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Induction of Polyploidy and Its Effect on Cannabis sativa L.

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Abstract

Polyploids are organisms with three or more complete chromosome sets. Polyploidization is widespread in plants, and is an important mechanism of speciation. Polyploids can be formed in various ways. The study of polyploids has both important theoretical significance and valuable applications. The production and application of polyploidy breeding have brought remarkable economic and social benefits. We reported the production of putative tetraploid plants of Cannabis sativa L., with the ultimate aim of improving the medicinal and physiological traits of this widely distributed cultivated plant. The production of tetraploid plant was improved with colchicine at different concentrations and time through dropping method. Flow cytometry analysis was used to confirm the ploidy level. Morphologic, anatomic and biochemical characteristics were compared between tetraploid and diploid control plants. The results showed that 0.2% colchicine for 24 h was the most efficient for production of polyploid plants. The percentage of tetraploid plants and the survival rate were lowered by the increasing the treatment time. In addition, the leaf index and height of tetraploid plants exhibited a significant decrease compared to the diploid plants. The size of stomata on epidermis of leaves were larger in tetraploid plant compared to the diploid ones, in spite of the tetraploid plants have less stomata density. However, the amount of total chlorophyll and carotenoids were almost the same in both tetraploid and diploid plants. In addition, some differences were also observed in the cross section of stem of these plants from a descriptive structural point of view. Overall, the results introduced usage of the stomata parameters as an effective, fast and convenient method for detecting the tetraploid plants. We also investigated polyploidy effects on some primary and secondary metabolites. The results of biochemical analyzes showed that soluble sugars and total protein content increased significantly into mixoploid plants compared to tetraploid and diploid plants. Tetraploid plants had higher amount of total proteins compared with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabionol only in mixoploid plants but tetraploid plants had lower amounts of this substance in comparison with diploids. Our results suggest that tetraploidization was not useful for production of tetrahydrocannabinol for commercial use but mixoploids were found suitable.

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... Shorter plants; larger stomata cells, guard cells and male flowers; thicker xylem tissue, fewer primary and secondary fibre; lower leaf index; decrease in the density of stomata cells and trichomes 2n = 4x [49,101] Intermediate stomata size between 2x 1 and 4x 3 2n = 3x [49] Larger stomata by about 30% and decrease in their density; larger leaves; increase in trichome density by about 40% 2n = 4x [74] Eucommia Salix viminalis L. Larger leaves and stem diameter; enlarged bark and wood layers; better developed root system 2n = 4x [75] Taraxacum kok-saghyz Rodin. ...
... In some species, polyploid forms are larger and stronger compared to diploid forms, e.g., in T. vulgaris [79], T. ammi [105], M. chamomilla [3], A. vera [106,109], Z. officinale [111]. In many other species, such as C. sativa [74,101], C. intybus [90], E. purpurea [104,115], polyploid plants are lower, or changes are hardly noticeable. The optimum degree of ploidy of a particular species, beyond which there are disturbances in tissue structure, development, a decrease in viability and productivity, is determined experimentally. ...
... For example, tetraploids of P. tomentosa, whose flowering takes place similarly to diploids in the second year after planting, have the same course [82]. In contrast, tetraploids of C. sativa reach the flowering stage two weeks earlier than the control diploids [101]. ...
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Article
Polyploidisation is an important process in the evolution of many plant species. An additional set of chromosomes can be derived from intraspecific genome duplication (autopolyploidy) or hybridising divergent genomes and chromosome doubling (allopolyploidy). Special forms of polyploidy are autoallopolyploidy and segmental allopolyploidy. Polyploidy arises from two basic processes: spontaneously occurring disturbances of meiotic division and induced by antimitotic agents’ disruption of mitosis. The first involves the induction and fusion of unreduced gametes, resulting in the formation of triploids and tetraploids. The second process uses antimitotics that disrupt cellular microtubules and prevent chromosome’s sister chromatids motion during anaphase. Colchicine, oryzalin, and trifluralin are the most commonly used antimitotics for inducing polyploids in plants. The exposure time and concentration of the antimitotics and the species, cultivar, genotype, and tissue type affect the efficiency of genome duplication. Polyploids are distinguished from diploids by increased cell size and vegetative parts of plants and increased content of secondary metabolites. Genome duplication generates several changes at the epigenetic level resulting in altered gene expression. Polyploidisation is used in plant breeding to overcome the non-viability and infertility of interspecific hybrids, obtain seedless polyploid cultivars and increase resistance/tolerance to biotic and abiotic factors.
... The above-ground shoot weight of the tetraploid plants was almost twice the mass of the diploids. At the cellular level, it is noted that tetraploid individuals have larger mesophyll cells and less intercellular space [162]. A recent study [161] reported a successful in vitro polyploidy induction in a drug type cannabis using oryzalin. ...
... In recent literature, it has been reported that polyploid cannabis plants had lower THC production compared to diploid controls but also had increased CBD production [161]. In hemp, the polyploid individuals produced on average 50% less THC in the female flowers compared to the control, but CBD production in the female leaves was more than three times greater in the polyploid population [162]. This study utilized a hemp variety of cannabis, which is bred for low secondary metabolite production [162]. ...
... In hemp, the polyploid individuals produced on average 50% less THC in the female flowers compared to the control, but CBD production in the female leaves was more than three times greater in the polyploid population [162]. This study utilized a hemp variety of cannabis, which is bred for low secondary metabolite production [162]. Duplication of some deleterious recessive alleles may be responsible for the decreased THC concentration observed in polyploid cannabis plants. ...
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For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.
... Doubling the chromosome set should allow more flexibility to increase potency or tailor the cannabinoid ratios. A handful of studies support the theory that polyploid Cannabis might have higher potency, although the results are mixed, with some studies finding decreases in THC (Clarke, 1981;Bagheri and Mansouri, 2015;Mansouri and Bagheri, 2017). However, these studies were conducted with hemp. ...
... Here, we show that treatment of axillary buds with the dinitroaniline herbicide oryzalin is an effective method for chromosome doubling. Past studies on the polyploidization of hemp (Bagheri and Mansouri, 2015;Mansouri and Bagheri, 2017) and its closest relative hops (Humulus lupulus L.) used colchicine for doubling (Roy et al., 2001;Trojak-Goluch and Skomra, 2013). However, oryzalin has greater specificity for plant tubulins (Morejohn et al., 1987) and is considered a more effective and less toxic alternative to colchicine (Petersen et al., 2003;Stanys et al., 2006;Ascough et al., 2008;Dhooghe et al., 2009;Sakhanokho et al., 2009;Viehmannová et al., 2009;Rêgo et al., 2011). ...
... Stomata were also about 30% larger (length and width) and less than half as dense (46%) compared to diploid leaves. Tetraploids of hemp also exhibit a lower density of stomata and stomata guard cells with larger length and diameter, and leaves are shorter and wider compared to diploids (Mansouri and Bagheri, 2017). Changes in stomata size and density are common among tetraploids (Ascough et al., 2008;Sakhanokho et al., 2009;Rêgo et al., 2011;Talebi et al., 2017). ...
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Cannabis sativa L. is a diploid species, cultivated throughout the ages as a source of fiber, food, and secondary metabolites with therapeutic and recreational properties. Polyploidization is considered as a valuable tool in the genetic improvement of crop plants. Although this method has been used in hemp-type Cannabis, it has never been applied to drug-type strains. Here, we describe the development of tetraploid drug-type Cannabis lines and test whether this transformation alters yield or the profile of important secondary metabolites: Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), or terpenes. The mitotic spindle inhibitor oryzalin was used to induce polyploids in a THC/CBD balanced drug-type strain of Cannabis sativa. Cultured axillary bud explants were exposed to a range of oryzalin concentrations for 24 h. Flow cytometry was used to assess the ploidy of regenerated shoots. Treatment with 20-40 μM oryzalin produced the highest number of tetraploids. Tetraploid clones were assessed for changes in morphology and chemical profile compared to diploid control plants. Tetraploid fan leaves were larger, with stomata about 30% larger and about half as dense compared to diploids. Trichome density was increased by about 40% on tetraploid sugar leaves, coupled with significant changes in the terpene profile and a 9% increase in CBD that was significant in buds. No significant increase in yield of dried bud or THC content was observed. This research lays important groundwork for the breeding and development of new Cannabis strains with diverse chemical profiles, of benefit to medical and recreational users.
... In particular, it is a negative regulator of tolerance to pathogens by targeting conserved sequences encoding the P-loop of NBS-LRR resistance proteins. As far as miR2916 is concerned, it has been reported to be responsive to boron stress in barley [49] and related to salinity tolerance in rice [50]. However, its role has not been investigated yet. ...
... This is particularly interesting since it has been long recognized that polyploidization can change the quality and quantity of secondary metabolites. Recently, Gaynor et al. [49] conducted a meta-analysis aimed at comparing the composition and concentration of secondary metabolites among closely related plant species differing in ploidy level. Although the authors suggested that it was not possible to determine the overall effect of whole-genome doubling on secondary metabolites, mainly due to the heterogeneity in methodological and analytical approaches used, targeted studies proved their increase in many plant species. ...
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Whole-genome doubling (polyploidy) is common in angiosperms. Several studies have indicated that it is often associated with molecular, physiological, and phenotypic changes. Mounting evidence has pointed out that micro-RNAs (miRNAs) may have an important role in whole-genome doubling. However, an integrative approach that compares miRNA expression in polyploids is still lacking. Here, a re-analysis of already published RNAseq datasets was performed to identify microRNAs’ precursors (pre-miRNAs) in diploids (2x) and tetraploids (4x) of five species (Arabidopsis thaliana L., Morus alba L., Brassica rapa L., Isatis indigotica Fort., and Solanum commersonii Dun). We found 3568 pre-miRNAs, three of which (pre-miR414, pre-miR5538, and pre-miR5141) were abundant in all 2x, and were absent/low in their 4x counterparts. They are predicted to target more than one mRNA transcript, many belonging to transcription factors (TFs), DNA repair mechanisms, and related to stress. Sixteen pre-miRNAs were found in common in all 2x and 4x. Among them, pre-miRNA482, pre-miRNA2916, and pre-miRNA167 changed their expression after polyploidization, being induced or repressed in 4x plants. Based on our results, a common ploidy-dependent response was triggered in all species under investigation, which involves DNA repair, ATP-synthesis, terpenoid biosynthesis, and several stress-responsive transcripts. In addition, an ad hoc pre-miRNA expression analysis carried out solely on 2x vs. 4x samples of S. commersonii indicated that ploidy-dependent pre-miRNAs seem to actively regulate the nucleotide metabolism, probably to cope with the increased requirement for DNA building blocks caused by the augmented DNA content. Overall, the results outline the critical role of microRNA-mediated responses following autopolyploidization in plants.
... Tsuro et al. [32] have used callus for in vitro autoployploidy induction in Chamaemelum nobile L. Explant tissues should always receive uniform treatment otherwise it may lead to production of mixoploids having variable cytology [79]. Such results were found in the findings of Mansouri and Bagheri [36], although an phytochemical production and end-product realization. Divergent explants being in their divergent stages of physiological development ramify divergent responses. ...
... Occasionally appearing mixoploids (organisms with differential chromosome numbers across cells; chimera) express enrichment of pharmaceutical metabolites as seen in Stevia rebaudiana (Bertoni) (13.5% increase in steviosides content) [24]. Similarly, Mansouri and Bagheri [36] in their study reported an increase in cannabinoids in induced mixoploids over tetraploids. Such results were due to decreased trichomes in tetraploids than mixoploids, hinting at probable negatively correlated gene interactions. ...
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Background Induced polyploidy serves as an efficient approach in extricating genetic potential of cells. During polyploidization, multiple sets of chromosomes are derived from the same organism resulting in the development of an autopolyploid. Alterations owing to artificially induced polyploidy level significantly influence internal homeostatic condition of resultant cells. Main text Induced autopolyploidy transpires as a result of an increase in the size of genome without any change in elementary genetic material. Such autopolyploidy, artificially induced via application of antimitotic agents, brings about a lot of beneficial changes in plants, coupled with very few detrimental effects. Induced autopolyploids exhibit superior adaptability, endurance to biotic and abiotic stresses, longer reproductive period and enzyme diversity coupled with enhanced rate of photosynthesis and gene action in comparison to their diploid counterparts. However, reduced rate of transpiration and growth, delay in flowering are some of the demerits of autopolyploids. Inspite of these slight unfavourable outcomes, induced autopolyploidization has been utilized in an array of instances wherein genetic improvement of plant species is concerned, since this technique usually boosts the biomass of concerned economic parts of a plant. In other way, it is also evident that multiplication of genome bestows enhanced production of secondary metabolites, which has contributed to a significant commercial value addition especially for plants with medicinal importance, in particular. Conclusion This review makes an attempt to explore the system and success of antimitotic agents vis-à-vis artificial autopolyploidization, interfered with the biosynthesis-cum-production of secondary metabolites having cutting-edge pharmaceutical importance.
... Recent research has reported many cannabinoid pharmacodynamic and pharmacokinetic properties, expanding the potential use of cannabinoids in medical therapies (Urits et al., 2019), and promoting the development of cannabis improved varieties with specific biochemical profiles. In this respect, in vitro culture is a useful tool that has been employed to complement cannabis conventional breeding through largescale micropropagation of selected elite clones (Lata et al., 2017), development of polyploid varieties with enhanced levels of secondary metabolites (Mansouri and Bagheri, 2017;Parsons et al., 2019) or genetic transformation of non-regenerating tissues (Feeney and Punja, 2003Wahby et al., 2013Wahby et al., , 2017. However, there is still a lack of an in vitro regeneration protocol efficient in the broad range of genetically diverse materials in the species. ...
... While Clarke (1981) found that polyploidization increased THC levels while decreased CBD and cannabinol (CBN) content, and that triploids proved to be inferior to both diploids and tetraploids in terms of cannabinoid production, Parsons et al. (2019) got a similar chemical profile between tetraploids and diploids, although notable increases in CBD and sesquiterpenes were associated with tetraploids. In relation to mixoploids and their cannabinoid content, Mansouri and Bagheri (2017) demonstrated that polyploidization increased significantly the content of THC in mixoploid plants compared with tetraploid and diploid plants, and that tetraploid plants had lower amounts of this substance in comparison with diploids, suggesting that mixoploids could be useful to produce THC for commercial use. Finally, it should be emphasized how polyploidization in C. sativa has always been induced by treating apical meristems of the plant with chemical microtubule disruptors with a high toxicity grade, such as colchicine or oryzalin (a less toxic alternative to colchicine), and that chemically induced polyploid plants often revert back to the diploid condition (Clarke, 1981), forcing to test the ploidy level of polyploid plants throughout generations. ...
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In vitro shoot regeneration can efficiently contribute to the improvement of recalcitrant Cannabis sativa L. We aimed at developing a highly efficient protocol for in vitro direct regeneration of C. sativa plants from different explants (cotyledon, hypocotyl, and true leaf) from seedlings of monoecious C. sativa short-day varieties Ferimon, Felina32, Fedora17, and USO31, together with dioecious neutral-day variety Finola. Ten regeneration media, including already published protocols, and self-designed combinations of plant growth regulators were tested. The developmental morphology since germination of seeds to the development of rooted plantlets was followed. Additionally, the ploidy level of explants and in vitro regenerants was analyzed. We concluded that hypocotyl is the best explant for in vitro direct regeneration of C. sativa plants with 49.45% of responding explants, while cotyledon and true leaf had a poor response with, respectively, 4.70 and 0.42% of explants developing plantlets. In terms of shoot regeneration, we found significant differences among the culture media evaluated and the varieties studied. Overall, the best regeneration media were ZEARIB 2.0 (mg/L) and ZEARIB 1.0 (mg/L) + NAA 0.02 (mg/L) with 66.67% of responding hypocotyls. Amazingly, hypocotyls cultured in medium without plant growth regulators showed an excellent response (61.54% of responding hypocotyls) and spontaneous rooting of regenerants (17.94%). In vitro regenerated plants were acclimatized just 6 weeks after culture initiation. The developmental morphology study suggests that regenerated shoots originate from pericycle cells adjacent to xylem poles. Polysomaty was detected in hypocotyls and cotyledons of all varieties studied, and diploid (>80%) and mixoploid (with diploid and tetraploid cells) plants were regenerated. Our protocol allows a high shoot organogenesis efficiency in different C. sativa varieties. The fact that a significant percentage of plants are mixoploid may provide an alternative way to develop polyploids in C. sativa. Our results show that direct in vitro regeneration may make a significant contribution to the development of improved C. sativa materials for medical applications.
... Cannabis, scientifically known as Cannabis sativa L. from the family Cannabaceae, has been cultivated for ages and utilized as a potential source of fiber, oil, protein, and popularly, psychoactive and medicinal purposes [1]. Historically, cannabis products have been illicitly grown for recreational and social activities to appreciate their euphoric effects [2]. ...
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In recent years, cannabis (Cannabis sativa L.) has been legalized by many countries for production, processing, and use considering its tremendous medical and industrial applications. Cannabis contains more than a hundred biomolecules (cannabinoids) which have the potentiality to cure different chronic diseases. After harvesting, cannabis undergoes different postharvest operations including drying, curing, storage, etc. Presently, the cannabis industry relies on different traditional postharvest operations, which may result in an inconsistent quality of products. In this review, we aimed to describe the biosynthesis process of major cannabinoids, postharvest operations used by the cannabis industry, and the consequences of postharvest operations on the cannabinoid profile. As drying is the most important post-harvest operation of cannabis, the attributes associated with drying (water activity, equilibrium moisture content, sorption isotherms, etc.) and the significance of novel pre-treatments (microwave heating, cold plasma, ultrasound, pulse electric, irradiation, etc.) for improvement of the process are thoroughly discussed. Additionally, other operations, such as trimming, curing, packaging and storage, are discussed, and the effect of the different postharvest operations on the cannabinoid yield is summarized. A critical investigation of the factors involved in each postharvest operation is indeed key for obtaining quality products and for the sustainable development of the cannabis industry.
... Chlorophyll and carotenoids are two of the main pigments of plant chloroplasts. Previously, Mansouri and Bagheri [35] reported no differences in chlorophyll and carotenoid contents between diploid and tetraploid Cannabis sativa plants. However, in L. japonica, Li et al. [11] showed that the chlorophyll content of diploid plants is higher than that of tetraploids. ...
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Article
Cnidium officinale is an important medicinal crop grown in Asia for its pharmacological properties. In this study, tetraploid breeding was conducted to increases the content of medicinal compound and tolerance to the environmental conditions using in vitro shoot culture of C. officinale. For this, we generated tetraploid C. officinale plants using oryzalin, a chromosome doubling agent, and compared the morphological traits, cytological characteristics, and heat stress-responsive gene expression levels between tetraploid and diploid genotypes. Chromosome doubling efficiency was the highest in plantlets treated with 4.0 mg∙L−1 oryzalin for 2 days. Compared with diploids, the plant height of tetraploids was reduced, while the petiole diameter was increased by approximately 39%. The dry matter of tetraploid leaves was significantly higher than that of diploid leaves. Compared with diploids, tetraploids showed higher chloroplast number and stomatal complex size but lower chlorophyll and carotenoid contents. The phenolic content of tetraploid plantlets was significantly higher than that of diploid plantlets. Contents of naringin as well as salicylic acid and gentisic acid, which are strong antioxidant compounds, were dramatically increased upon tetraploidization. Interestingly, liquid chromatography–mass spectrometry (LC–MS) analyses revealed increased levels of senkyunolide F and phthalide in tetraploid roots but not in tetraploid or diploid leaves.
... There is only one report of a natural tetraploid of C. sativa, from India (Sharma et al., 2015). Tetraploid C. sativa has been produced using colchicine on seedling shoot tips (Bagheri and Mansouri, 2015;Mansouri and Bagheri, 2017), and by using oryzalin on in vitro nodal explants (Parsons et al., 2019). Tetraploid plants produced in these studies exhibited traits such as larger leaves and greater shoot fresh weight and flavonoid content. ...
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To maximize yield, cannabidiol (CBD) hemp producers prefer female plants, and this is accomplished by using expensive feminized seed, vegetatively propagated female clones, or by removing male plants from dioecious seed lots. Hemp pollen drifts long distances on wind, and pollination of females reduces CBD content. Induction of triploidy is a common strategy used by plant breeders to produce sterile cultivars of agricultural crops. Triploid (3n) hemp, with three sets of chromosomes, was developed by crossing naturally diploid (2n) hemp with tetraploid (4n) hemp. Tetraploid plants used to create triploids were produced using pregerminated seeds and the mitotic spindle inhibitor colchicine. Seedlings from seeds of ‘Abacas’ × [(‘Otto2’ × ‘BaOx’) × (‘BaOx’ × ‘Colorado Cherry’)] treated with 0.05% colchicine or 0.02% colchicine for 12 hours and longer were significantly shorter than controls and ≤1 cm tall at 10 days after sowing. Surviving seedlings exhibited thickened cotyledons and hypocotyls, which indicated a potential change in ploidy. Tetraploid induction ranged from 26% to 64% for pregerminated seeds of five different hemp cultivars (Abacus × Wife, Cherry Wine, Mountain Mango, Wife, and Youngsim10) treated with 0.05% colchicine for 12 hours. Tetraploids had nearly twice the DNA content as diploids according to flow cytometric analysis. Tetraploid ‘Wife’ had larger stomates and reduced stomatal density compared with diploid ‘Wife’. Four triploid ‘Wife’ genotypes produced from crossing tetraploid ‘Wife’ with diploid ‘Wife’ were acclimated to greenhouse conditions after embryo rescue. DNA content and stomate size of triploid ‘Wife’ was intermediate between the parents. This is the first report of triploid plants of hemp. Future research will evaluate the sterility of triploid hemp.
... Therefore, manipulation with ploidy, as a biotechnological tool, could be useful in improving pharmaceutical value of medicinal plants, since synthetic autopolyploids usually exhibit increases in bioactive metabolite concentration compared to their diploid progenitors (Javadian et al., 2017;Kong et al., 2017;Lavania et al., 2012;Sadat Noori et al., 2017;Tavan et al., 2015;Xu et al., 2014). However, several studies have shown that polyploidization did not affect the metabolite production (Hull-Sanders et al., 2009;Wohlmuth et al., 2005) or even that the effect of polyploidy on the production of specialized metabolites was negative (Caruso et al., 2013;Cohen et al., 2013;Mansouri and Bagheri, 2017;Tan et al., 2017). Furthermore, in comparison with diploids, polyploids generally produce larger cells which can result in an increased size of plant structures such as leaves, flowers and fruit, increased biomass and often show greater resistance to biotic and abiotic stress (Lavania et al., 2012;Yildiz, 2013). ...
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The impact of ploidy level on both the regenerative potential under in vitro conditions and the production of major bioactive specialized metabolites, such as iridoids and xanthones, was examined in Centaurium erythraea Rafn. Shoot regeneration frequency was genotype dependent, but not affected by explant ploidy level. In most cases, the regenerated shoots of autotetraploid (4x) genotypes were more robust than diploid (2x) ones. Regeneration efficiency of root explants declined from the apical to the basal root segment. Shoot and root biomass production of two month-old plants was not significantly different between 2x and 4x genotypes. Both 4x and 2x genotypes were characterized by the predominance of secoiridoid glucoside gentiopicrin in shoots and roots, which is followed by sweroside and swertiamarin. Loganic acid, loganin and secologanin were much less abundant. Methylbellidifolin was the major xanthone in both shoots and roots. Diploid plants showed higher biosynthetic capacity for the production of secoiridoids and xanthones in both shoots and roots. Results highlight a higher potential of diploid C. erythraea genotypes for biotechnology-based sustainable production of secoiridoids in comparison to tetraploid genotypes.
... Polyploidy involves the duplication of same genome in case of autopolyploids or different genomes in case of allopolyploids. Normally polyploids show enlargement in different plant parts or increase in secondary metabolites (Berkov 2001;Sanwal et al. 2010;Sun et al. 2011;Van Laere et al. 2011;Sattler et al. 2016), but there are many reports where there may be no effect or negative effect on the amount of secondary metabolites (Cohen et al. 2013;Mansouri and Bagheri 2017). Polyploids use shorter metabolic pathways for the utilisation of their resources. ...
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Siegesbeckia orientalis L. is an annual herb widely distributed throughout the world and has many medicinal properties. In Chinese traditional system, it is popularly known as Xi-Xian and used for its anti-inflammatory properties. In the present study, two cytotypes (diploid and tetraploid) have been investigated for their secondary metabolites. The different plant parts have been explored in terms of total phenolics, total flavonoids, DPPH radical scavenging acitivity and total antioxidant capacity. Out of different plant parts, leaves have the maximum amount of secondary metabolites and antioxidant potential. HPTLC technique has been applied to quantify six marker compounds in the two cytotypes. Tetraploid cytotype has been compared with diploid cytotype, which shows that tetraploid has the maximum amount of studied secondary metabolites with high antioxidant potential.
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Cannabis sativa has been used for millennia in traditional medicine for ritual purposes and for the production of food and fibres, thus, providing important and versatile services to humans. The species, which currently has a worldwide distribution, strikes out for displaying a huge morphological and chemical diversity. Differences in Cannabis genome size have also been found, suggesting it could be a useful character to differentiate between accessions. We used flow cytometry to investigate the extent of genome size diversity across 483 individuals belonging to 84 accessions, with a wide range of wild/feral, landrace, and cultivated accessions. We also carried out sex determination using the MADC2 marker and investigated the potential of flow cytometry as a method for early sex determination. All individuals were diploid, with genome sizes ranging from 1.810 up to 2.152 pg/2C (1.189-fold variation), apart from a triploid, with 2.884 pg/2C. Our results suggest that the geographical expansion of Cannabis and its domestication had little impact on its overall genome size. We found significant differences between the genome size of male and female individuals. Unfortunately, differences were, however, too small to be discriminated using flow cytometry through the direct processing of combined male and female individuals.
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We profiled and quantified primary (amine, organic acids, tricarboxylic acid cycle intermediates, amino acids, and carbohydrates) and secondary metabolites (triterpenoids, phenolic acids, carotenoids, flavonoids, and anthocyanins) in the edible parts (leaves and fruits) of the diploid and tetraploid cultivar Morus alba L. 'Cheongil.' Through comprehensive metabolic profiling, the tetraploid mulberry cultivar was able to produce diverse metabolites supported by higher accumulation patterns of primary and secondary metabolites in their edible parts. In particular, the edible parts of the tetraploid showed higher accumulation patterns of most metabolites (amino acids, carbohydrates, carotenoids, and anthocyanins) than the diploid, which was supported by the results of principal component analyses (PCAs) showing a clear separation between the diploid and tetraploid groups. Additionally, this metabolome study comprehensively described the correlation between primary and secondary metabolites in the edible parts of diploid and tetraploid mulberry cultivars and provided information useful for plant breeding strategies to improve metabolite biosynthesis using polyploidy.
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Main conclusion Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles. For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.
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An efficient colchicine-mediated chromosome doubling of diploid feverfew followed by the morphophenological, physiological, phytochemical, and cytological changes of the obtained tetraploid plants was conducted. One-week-old seedlings of feverfew were treated with 0.05% (w/v) colchicine for 2, 4, 6, 8, and 24 h. Tetraploid plants were regenerated after 4 months, showing significant changes in stomatal size and density; sizes of seed; flower, pollen, leaf, trichome, cell, nucleus, and parthenolide content; chromosome number; ploidy level; chlorophyll content index; and quantum efficiency of photosystem II. Such characteristics of induced tetraploid feverfews can be useful in medicinal and ornamental applications, e.g., the study of flower morphogenesis, trichome differentiation, and parthenolide biosynthesis. The increase in parthenolide in tetraploids of the next generation (selfed T0 plants) showed that ploidy induction is a good breeding method for feverfew.
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INTRODUCTION Polyploidy is an intriguing phenomenon in plants that has provided an important pathway for evolution and speciation. Although the first polyploid was discovered over a century ago, the genetic and evolutionary implications of polyploidy are still being elucidated (Bennett, 2004; Soltis et al., 2003). On a more practical level, there are many opportunities for utilizing polyploidy as a valuable tool in traditional plant breeding programs. WHAT IS POLYPLOIDY AND HOW DOES IT ARISE? A polyploid is simply an organism that contains more than two complete sets of chromosomes. For animals, this is a fairly rare occurrence (though a polyploid rat, the first polyploid mammal ever identified, was recently discovered in Argentina). In plants, however, polyploidy occurs naturally and is very common. The term "ploidy" or "ploidy level" refers to the number of complete sets of chro-mosomes and is notated by an "x." An individual with two sets of chromosomes is referred to as a diploid (2x), three sets would be a triploid (3x), and so on with tetraploid (4x), pentaploid (5x), hexaploid (6x), etc. It is sometimes also important to identify if one is referring to the reduced (gametophytic) chromosome number following meiosis as would be found in egg and sperm (denoted as "n") or in nonre-duced (sporophytic) tissue (denoted as "2n"). Thus, for example, a tetraploid birch tree would be presented as 2n = 4x = 56. Polyploidy can naturally arise in a number of different ways. In some cases a so-matic (nonreproductive) mutation can occur, due to a disruption in mitosis, resulting in chromosome doubling in a meristematic cell(s) that will give rise to a polyploid shoot. These sports are sometimes evident on a plant by their enlarged "gigas" con-dition. Polyploids can also result from the union of unreduced gametes — eggs and sperm that have not undergone normal meiosis and still have a 2n constitution. The origin of a polyploid can often determine if it will be fertile and may further indicate how it can best be used in a plant improvement program. If a tetraploid arises from spontaneous doubling in a shoot or from the union of unreduced gam-etes from two closely related (e.g., same species) diploid individuals, it will have four similar (homologous) versions of each chromosome. Despite different origins, both of these polyploids behave similarly reproductively and are often referred to as autotetraploids (or polysomic tetraploids). Autopolyploids may or may not be fertile. In diploids, meiosis involves the pairing of homologous chromosomes, which even-tually segregate to form two separate gametes, each with one set of chromosomes. Infertility can arise in autopolyploids due to the fact that there are more than two homologous chromosomes. The presence of multiple homologous chromosomes of-ten results in spurious pairing between multiple chromosomes, unpaired chromo-somes, and gametes with unbalanced chromosome numbers (anueploids).
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Seven tetraploid watermelon lines developed by colchicine treatments were compared with their diploid counterpart for plant, flower, fruit, seed and qualitative characteristics. Tetraploid genotypes attained statistically higher vine thickness (8.04 mm), leaf area (298.9 cm2) and chlorophyll content (55.6) while internode length and chlorophyll fluorescence was similar to their corresponding diploid. Both pistillate and staminate flower organs (pedicel, anther, ovary, stigma, petals) were larger in tetraploid plants; however, the percent increase in flower components varied across the tetraploid lines. Fruit weight and total sugar content (∘Brix) in both ploidy fruits was similar. Rind thickness in fruits varied significantly and averaged 12.7 and 17.2 mm in diploid and tetraploid fruits, respectively. Tetraploid genotypes showed sterility, yielded lower number of seed per fruit (37.9), and tetraploid seed was larger and thicker than diploid seed. Overall β-carotene (0.89), lycopene (1.16), fructose (5.43%) and glucose (2.38%) contents were higher in tetraploid than diploid fruits.
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Tetraploidy was induced in vitro in mat rush (Juncus effusus L.) cultivar Nonglin-4 by exposure to colchicine (0, 50, 100 and 500 mg dm−3) for 6, 12 and 24 h. Flow cytometric analysis was used to confirm the ploidy level. Anatomical and ultrastructural analyses at cellular and subcellular levels in tetraploid and diploid control plants revealed differences between diploid and tetraploid plants. The leaf epidermis had larger stomata but lower stomatal density in tetraploid plants. In addition, mesophyll cells in tetraploid plants appeared more compact and showed less intercellular spaces along with increased size of vascular bundles. However, a significant reduction of chlorophyll content was observed in tetraploid plants that might be the result of structural modification in the lamellar membranes of chloroplasts. Additional key wordschlorophyll-chloroplast ultrastructure-diploid- in vitro mutation-vascular bundle
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A high frequency of tetraploidy was induced in mulberry (Morus alba L.) through apical bud treatment under in vitro conditions. Apical buds from in vitro-grown plants were treated with three different concentrations (0.05, 0.1 and 0.2%) of colchicine in MS medium for 24 h. Tetraploidy at a frequency of 39.4±4.8% was obtained using 0.1% colchicine, whereas the frequency of tetraploidy was significantly reduced to 16.7±2.3% when 0.2% colchicine was used. Morphological, histological and cytological evidence indicated a phenotypic and genomic similarity of in vitro- with ex vitro-induced tetraploids. Rooting of tetraploids was on basal medium containing 2.6 μm NAA. The recovery of tetraploids was 80.8% more efficient using the in vitro method instead of the ex vitro method. The use of the same colchicine medium for up to 4 weeks with additional explants was found to be equally effective for the induction of tetraploidy.
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We report the production of tetraploid plants of Platanus acerifolia, with the ultimate aim of improving the ornamental qualities of this important urban landscaping tree. Chromosome doubling was achieved by the application of colchicine to either pre-soaked seed or to the apical meristems of young seedlings. Treatment of the ungerminated seed was the more efficient method in terms of numbers of tetraploid seedlings (up to 40%, as determined by chromosome counting of the root-tip nuclei) but this method produced no mature tetraploid plants due to the deleterious effect of colchicine on subsequent root growth. When colchicine was applied directly to the apical growing tip of cotyledon-stage seedlings, leaf and stem growth was temporarily affected but the plants eventually recovered. We conducted a preliminary screen for putative tetraploids based on the observation in other plant species of a correlation of stomatal size and distribution with ploidy. Plants containing significantly larger stomata and at a lower density across the lower leaf epidermis, were selected for further analysis by flow cytometry and chromosome counting. These techniques confirmed that, of the 12 putative polyploids, four were tetraploid, five were mixoploid and three were, in fact, diploid. Morphological differences of the tetraploids included a more compact growth habit and broader, thicker leaves. These plants are being grown to full maturity in order to test their potential for use in a breeding programme aimed at producing sterile triploid lines.
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Polyploids are organisms with three or more complete chromosome sets. Polyploidization is widespread in plants and animals, and is an important mechanism of speciation. Genome sequencing and related molecular systematics and bioinformatics studies on plants and animals in recent years support the view that species have been shaped by whole genome duplication during evolution. The stability of polyploids depends on rapid genome recombination and changes in gene expression after formation. The formation of polyploids and subsequent diploidization are important aspects in long-term evolution. Polyploids can be formed in various ways. Among them, hybrid organisms formed by distant hybridization could produce unreduced gametes and thus generate offspring with doubled chromosomes, which is a fast, efficient method of polyploidization. The formation of fertile polyploids not only promoted the interflow of genetic materials among species and enriched the species diversity, but also laid the foundation for polyploidy breeding. The study of polyploids has both important theoretical significance and valuable applications. The production and application of polyploidy breeding have brought remarkable economic and social benefits.
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Polyploidy is widely acknowledged as a major mechanism of adaptation and speciation in plants. The stages in polyploid evolution include frequent fertility bottlenecks and infrequent events such as gametic nonreduction and interspecific hybridization, yet little is known about how these and other factors influence over- all rates of polyploid formation. Here we review the literature regarding polyploid origins, and quantify parameter values for each of the steps involved in the princi- pal pathways. In contrast to the common claim that triploids are sterile, our results indicate that the triploid bridge pathway can contribute significantly to autopoly- ploid formation regardless of the mating system, and to allopolyploid formation in outcrossing taxa. We estimate that the total rate of autotetraploid formation is of the same order as the genic mutation rate (10¡5), and that a high frequency of interspecific hybridization (0.2% for selfing taxa, 2.7% for outcrossing taxa) is required for the rate of tetraploid formation via allopolyploidy to equal that by autopolyploidy. We conclude that the rate of autopolyploid formation may often be higher than the rate of allopolyploid formation. Further progress toward under- standing polyploid origins requires studies in natural populations that quantify: (a) the frequency of unreduced gametes, (b) the effectiveness of triploid bridge pathways, and (c) the rates of interspecific hybridization.
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The role of polyploidy in facilitating invasiveness of introduced plants has not been well explored. Examination of traits of diploid and polyploid plants in both their native and introduced ranges can shed light on evolutionary processes occurring postintroduction in invasive plants. We determined the distribution and prevalence of cytotypes of Solidago gigantea in both its native range (USA) and introduced range (Europe), and measured a suite of biochemical, physiological, and reproductive characters for plants from both continents. Tetraploids were the most frequent cytotype encountered on both continents, while hexaploids were found only in the USA. Hexaploids were the most distinctive cytotype, with fewer differences observed between diploids and tetraploids. Comparison of diploids and tetraploids in the USA and Europe showed that traits changed in concert for both cytotypes. Both diploids and tetraploids in Europe had reduced concentrations of three classes of secondary chemical and invested relatively more into rhizomes than into flowers. The same changes occurring in both cytotypes in the introduced range show that altered phenotypes of European plants are not due to shifts in the proportions of cytotypes but instead occur within them. There was no evidence that polyploids evolve more quickly in the introduced range.
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Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P< or =0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of "true-to-type" propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.
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Chromosome doubling was induced in vitro in a diploid hybrid of Rosa rugosa Thunb. using oryzalin as the spindle inhibitor. Nodal sections, 2 mm long, were exposed to 2.5 or 5 microM oryzalin and 10 mm nodal sections were exposed to 5 microM oryzalin for 0 (controls), 6, 12, 24 and 48 h. The ploidy of the emergent shoots was determined by flow cytometry. The frequency of tetraploid and mixoploid leaves that developed from 2 mm nodal sections exposed to 5 microM oryzalin peaked at 12 h exposure, when 35% of the leaves were tetraploid, but fell after longer exposures. Fewer tetraploid and mixoploid leaves were found when 2 mm nodes were exposed to 2.5 microM oryzalin for 6 and 12 h, indicating that it took longer for a spindle inhibiting concentration of oryzalin to build up in the meristem. However, the frequencies of tetraploid and mixoploid leaves continued to rise after 12 h and were highest at 48 h, when 44% were tetraploid. In treatments with 5 microM oryzalin, the frequencies of tetraploid and mixoploid leaves were lower, at equivalent exposure times, in 10 mm nodes than 2 mm nodes. This suggests that oryzalin diffused to the meristem mainly via the cut surfaces and that access via the epidermis and cuticle was impeded.
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The glandular secretory system in Cannabis sativa L. (marihuana) consists of three types of capitate glandular hairs (termed bulbous, capitate-sessile, and capitate-stalked) distinguishable by their morphology, development, and physiology. These gland types occur together in greatest abundance and developmental complexity on the abaxial surface of bracts which ensheath the developing ovary. Bulbous and capitate-sessile glands are initiated on very young bract primordia and attain maturity during early stages of bract growth. Capitate-stalked glands are initiated later in bract growth and undergo development and maturation on medium, to full sized bracts. Glands are epidermal in origin and derived, with one exception, from a single epidermal initial. The capitate-stalked gland is the exception and is of special interest because it possesses a multicellular stalk secondarily derived from surrounding epidermal and subepidermal cells. Glands differentiate early in development into an upper secretory portion and a subtending auxiliary portion. The secretory portion, depending on gland type, may range from a few cells to a large, flattened multicellular disc of secretory cells. The secretory portion produces a membrane-bound resinous product which caps the secretory cells. Capitate-stalked glands are considered to be of particular evolutionary significance because they may represent a gland type secondarily derived from existing capitate-sessile glands.
Book
Preface. Acknowledgments. Introduction. Fatty acids. Acetylenic compounds in plants. Plant waxes. Polyketides. Benzoquinones. Naphthoquinones, and anthraquinones. Shikimic acid pathway. Phenylpropanoids. Coumarins. 2-pyrones, stilbenes, dihydrophenanthrenes, and xanthones. Flavonoids. Tannins. Non-protein amino acids. Peptides. Carbohydrates. Cyanogenic glycosides, and Cyanolipids. Glucosinolates. Introduction to terpenes. Monoterpenes. Iridoid monoterpenes. Sesquiterpenes. Diterpenes and sesterterpences. Triterpenes and steroids. Saponins and cardenolides. Limonoids, Quassinoids and related compounds. Tetraterpenes or cartenoids. Limonoids, quassinoids, and related compounds. Simple aimines, simple aromatic and pyridine alkaloids. Pyrrolidine, tropane, piperidine, and polyeketide alkaloids. Pyrrolizidine, quinolizidine and indolizidine alkaloids. Alkaloids derived from anthranilic acid. Isoquinoline and benzylisoquineoline alkaloids. Alkaloids derived from both tyrosine and phenylalanine. Indole alkaloids. Ergot and other indole alkaloids. Alkaloids of terpenoid orgin. Miscellaneous types of alkaloids.
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The growth of medicinal plants and biosynthesis of secondary metabolites is influenced by plant growth regulators. In this study we investigated influence offour levels (0, 500, 1000, and 1500 mg l -1 ) of2- chloroethyltrimethylammonium chloride (Cycocel), a plant growth retardant, on growth parameters and some biochemical parameters of cannabis plants. Cycocel only at 500 mg l -1 decreased shoot length of male plants. The fresh weight of leaves in female plants decreased with 1500 mg l -1 Cycocel. The other Cycocel treatments increased the fresh weight of leaves in female and male plants. Root and stem fresh weight of male and female plants showed an increase in most treatments (except for 1500 mg l -1 Cycocel). Cycocel at 1000 mg l -1 had enhancing effect on the fresh weight of male and female flowers. Also, the plants treated with Cycocel had a higher content of soluble carbohydrates and protein. Malondialdehyde content was decreased in male and female plants by 500 mg l -1 Cycocel treatment. Tetrahydrocannabinol (THC) content increased in male plant leaves under 1000 and 1500 mg l -1 Cycocel treatment, but in female plants only 500 mg l -1 Cycocel caused an increase in THC content in leaves. While Cycocel decreased cannabidiol (CBD) content in male leaves, in female leaves it increased CBD concentration. In fact, only 500 mg l -1 Cycocelled to more CBD content in female flowers while the other treatments declined CBD content in female flowers. The findings showed that the response to Cycocel depended on the sex in cannabis plants. Ineffectiveness of Cycocel in reducing shoot length suggests that cannabis is insensitive to Cycocel as an inhibitor of gibberellin biosynthesis and its application at specific concentrations can be used to improve growth.
Article
Tetraploid forms of lisianthus [ Eustoma grandiflorum (Grisebach) Schinners] were produced by applying 0.5% (w/v) aqueous colchicine to seedling apices. The tetraploids had stronger stems and reduced plant stature, which provided better support to flowers than diploids.
Chapter
Quinones, naphthoquinones, and anthraquinones are found in many types of higher plants and fungi. About 600 naturally occurring quinones have been described. More than half of these are from one plant family, the Rubiaceae (Haslam, 1974; Harborne, 1982). Quinones are cyclic α,ß-diketones of such a structure that they are converted by reduction into hydroquinones, that is, phenols containing two hydroxyl groups such as (2) (Fig. 6.1) (Morrison and Boyd, 1973). The reduced and oxidized forms are closely balanced energetically and are easily interconverted.
Article
This study is aimed at testing the efficiency of colchicine on inducing polyploidy in Cannabis sativa L. and investigation of effects of polyploidy induction on some primary and secondary metabolites. Shoot tips were treated with three different concentrations of colchicine (0, 0.1, 0.2 % w/v) for 24 or 48 h. The biggest proportion of the almost coplanar tetraploids (43.33 %) and mixoploids (13.33 %) was obtained from the 24-h treatment in 0.2 and 0.1 % w/v, respectively. Colchicine with 0.2 % concentration and 48 h duration was more destructive than 24 h. The ploidy levels were screened with flow cytometry. The biochemical analyses showed that reducing sugars, soluble sugars, total protein, and total flavonoids increased significantly in mixoploid plants compared with tetraploid and diploid plants. Tetraploid plants had a higher amount of total proteins, total flavonoids, and starch in comparison with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabinol in mixoploid plants only, but tetraploid plants had lower amounts of this substance in comparison with diploids. Also, we found such changes in protein concentration in electrophoresis analysis. In overall, our study suggests that tetraploidization could not be useful to produce tetrahydrocannabinol for commercial use, and in this case, mixoploids are more suitable.
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... In Plant Speciation , Grant (1981) devoted five chapters (15% of the total text) to polyploidy, reflecting the importance of the topic both to the author and to plant biologists. We update Grant&apos;s (1981) coverage by highlighting some ...
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Seed set in triploid Musa spp. L. is very low due to high levels of sterility. This makes cross breeding of plantain and banana difficult. Nonetheless, several triploid plantain and banana cultivars produce seeds after hand pollination with diploid parents. This paper reports results about the pattern of seed variation, success of in vitro germination and hybrid production after triploid-diploid crosses. Between-and within-clones differences in seed set were observed. ABB cooking bananas had the highest rates of seed production followed by AAB French plantains. Plantains produced more diploid than tetraploid offsprings after triploid-diploid crosses, which suggests that meiosis resulted in the production of balanced haploid gametes (n = x) due to normal chromosome behaviour between homologous genomes. Seed set variation within clones was influenced by both the production of 2n (= 3x) gametes in the triploid parent and climatic factors. Indeed, seed production, embryo and seed germination success, and euploid and tetraploid hybrid production followed a pattern of seasonal variation reaching a peak in September. High frequency of seed with tetraploid embryos was obtained in the period from January to mid-March. The results demonstrate that production of desirable tetraploids would be more efficient when hand pollinations are done under high temperature, high solar radiation and low relative humidity. These seem to be the most convenient conditions for relatively high production of 2n eggs as compared to haploid gametes.
Article
Parry, M. A. J., Schmidt, C. N. G., Cornelius, M. J., Millard, B. N., Burton, S., Gutteridge, S., Dyer, T. A. and Keys, A. J. 1987. Variation in properties of ribulose-l,5-bisphosphate carboxylase from various species related to differences in amino acid sequences.—J. exp. Bot. 38: 1260–1271. Properties of ribulose-P2-carboxylases from wheat, maize, tobacco, spinach and pea and one photosynthetic bacterium (Rhodospirillum rubrum) were compared. Electrophoresis showed no differences in mass of the large subunit polypeptides of the higher plant carboxylases. The small subunit polypeptides from spinach, pea and tobacco carboxylases had similar molecular weights of about 14000. This value is smaller than that of the small subunit of wheat and larger than that of the small subunit of maize (carboxylase). The purified carboxylases differed in their content of a rapidly activating form of the enzyme and in their sensitivity to Mg²⁺. The differences were not easily fitted into discrete categories so that no clear classification was possible on the basis of activation characteristics. Relative activities of the various enzymes as carboxylases and oxygenases were quantified as specificity factors. The carboxylase from R. rubrum, was much less effective as a carboxylase compared to an oxygenase than carboxylases from the higher plants. Specificity factors for the higher plant enzymes were between 77 and 104. The highest values were recorded for the carboxylase of wheat and the lowest for carboxylases of maize and tobacco. The differences in specificity factors between carboxylases from wheat, maize and tobacco are discussed in relation to differences in amino acid sequences of the large subunit polypeptide. Four differences in residues within the active site may be relevant with respect to the observed differences in enzyme properties. Mild trypsinolysis very rapidly decreased the activity of carboxylases from maize, wheat and spinach but pea, barley and tobacco carboxylases were inactivated more slowly and the enzyme from R. rubrum was not affected at all.
Article
The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n=2x=26) but nine were tetraploid (2n=4x=52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.
Article
Enormous strides have been made in the study of polyploidy over the last 20 yr. Here, we highlight some of these discoveries and note where our understanding of polyploid evolution has changed. Genetic and genomic studies have dramatically altered the polyploidy paradigm. The estimated frequency of polyploidy has increased, and it is now recognized that multiple origins are the rule for most polyploids. Likewise, autopolyploidy is much more common than traditionally maintained. Rapid genomic rearrangements, genomic downsizing, movement of genetic elements across genomes, and the movement of foreign genetic materials into the polyploid genome illustrate the complex dynamics of polyploid genomes. Following polyploidization, both genetic and epigenetic mechanisms may play an important role in altering gene expression. Ecological studies reveal that plant polyploidy can have profound effects on interactions with animal herbivores and pollinators and that polyploidy may trigger changes in the reproductive biology of a species. Despite the recent advances in our understanding of polyploid evolution, many exciting aspects remain under-investigated. Some of these include the consequences of genetic and genomic changes in natural polyploid populations, the physiological and ecological effects of polyploidy, and whether recurrent polyploidy prompts evolution to repeat itself.
Article
The University of Mississippi has a contract with the National Institute on Drug Abuse (NIDA) to carry out a variety of research activities dealing with cannabis, including the Potency Monitoring (PM) program, which provides analytical potency data on cannabis preparations confiscated in the United States. This report provides data on 46,211 samples seized and analyzed by gas chromatography-flame ionization detection (GC-FID) during 1993–2008. The data showed an upward trend in the mean Δ9-tetrahydrocannabinol (Δ9-THC) content of all confiscated cannabis preparations, which increased from 3.4% in 1993 to 8.8% in 2008. Hashish potencies did not increase consistently during this period; however, the mean yearly potency varied from 2.5–9.2% (1993–2003) to 12.0–29.3% (2004–2008). Hash oil potencies also varied considerably during this period (16.8 ± 16.3%). The increase in cannabis preparation potency is mainly due to the increase in the potency of nondomestic versus domestic samples.
Article
Tetraploids were induced successfully from in vitro plants of diploid Alocasia `Green Velvet' (2n=28) by treating shoot tips with colchicine or oryzalin. Colchicine at a concentration of 0.01, 0.05, 0.1% and oryzalin at 0.005, 0.01, 0.05% (both were dissolved in 1% DMSO, dimethylsulfoxide) were tested for 24, 48 and 72 h. Ploidy levels could be easily determined by flow cytometry. In total, 396 plantlets were investigated. Controls with DMSO maintained the diploid status; 22 tetraploids and 22 chimeras were obtained from treatments with colchicine and oryzalin. The most efficient condition for inducing tetraploids seemed to be treatment with 0.01% oryzalin for 24 h. Variation in the morphological characteristic of leaf shape was found among the multiplied plants under the same growing condition. While the leaves of diploids had elongated-heart shape, the leaves of tetraploids and chimeras tended to be round.
Article
An autotetraploid population of caraway (Carum carvi L.) was produced by treating the growing points of young plants with a 0.2% colchicine solution. Selection on morphology, pollen size and pollen shape, followed by a selection on seed size proved to be an effective way to identify the mixoploid C0 plants. Three percent of the treated plants produced a tetraploid progeny. Moderate selection in the C1 and the C2 population resulted in an improvement of the average seed-set to the amount of 50% of the diploid control, with a range of 21–78%. The 1000-kernel weight of the tetraploid material was 7 g, compared to 3 g in the diploid control. The oil content of the autotetraploid seed was increased with 35.6%; the increase in carvone amounted to 6.9%. The results of the experiment indicate that breeding work in autotetraploid caraway may result in a new cultivar.
Article
Artificial polyploidy generally enhances the vigour of determinate plant parts and may be favourable where vegetative organs and biomass constitute the economic product. Giving suitable examples, it is shown that genomic multiplication can confer enhanced production and/or qualitative improvement in the biochemical profile of secondary metabolites. Allopolyploidisation can bring about natural complementation of biosynthetic pathways to harness the useful metabolites. A plea is made to utilise the induced polyploidy approach as a rapid means to attain enhanced production of secondary metabolites: pharmaceuticals, aroma chemicals, etc. The necessary prerequisites that may be needed for achieving genetic stability and reproductive success of the induced polyploids are outlined.
Article
This paper reports the use of liquid chromatography for the separation and determination of the major cannabinoids extracted from hashish samples. The direct coupling to the mass spectrometer enables the selective identification both of neutral and acidic cannabinoids. The developed method does not require any preliminary derivatization and should, therefore, be of interest in forensic analysis for simple and unequivocal determination of hashish constituents.
Article
Tetraploids plants of Anthurium andraeanum “Arizona” were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e., 0.1, 0.2, 0.3%) for about 3, 5 and 7h, and then were transferred into Murashige and Skoog medium containing 3mg/l BAP+0.2mg/l 2,4-D. After 60days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast, the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2 to 7.6%. The best induction was obtained with a 5-h, treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation. Keywords Anthurium andraeanum –Tetraploid–Colchicine–Flow cytometry–Variation
Article
The root ofSalvia miltiorrhiza is a traditional Chinese medicinal plant used as an important drug to cure cardiovascular diseases. Research on the technology of induction, identification and chemical analysis of polypoid plants is reported. The obtained results indicated that basal MS media plus 10 ppm colchicine can induce polyploid mutants effectively. Tetraploid plants were transferred to the fields so that comparative experiment for further identification, observation and screening of 15 agronomic characteristics could be made. The major chemical compounds, tanshinones, in two tetraploid plants were higher than that in the control. An excellent plant 61-2-22 may develop into a new variety for large scale production.
Article
Sex chromosomes in land plants can evolve as a consequence of close linkage between the two sex determination genes with complementary dominance required to establish stable dioecious populations, and they are found in at least 48 species across 20 families. The sex chromosomes in hepatics, mosses, and gymnosperms are morphologically heteromorphic. In angiosperms, heteromorphic sex chromosomes are found in at least 19 species from 4 families, while homomorphic sex chromosomes occur in 20 species from 13 families. The prevalence of the XY system found in 44 out of 48 species may reflect the predominance of the evolutionary pathway from gynodioecy towards dioecy. All dioecious species have the potential to evolve sex chromosomes, and reversions back from dioecy to various forms of monoecy, gynodioecy, or androdioecy have also occurred. Such reversals may occur especially during the early stages of sex chromosome evolution before the lethality of the YY (or WW) genotype is established.
Article
Alternative breeding strategies, based on colchicine-induced autotetraploids, have been proposed as a means of introducing disease resistance into banana breeding programs. This paper describes techniques for the in vitro induction of banana autotetraploids by the use of colchicine on cultured explants. The technique can be readily applied and large numbers of autotetraploids produced. The optimum treatment involved immersing shoot tips in a 0.5% w/v colchicine solution for 2 h under aseptic conditions. Dimethyl sulfoxide (DMSO) was applied with the colchicine treatments to increase cell permeability and so absorption of colchicine, resulting in the optimum treatment unchanged at 0.5% colchicine, but including the addition of 2% v/v DMSO. Of the shoot tips treated over 30% were induced to the autotetraploid level. Methods for in vitro selection of induced tetraploids from treated diploid plantlets were also developed. Tetraploid plants were more robust with thicker pseudostems, roots and broader leaves than diploids and they could be selected on these morphological characteristics. Mean stornatal lengths of diploid banana plants growing in vitro were significantly smaller (16.0 pm) than the tetraploids (26.9pm) and were used as a more reliable indicator of ploidy than morphological criteria alone. A root tip squash technique using carbol fuchsin was developed for positive confirmation of ploidy change by chromosome counts. Although chimerism and reversion to the diploid form occurred, it was not considered a problem because of the large number of autotetraploids induced. Stable autotetraploids were recovered and established in the field and were characterised by their large, drooping leaves and thick pseudostems. They have retained these characteristics for more than 3 years in the field.
Article
Polyploidy is often assumed to increase the spread and thus the success of alien plant species, but few empirical studies exist. We tested this hypothesis with Centaurea maculosa Lam., a species native to Europe and introduced into North America approximately 120 years ago where it became highly invasive. We analyzed the ploidy level of more than 2000 plants from 93 native and 48 invasive C. maculosa populations and found a pronounced shift in the relative frequency of diploid and tetraploid cytotypes. In Europe diploid populations occur in higher frequencies than tetraploids and only four populations had both cytotypes, while in North America diploid plants were found in only one mixed population and thus tetraploids clearly dominated. Our results showed a pronounced shift in the climatic niche between tetraploid populations in the native and introduced range toward drier climate in North America and a similar albeit smaller shift between diploids and tetraploids in the native range. The field data indicate that diploids have a predominately monocarpic life cycle, while tetraploids are often polycarpic. Additionally, the polycarpic life-form seems to be more prevalent among tetraploids in the introduced range than among tetraploids in the native range. Our study suggests that both ploidy types of C. maculosa were introduced into North America, but tetraploids became the dominant cytotype with invasion. We suggest that the invasive success of C. maculosa is partly due to preadaptation of the tetraploid cytotype in Europe to drier climate and possibly further adaptation to these conditions in the introduced range. The potential for earlier and longer seed production associated with the polycarpic life cycle constitutes an additional factor that may have led to the dominance of tetraploids over diploids in the introduced range.
Article
Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA(3)) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA(3) caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Delta(9)-tetrahydrocannabinol (THC) contents and an increase in alpha-tocopherol content. The treated plants with GA(3) showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and beta-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA(3) can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
This chapter discusses the use of 4', 6-diamidino-2-phenylindole (DAPI) for flow cytometry (FCM). DAPI has been used from many years. It proved to be a specific, highly fluorescent stain, very well suited for FCM of DNA in whole cells, in nuclei, and in chromosomes. The chapter presents the staining protocol that has been developed for use in cell suspensions after fixation with 70% ethanol. It is applicable in specimens that have to be stored before measurement. The procedure consists of a pretreatment of cells with citric acid plus Tween 20 to produce a suspension of isolated nuclei, followed by the addition of a solution of disodium phosphate or trisodium citrate with DAPI, which raises the pH and stains the DNA. This protocol is recommended for the accurate and reproducible measurement of the nuclear DNA, especially for the assessment of DNA content variations in cell populations affected by chemical or physical mutagens and for the discrimination of spermatogenic cells.
Article
Changes in ploidy occurred early in the diversification of some animal and plant lineages and represent an ongoing phenomenon in others. While the prevalence of polyploid lineages indicates that this phenomenon is a common and successful evolutionary transition, whether polyploidization itself has a significant effect on patterns and rates of diversification remains an open question. Here we review evidence for the creative role of polyploidy in evolution. We present new estimates for the incidence of polyploidy in ferns and flowering plants based on a simple model describing transitions between odd and even base chromosome numbers. These new estimates indicate that ploidy changes may represent from 2 to 4% of speciation events in flowering plants and 7% in ferns. Speciation via polyploidy is likely to be one of the more predominant modes of sympatric speciation in plants, owing to its potentially broad-scale effects on gene regulation and developmental processes, effects that can produce immediate shifts in morphology, breeding system, and ecological tolerances. Theoretical models support the potential for increased adaptability in polyploid lineages. The evidence suggests that polyploidization can produce shifts in genetic systems and phenotypes that have the potential to result in increased evolutionary diversification, yet conclusive evidence that polyploidy has changed rates and patterns of diversification remains elusive.
Article
It is often anticipated that many of today's diploid plant species are in fact paleopolyploids. Given that an ancient large-scale duplication will result in an excess of relatively old duplicated genes with similar ages, we analyzed the timing of duplication of pairs of paralogous genes in 14 model plant species. Using EST contigs (unigenes), we identified pairs of paralogous genes in each species and used the level of synonymous nucleotide substitution to estimate the relative ages of gene duplication. For nine of the investigated species (wheat [Triticum aestivum], maize [Zea mays], tetraploid cotton [Gossypium hirsutum], diploid cotton [G. arboretum], tomato [Lycopersicon esculentum], potato [Solanum tuberosum], soybean [Glycine max], barrel medic [Medicago truncatula], and Arabidopsis thaliana), the age distributions of duplicated genes contain peaks corresponding to short evolutionary periods during which large numbers of duplicated genes were accumulated. Large-scale duplications (polyploidy or aneuploidy) are strongly suspected to be the cause of these temporal peaks of gene duplication. However, the unusual age profile of tandem gene duplications in Arabidopsis indicates that other scenarios, such as variation in the rate at which duplicated genes are deleted, must also be considered.
Article
Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 degrees C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3-4 days later) than diploid plants.
Article
The cannabis plant (Cannabis sativa L.) and products thereof (such as marijuana, hashish and hash oil) have a long history of use both as a medicinal agent and intoxicant. Over the last few years there have been an active debate regarding the medicinal aspects of cannabis. Currently cannabis products are classified as Schedule I drugs under the Drug Enforcement Administration (DEA) Controlled Substances act, which means that the drug is only available for human use as an investigational drug. In addition to the social aspects of the use of the drug and its abuse potential, the issue of approving it as a medicine is further complicated by the complexity of the chemical make up of the plant. This manuscript discusses the chemical constituents of the plant with particular emphasis on the cannabinoids as the class of compounds responsible for the drug's psychological properties.