micropropagation and for the development of polyploid varieties with enhanced levels of cannabinoids without using toxic chemical inductors.
[0109] The method for in vitro direct regeneration of a polyploid Cannabis plant comprises the following steps: [0110] a) Collecting seeds of a donor Cannabis plant; [0111] b) Surface sterilization of the seeds with ethanol, bleach, mercuric chloride or other chemical or physical disinfectant agent. In a preferred embodiment, seeds are surface sterilized in 75% (v/v) ethanol during two minutes and 30 seconds, followed by immersion in 30 g/L of NaClO with 0.1% (v/v) of Tween 20 during 25 minutes, being finally washed three times (1 minute, 4 minutes and 5 minutes) in autoclaved deionized water; [0112] c) Germination of the seeds in a Petri dish containing culture medium with plant growth regulators. In a preferred embodiment, the culture medium is composed of ½ MS basal salts and vitamins (Murashige and Skoog, 1962)+1.5% (w/v) sucrose+3.5 g/L Gelrite® with a pH value of 5.8. In some embodiments of the present invention the culture medium is hormone free; [0113] d) Dissection of cotyledons, hypocotyls and/or epicotyls comprising pericycle cells adjacent to xylem poles from a plant in a phenological growth stage coded from 00 to 99, more preferably 05 to 19 according to BBCH-scale of Mishchenko et al. (2017), more preferably, from seven-days-old Cannabis seedlings. In some embodiments, hypocotyls or epicotyls are dissected from seedlings at a phenological growth stage coded by number 11 of BBCH-scale for C. sativa (Mishchenko et al., 2017); [0114] e) Culturing of the explants in a culture media under controlled conditions of temperature and relative humidity during 2-3 weeks. In a preferred embodiment, explants are cultured at 22° C.±1° C. and 60%±1% relative humidity with a photoperiod of 16 hours of light per day. In a preferred embodiment, plant growth regulators and/or hormones are absent in the culture medium. In some preferred embodiments, polyploid plants are regenerated from cotyledon, hypocotyl and/or epicolyl explants without using any chemical microtubule disruptor with a high toxicity grade in the culture medium; [0115] f) Selection of embryos, shoots and/or shoots with roots; [0116] g) Sub-culturing the specimens of f) individually to glass-tubes or other containers of different volumes with the culture media used in step e) until spontaneously rooted plants are generated or until spontaneously rooted plants develop enough to start the acclimatization process; [0117] h) Transplanting spontaneously rooted plants in pots with fertilized substrate and acclimatizing as needed;
[0118] Culture Medium Used in Steps c), e) and g)
[0119] The present invention describes semi-solid or liquid media including, but not limited to macronutrients like CaCl.sub.2), Ca(NO.sub.3).sub.24H.sub.2O, MgSO.sub.4, NaH.sub.2PO.sub.4, (NH.sub.4).sub.2SO.sub.4, NH.sub.4NO.sub.3, KNO.sub.3, KH.sub.2PO.sub.4 and K.sub.2SO.sub.4, in concentrations ranging, but not limited to between 50 and 5,000 mg/L, including, but not limited to micronutrients like CoCl.sub.26H.sub.2O, CuSO.sub.45H.sub.2O, FeNaEDTA, H.sub.3BO.sub.3, KI, MnSO.sub.4H.sub.2O, Na.sub.2MoO.sub.42H.sub.2O and ZnSO.sub.47H.sub.2O, in concentrations ranging, but not limited to between 0.001 and 40 mg/L, including, but not limited to vitamins like glycine, myo-inositol, nicotinic acid, pyridoxine HCl, thiamine HCl, biotin, D(+)-biotine, folic acid, L-glutamine, gluthatione (reduced) and L-serine in concentrations ranging, but not limited to between 0.01 and 1,000 mg/L, including, but not limited to carbon sources like sucrose, D-fructose, D-galactose, D-glucose monohydrate, lactose monohydrate, maltose monohydrate, D-mannose, D-mannitol or D-sorbitol, including, but not limited to concentrations ranging from 1 g/L to 300 g/L, with or without gelling agents including, but not limited to plant agar, Daishin agar or Gelrite®, including, but not limited to concentrations ranging from 0 g/L to 10 g/L, with or without active charcoal, including, but not limited to concentrations ranging from 0 to 100 g/L, without plant growth regulators and without chemical microtubule disruptors. The pH of the media is adjusted to a value between 4.0 and 8.0.
[0120] In some embodiments, hypocotyls or epicotyls are preferably cultured in medium without plant growth regulators or hormones due spontaneous rooting of around 20% of in vitro regenerated plants.
[0121] In some embodiments, any medium, or combinations thereof, of the present disclosure may be utilized for the in vitro culture of Cannabis sativa L. explants in plastic or glass tubes, vessels or containers of multiple volumes.
[0122] The present invention includes a unique culture in which in vitro plants are regenerated and rooted in the same step or multiple and sequential subcultures of the original explant or in vitro regenerated plantlets in a different container with the same or a different medium (e.g. culturing the original explant or in vitro regenerated plants in a different container with the same or a different medium for promoting growth of regenerated shoots, continuous, exponential and unlimited regeneration of shoots or the induction of rooting of in vitro regenerated plantlets).
EXAMPLES
Example 1: Plant Material and Growth Conditions
[0123] Seeds from monoecious C. sativa short-day varieties Ferimon, Felina32, Fedora17 and USO31, together with seeds from dioecious neutral-day variety Finola were surface sterilized in 75% (v/v) ethanol during two minutes and 30 seconds, followed by immersion in 30 g/L of NaClO with 0.1% (v/v) of Tween 20 during 25 minutes, and finally washed three times in autoclaved deionized water. Once sterilized, seeds were germinated in 9 cm diameter Petri dishes containing previously autoclaved germination medium whose composition was ½ MS basal salts and vitamins (Murashige and Skoog, 1962)+1.5% (w/v) sucrose+3.5 g/L Gelrite® with a pH value of 5.8. After germination, explants (cotyledons, hypocotyls, leaf and epicotyls) dissected from seven-days-old seedlings, which is equivalent to the phenological growth stage coded in this species by number 11 in BBCH-scale (Mishchenko et al., 2017), were cultured in different media described in Table 1 below.
[0124] The culture medium is composed of ½ MS basal salts and vitamins (Murashige and Skoog, 1962)+1.5% (w/v) sucrose+3.5 g/L Gelrite®. The different culture media referred in Table 1 (see below) contain such medium composition without plant growth regulators (medium 0) and with different plant growth regulators (media 1 to 9).
TABLE-US-00001 TABLE 1 Media tested for in vitro shoot induction from cotyledons, hypocotyls, true leaves and epicotyls of C. sativa, including plant growth regulators composition and their respective concentrations. Plant growth regulators and Medium concentrations (mg/L) Reference 0 Without plant growth — regulators (Control) 1 TDZ (0.4) + NAA (0.2) (Chaohua et al., 2016) 2 BAP (2.0) + IBA (0.5) (Movahedi et al., 2015) 3 BAP (0.5) + 2,4-D (0.1) (Movahedi et al., 2016a) 4 ZEA.sup.RIB (2.0) (García-Fortea et al., 2019) 5 BAP (1.0) + NAA (0.02) — 6 BAP.sup.RIB (1.0) + NAA (0.02) — 7 TDZ (1.0) + NAA (0.02) — 8 4-CPPU (1.0) + NAA (0.02) — 9 ZEA.sup.RIB (1.0) + NAA (0.02) —
[0125] Seedlings and explants were grown under controlled conditions at 22° C.±1° C. and 60%±1% relative humidity. Photoperiod consisted of 16 hours of light and 8 hours of dark. Light was provided by LED tubes of 18 W with a color temperature of 6,000K, which was translated in 6,010 lux and 90.1 μmol m.sup.−2 s.sup.−1. Explants producing shoots and roots, and number of shoots developed on each of responding explants were counted periodically during two weeks of culture. After that time, in vitro regenerants were subcultured individually to glass-tubes of 2.5 cm of diameter and 15 cm long, containing the same medium in which shoots were generated.
[0126] When roots were visible, spontaneously-rooted plants were cultured in pots (2 L) with fertilized commercial substrate with a pH value of 6 and a conductivity of 1 mS/cm. Previously, gelled medium was carefully washed from roots. After transplant and during the whole process of acclimatization, the substrate was maintained slightly moist and, twice per day, regenerants received foliar pulverization with water. To avoid desiccation, the small plants were covered with plastic vessels and were progressively exposed to the environmental humidity. Until complete acclimatization, plants were grown under identical conditions of temperature, photoperiod and light as described above.
Example 2: In Vitro Shoot Organogenesis Experiments
[0127] In order to promote in vitro shoot organogenesis in C. sativa, cotyledons, hypocotyls, true leaves and epicotyls dissected from seven-days-old seedlings of the short and neutral-day varieties, were cultured in culture medium with the same composition as described in Example 1, except for the addition of different plant growth regulators. As a part of this study, the author of the present invention aimed at evaluating with their own genotypes the efficiency of different in vitro shoot regeneration published protocols developed for C. sativa. Therefore, he selected studies in which different explants, cytokinins and auxins and their ratios were successfully tested. In this way, the following medium used in a study regarding the regenerative capacity of cotyledons was tested through addition of thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA) to the culture medium (Chaohua et al., 2016), one work concerning in vitro plant regeneration from cotyledons and epicotyls by means of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) (Movahedi et al., 2015), and another one from leaves and hypocotyls through BAP and 2,4-dichlorophenoxyacetic acid (2,4-D) (Movahedi et al., 2016a). Additionally, it was added to the present study an effective and newly released protocol developed for eggplant in which the use of zeatin riboside (ZEA.sup.RIB), provided good results not only in terms of shoot organogenesis, but also in polyploidization of regenerants (Garcia-Fortea et al., 2019).
[0128] Finally, as it is known that root and shoot development depends on cytokinin:auxin ratio, and that high levels of cytokinin supports shoot formation (Skoog and Miller, 1957; Su et al., 2011), the author of the present invention tested the effect of a cytokinin:auxin ratio of 50:1 with different adenine and phenylurea derivatives like BAP, 6-benzylaminopurine riboside (BAP.sup.RIB), TDZ, forchlorfenuron (4-CPPU) and ZEA.sup.RIB plus NAA, an auxin commonly employed in protocols for in vitro regeneration of shoots (Plawuszewski et al., 2006; Wielgus et al., 2008; Lata et al., 2010; Chaohua et al., 2016) and in vitro rooting of C. sativa(Lusarkiewicz-Jarzina et al., 2005; Wang et al., 2009; Movahedi et al., 2016a; Parsons et al., 2019). Results obtained from evaluation of all these hormonal combinations were compared with results extracted from cultures in medium without plant growth regulators. The different hormonal combinations present in the different shoot induction media evaluated in this work, are detailed in Table 1.
[0129] Results: Effect of Genotype, Explant and Medium on In Vitro Shoot Organogenesis of C. sativa
[0130] The effect of explant on direct in vitro shoot organogenesis of C. sativa is shown in Table 2 (see below). As can be seen from these results, hypocotyl and epicotyl are shown to be preferred explants for the method of direct in vitro regeneration of Cannabis plants according to the present invention.
TABLE-US-00002 TABLE 2 Effect of explant on direct in vitro shoot organogenesis rate of different explants from C. sativa. Mean of responding explants (%), significance and sample size (n) are presented in different columns. Mean of responding explants is expressed as a percentage (±SE) relative to the total amount of cultured explants. Responding Factor explants (%) Significance.sup.a n Explant Cotyledon 4.70 ± 0.66 c 1000 Hypocotyl 49.45 ± 3.02 a 275 Leaf 0.42 ± 0.18 d 1188 Epicotyl 22.22 ± 3.86 b 117 .sup.aDifferent letters among the factor levels indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
[0131] Shoot in vitro regeneration was observed in all C. sativa varieties, explant types and media tested, resulting in a total of 294 in vitro regenerated shoots, although significant differences (p<0.05) for the three main factors (variety, explant and culture medium) were observed. Regarding the factor explant and its effect on the percentage of explants developing shoots, significant differences were detected between cotyledons, hypocotyls, epicotyls and true leaves. On average, hypocotyl showed the best response in terms of direct plant regeneration, reaching 49.45% of explants with shoot formation, followed by epicotyl reaching 22.22%, cotyledon with 4.70% and true leaf with 0.42% (see Table 2 above).
[0132] Since true leaves displayed a weak capacity to induce in vitro direct shoot organogenesis, the author of the present invention analyzed separately data from epicotyls (see Table 3 below), cotyledons (see Table 4 below) and hypocotyls (see Table 5 below). When epicotyls were used as explant, they gave best performance in a culture medium free of plant growth regulators, as shown in Table 3 below.
TABLE-US-00003 TABLE 3 Effect of medium on direct in vitro shoot organogenesis rate of epicotyls from C. sativa. Mean of responding explants (%), significance and sample size (n) are presented in different columns. Mean of responding explants is expressed as a percentage (±SE) relative to the total amount of cultured explants. Responding Factor explants (%) Significance.sup.a n Medium (mg/L) 0 .fwdarw. Without plant growth 42.86 ± 13.73 a 14 regulators 1 .fwdarw. TDZ 0.4 + NAA 0.2 22.73 ± 9.14 b 22 2 .fwdarw. BAP 2 + IBA 0.5 0.00 ± 0.00 c 5 3 .fwdarw. BAP 0.5 + 2,4-D 0.1 0.00 ± 0.00 c 7 4 .fwdarw. ZEA.sup.RIB 2 22.22 ± 14.70 b 9 5 .fwdarw. BAP 1 + NAA 0.02 11.11 ± 11.11 bc 9 6 .fwdarw. BAP.sup.RIB 1 + NAA 0.02 11.11 ± 11.11 bc 9 7 .fwdarw. TDZ 1 + NAA 0.02 28.57 ± 12.53 ab 14 8 .fwdarw. 4-CPPU 1 + NAA 0.02 28.57 ± 12.53 ab 14 9 .fwdarw. ZEA.sup.RIB 1 + NAA 0.02 21.43 ± 11.38 b 14 .sup.aDifferent letters among the factor levels indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
[0133] Regarding cotyledons, significant differences were observed among the different media tested. Medium 1 (TDZ 0.4 mg/L+NAA 0.2 mg/L) was the best, achieving the highest shoot induction rate with a 22.32% of responding explants (see Table 4 below). Medium 0 (without plant growth regulators) and number 9 (ZEA.sup.RIB 1 mg/L+NAA 0.02 mg/L) were the worst treatments, without any explant showing response in terms of shoot organogenesis (see Table 4 below).
TABLE-US-00004 TABLE 4 Effect of medium on direct in vitro shoot organogenesis rate of cotyledons from C. sativa. Mean of responding explants (%), significance and sample size (n) are presented in different columns. Mean of responding explants is expressed as a percentage (±SE) relative to the total amount of cultured explants. Responding Factor explants (%) Significance.sup.a n Medium (mg/L) 0 .fwdarw. Without plant growth 0.00 ± 0.00 d 234 regulators 1 .fwdarw. TDZ 0.4 + NAA 0.2 22.32 ± 3.95 a 112 2 .fwdarw. BAP 2 + IBA 0.5 1.85 ± 1.30 c 108 3 .fwdarw. BAP 0.5 + 2,4-D 0.1 5.56 ± 2.42 bc 90 4 .fwdarw. ZEA.sup.RIB 2 1.28 ± 1.28 cd 78 5 .fwdarw. BAP 1 + NAA 0.02 1.92 ± 1.35 c 104 6 .fwdarw. BAP.sup.RIB 1 + NAA 0.02 6.25 ± 3.04 bc 64 7 .fwdarw. TDZ 1 + NAA 0.02 2.56 ± 1.80 c 78 8 .fwdarw. 4-CPPU 1 + NAA 0.02 14.29 ± 5.46 ab 42 9 .fwdarw. ZEA.sup.RIB 1 + NAA 0.02 0.00 ± 0.00 d 90 .sup.aDifferent letters among the factor levels indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
[0134] Concerning hypocotyl, significant differences were identified between the different varieties and media evaluated in this experiment. USO31 was the best variety evaluated, with 71.15% of its explants developing shoots (see Table 5 below), while Finola and Ferimon were the varieties with the lowest regeneration percentages with, respectively, 35.42% and 32.26% of its explants regenerating shoots (see Table 5 below). In relation to the effect of medium on shoot organogenesis, although media number 4 (ZEA.sup.RIB 2 mg/L) and number 9 (ZEA.sup.RIB 1 mg/L+NAA 0.02 mg/L) resulted in the highest rate of shoot induction with 66.67% of responding explants, followed by medium 0 (without plant growth regulators) and medium 1 (TDZ 0.4 mg/L+NAA 0.2 mg/L) with, respectively, 61.54% and 54.17% of shoot organogenesis rate, regarding percentage of responding explants, there were no significant differences among media with the best shoot induction rates and medium without plant growth regulators (see Table 5 below).
TABLE-US-00005 TABLE 5 Effect of genotype and medium on direct in vitro shoot organogenesis rate of hypocotyls from C. sativa. Mean of responding explants (%), significance and sample size (n) are presented in different columns. For each factor, mean of responding explants is expressed as a percentage (±SE) relative to the total amount of cultured explants. Responding Factor explants (%) Significance.sup.a n Variety Ferimon 32.26 ± 5.98 c 62 Felina32 62.50 ± 6.09 ab 64 Fedora17 44.90 ± 7.17 bc 49 USO31 71.15 ± 6.34 a 52 Finola 35.42 ± 6.97 c 48 Medium (mg/L) 0 .fwdarw. Without plant growth regulators 61.54 ± 7.89 ab 39 1 .fwdarw. TDZ 0.4 + NAA 0.2 54.17 ± 5.91 ab 72 2 .fwdarw. BAP 2 + IBA 0.5 36.67 ± 8.94 c 30 3 .fwdarw. BAP 0.5 + 2,4-D 0.1 38.71 ± 8.89 c 31 4 .fwdarw. ZEA.sup.RIB 2 66.67 ± 12.59 a 15 5 .fwdarw. BAP 1 + NAA 0.02 41.67 ± 10.27 bc 24 6 .fwdarw. BAP.sup.RIB 1 + NAA 0.02 43.75 ± 12.80 bc 16 7 .fwdarw. TDZ 1 + NAA 0.02 40.00 ± 13.09 c 15 8 .fwdarw. 4-CPPU 1 + NAA 0.02 38.89 ± 11.82 c 18 9 .fwdarw. ZEA.sup.RIB 1 + NAA 0.02 66.67 ± 12.59 a 15 .sup.aDifferent letters among the levels of each of the two factors indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
[0135] In addition, the number of shoots developed on each of the responding explants were statistically analyzed for the combinations of varieties and media with the best shoot induction rates identified in this study. In the case of cotyledons, as varieties USO31, Fedora17 and Ferimon and media 1 (TDZ 0.4 mg/L+NAA 0.2 mg/L) and 8 (4-CPPU 1 mg/L+NAA 0.02 mg/L) gave the best shoot induction rates, their number of shoots per responding explant were statistically compared (see Table 6 below). Although no significant differences were found between varieties and media in terms of number of shoots per responding cotyledon, Fedora17 showed the best results with 1.42 shoots per responding explant, while medium 1 (TDZ 0.4 mg/L+NAA 0.2 mg/L) reached 1.28 shoots per responding cotyledon (see Table 6 below).
TABLE-US-00006 TABLE 6 Effect of genotype and medium on the number of shoots per responding cotyledon of C. sativa. Mean number of shoots per responding explant (±SE), significance and sample size (n) are presented in different columns. Shoots per responding explant from varieties and media with the best shoot induction rates are statistically compared. Shoots per Factor responding explant Significance.sup.a n Variety Ferimon 1.09 ± 0.09 a 11 Fedora17 1.42 ± 0.15 a 12 USO31 1.00 ± 0.00 a 13 Medium (mg/L) 1 .fwdarw. TDZ 0.4 + NAA 0.2 1.28 ± 0.09 a 25 8 .fwdarw. 4-CPPU 1 + NAA 0.02 1.00 ± 0.00 a 6 .sup.aDifferent letters among the levels of each of the two factors indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
[0136] Regarding hypocotyls, since varieties USO31 and Felina32 and media 0 (without plant growth regulators), 1 (TDZ 0.4 mg/L+NAA 0.2 mg/L), 4 (ZEA.sup.RIB 2 mg/L) and 9 (ZEA.sup.RIB 1 mg/L+NAA 0.02 mg/L) attained the best shoot organogenesis rates, their number of shoots per responding explant were also statistically compared (see Table 7 below). Once again, USO31 exhibited the best response in terms of number of shoots per responding hypocotyl, reaching 1.70 shoots per responding explant (see Table 7 below). Furthermore, although no significant differences were found among media tested, medium 4 (ZEA.sup.RIB 2 mg/L), closely followed by medium 0 (without plant growth regulators), were the best media evaluated in this experiment with, respectively, 1.60 and 1.54 shoots per responding hypocotyl (see Table 7 below).
TABLE-US-00007 TABLE 7 Effect of genotype and medium on the number of shoots per responding hypocotyl of C. sativa. Mean number of shoots per responding explant (±SE), significance and sample size (n) are presented in different columns. Shoots per responding explant from varieties and media with the best shoot induction rates are statistically compared. Shoots per Factor responding explant Significance.sup.a n Variety Felina32 1.20 ± 0.06 b 40 USO31 1.72 ± 0.12 a 37 Medium (mg/L) 0 .fwdarw. Without plant growth 1.54 ± 0.12 a 24 regulators 1 .fwdarw. TDZ 0.4 + NAA 0.2 1.49 ± 0.11 a 39 4 .fwdarw. ZEA.sup.RIB 2 1.60 ± 0.16 a 10 9 .fwdarw. ZEA.sup.RIB 1 + NAA 0.02 1.30 ± 0.15 a 10 .sup.aDifferent letters among the levels of each of the two factors indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
[0137] Concerning epicotyls, since medium 0 (without plant growth regulators), medium 7 (TDZ 1 mg/L+NAA 0.02 mg/L) and medium 8 (4-CPPU 1 mg/L+NAA 0.02 mg/L) reached the highest percentages of responding explants, their data were employed to calculate the number of shoots per responding explant on each of them (see table 8 below). Although no significant differences were detected between the different media evaluated, medium 7 (TDZ 1 mg/L+NAA 0.02 mg/L) got the best results with 1.75 shoots per responding epicotyl (table 8).
TABLE-US-00008 TABLE 8 Effect of medium on the number of shoots per responding epicotyl of C. sativa. Mean number of shoots per responding explant (±SE), significance and sample size (n) are presented in different columns. Shoots per responding explant from media with the best shoot induction rates are statistically compared. Shoots per Factor responding explant Significance.sup.a n Medium (mg/L) 0 .fwdarw. Without plant growth 1.33 ± 0.21 a 6 regulators 7 .fwdarw. TDZ 1 + NAA 0.02 1.75 ± 0.25 a 4 8 .fwdarw. 4-CPPU 1 + NAA 0.02 1.5 ± 0.29 a 4 .sup.aDifferent letters among the factor levels indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
Example 3: Developmental Morphology of the In Vitro Regeneration Process
[0138] The whole developmental process of in vitro shoot organogenesis was followed since germination of seeds to the acclimatization of plants. The time needed for each of the different developmental stages was registered. High resolution images of the different developmental stages were recorded with a stereozoom-microscope equipped with a digital camera.
[0139] In order to estimate the time needed to obtain and acclimatize in vitro regenerated C. sativa plants, the duration of each of the different stages throughout whole culture process was recorded. Additionally, all the developmental process from germination of seeds until acclimatization of in vitro regenerated plants was registered with images. First of all, seeds of the different varieties (
[0140] At this stage of seedling development, cotyledons were easily dissected (
[0141] Alternatively, hypocotyls were cut from seven-days-old seedlings. Hypocotyls employed in this experiment measured approximately one centimeter in height (
[0142] On the other hand, the regenerative capacity of the first pair of true leaves from seven-days-old seedlings was also studied. For this, each leaf was carefully dissected (
[0143] Finally, the different developmental stages of shoot regeneration from epicotyls were carefully examined. Epicotyls were dissected from C. sativa seedlings with four pairs of true leaves (
Example 4: Rooting of Explants, Spontaneous Rooting of Hypocotyl-Derived Plants and Plant Acclimatization
[0144] Although the present study and its derived experiments were focused on in vitro shoot organogenesis, some of the cultured explants developed roots instead of shoots. Specifically, two weeks after culture initiation, 1.09% of cultured hypocotyls developed vigorous roots with root hairs on the lower zone of the explant, as illustrated in
[0145] After spontaneous rooting of in vitro regenerants, these plantlets were submitted to the acclimatization process. The first step consisted of carefully washing the remaining gellified medium from roots. After 28 days of in vitro culture, regenerants showed different root morphogenesis patterns as illustrated in
Example 5: Determination of Ploidy Level of Explants and In Vitro Regenerants
[0146] Ploidy level of cotyledons, hypocotyls, epicotyls and leaves from in vitro grown seven-days-old seedlings was evaluated to verify their polysomatic pattern. The four short-day varieties Ferimon, Felina32, Fedora17 and USO31, together with dioecious neutral-day variety Finola were analyzed in this experiment. Three seedlings coming from each variety were employed for this assay. On the other hand, young leaves from in vitro regenerated plants were also examined. Ploidy level of 38 in vitro regenerants (17 from cotyledons, 15 from hypocotyls, three from epicotyls and three from leaves) was determined. Cell nuclei of explants dissected were mechanically isolated. Sections of approximately 0.5 cm.sup.2 were chopped with a razor blade in a 6 cm diameter glass Petri dish containing 0.5 ml lysis buffer LB01 (pH 7.5) (Dpooležel et al., 1989), and incubated for 5 minutes. Subsequently, the suspension containing nuclei and cell fragments was filtered using a 30 μm CellTrics filter (Sysmex, Sant Just Desvern, Spain). The nuclei in the filtrate were stained with CyStain UV Ploidy (Sysmex) and incubated for 5 minutes. The fluorescence intensity of the homogenate was measured using a CyFlow® Ploidy Analyser Sysmex Partec GmbH, analyzing at least 4,000 nuclei for each sample. Young leaves of diploid plants from all varieties studied were used as control. A diploid control peak was established at 50 points of the arbitrary intensity value of the fluorescence in the histogram. By comparison with this peak, the ploidy of the other tissues evaluated was checked.
[0147] The analysis of the ploidy level of freshly dissected cotyledons, hypocotyls, epicotyls and true leaves of seven-days-old seedlings of the five varieties evaluated revealed that only true-leaves (green) showed a diploid pattern, while cotyledons (blue), hypocotyls (red) and epicotyls (black) exhibited a mixoploid pattern (with diploid and tetraploid cells) (
[0148] A total of 38 in vitro regenerated plants (17 from cotyledons, 15 from hypocotyls, three from epicotyls and three from leaves) were analyzed by means of flow cytometry at 28 days after tissue culture initiation. Only diploid and mixoploid plants (with diploid and tetraploid cells) were obtained. Differences in nuclear DNA histogram patterns between diploid (
TABLE-US-00009 TABLE 9 Effect of explant on ploidy level of in vitro regenerated plants coming from cotyledons, hypocotyls, epicotyls and leaves of C. sativa. Mean of diploid and mixopioid regenerants (%), significance and sample size (n) values are presented in different columns. For each explant, mean of diploid and mixopioid plants is expressed as a percentage (±SE) relative to the total amount of plants submitted to flow cytometry analysis. 2 X REGENERANTS 2 X + 4 X REGENERANTS Diploid Signifi- Mixopioid Signifi- Factor n regenerants (%) cance.sup.a regenerants (%) cance.sup.a Explant Cotyledon 17 82.35 ± 9.53 a 17.65 ± 9.53 a Hypocotyl 15 86.67 ± 9.08 a 13.33 ± 9.08 a Epicotyl 3 66.66 ± 33.33 a 33.33 ± 33.33 a Leaf 3 100.00 ± 0.00 a 0.00 ± 0.00 a .sup.aDifferent letters among the levels of explant factor indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
Example 6: Obtention of Transgenic or Gene-Edited Cannabis Plants from Cotyledons and Hypocotyls
[0149] Hypocotyls and cotyledons were exposed to Agrobacterium tumefaciens strain LBA4404 containing binary plasmid pB1121 carrying ß-glucuronidase (GUS) and kanamycin resistance neomycin phosphotransferase 11 (NPTII) genes. Hypocotyls dissected from seven-days-old C. sativa seedlings (
[0150] Regarding cotyledon-derived regenerants transformation, cotyledons dissected from seven-days-old C. sativa seedlings (
[0151] With respect to the response of regenerants after kanamycin exposure, all those hypocotyl and cotyledon derived transformed regenerants that showed expression of the GUS gene, and in which the presence of the GUS and kanamycin resistance neomycin phosphotransferase II (NPTII) genes was detected through polymerase chain reaction (PCR) (
[0152] It should be noted that cotyledon regeneration rate was severely affected after co-culture with A. tumefaciens. Cotyledon and hypocotyl regeneration and transformation rates are described in, respectively, table 10 and table 11.
TABLE-US-00010 TABLE 10 Effect of A. tumefaciens co-culture on regeneration rates of hypocotyls and cotyledons from Cannabis sativa L. Mean of responding explants (%), significance and sample size (n) are presented in different columns. For each factor, mean of responding explants is expressed as a percentage (±SE) relative to the total amount of cultured explants. Factor Responding explants (%) Significance.sup.a n Explant Cotyledon 1.02 ± 0.25 b 1569 Hypocotyl 25.06 ± 1.50 a 834 .sup.aDifferent letters among the levels of explant factor indicate significant differences between them (p < 0.05) according to non-parametric Kruskal-Wallis and pairwise Wilcoxon tests.
TABLE-US-00011 TABLE 11 Effect of explant on production of Cannabis transgenic plants after co-culture of hypocotyls and cotyledons coming from Cannabis sativa L with A. tumefaciens. Only were considered transformed regenerants those that showed a green phenotype after culture on selective regeneration medium, expression of the GUS gene after incubation in X-gluc, and in which the presence of the GUS and kanamycin resistance neomycin phosphotransferase II (NPTII) genes was detected through polymerase chain reaction (PCR). Factor Transformation rate (%) n Explant Cotyledon 100 ± 0.00 2 Hypocotyl 5.00 ± 2.00 120
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