•Efficient in vitro shoot regeneration from leaves using TDZ in an induction medium is proposed.•Leaf regeneration capacity can be increased by pre-culture of donor shoots with TDZ.•In vitro ...polyploidisation method for apple has been elaborated.•Higher tetraploid production can be achieved through the use of shoot explants and APM as antimitotic agent.
Triploids and tetraploids of Malus×domestica Borkh. are widely used in breeding programmes because they are characterised by lush growth, larger organs and greater resistance to biotic and abiotic stress. The aim of this study was to develop an in vitro method of apple polyploidisation using leaf and shoot explants of six cultivars. At first, the procedure of efficient in vitro shoot regeneration from leaves was optimised. The leaf regeneration capacity was generally increased by preculture of donor shoots with 4.5μM thidiazuron (TDZ) compared to a standard preculture in the presence of 4.5μM benzyladenine (BA). Therefore, for polyploidisation, the leaf explants were collected from four-week shoot cultures that were pretreated with TDZ and the shoot explants derived from standard four-week multiplication subculture. The explants were incubated for six days in darkness on induction medium containing 2.5μM 1-naphthaleneacetic acid (NAA) and 4.5μM TDZ or 18μM BA (leaves) or and 0.5μM indole-3-butyric acid (IBA) and 4.5μM BA (shoots) and one antimitotic agent: colchicine, trifluralin, oryzalin or amiprophos methyl (APM). Subsequently, the explants were cultured for four weeks in darkness on the medium without antimitotic agents and then subcultured on multiplication medium containing BA over a 16-h photoperiod. From leaf explants, 58, 38 and 6 tetraploids were obtained for three out of six cultivars, ‘Pinova’, ‘Redchief’ and ‘Sander’, respectively. For leaf explants, the highest polyploidisation efficiency, approximately 20%, was recorded for colchicine at 125 and 250mgl−1. For shoot explants, tetraploids were detected for all the cultivars with the higher tetraploid numbers – 13, 26 and 27, for Co-op 32, ‘Free Redstar’ and ‘Redchief’, respectively – and a few tetraploids were obtained for other genotypes (‘Gala Must., ‘Sander’ and ‘Pinova’). For shoot explants, treatment with 10mgl−1 APM resulted in the highest polyploidisation efficiency of 9.8%. Additionally, mixoploids were detected three times more than tetraploids when shoots were used for polyploidisation, compared to the sporadic occurrence of mixoploids when leaf explants were used.
Efficacy of oryzalin to generate novel genotypes of Mentha spicata L. with superior morphology and enhanced levels of essential oil via in vitro polyploidization was assessed. Micropropagated nodal ...segments were treated with oryzalin at 20, 40, and 60 μM for 24 and 48 h. The survival rate of the nodal segments dropped significantly with the increasing concentration and duration of oryzalin. Further, the ploidy level of the survived plants was confirmed using flowcytometry and chromosome counting. A total of six polyploids (2 n = 6x = 72) and six mixoploid plants were obtained. The highest frequency of polyploids (8 %) was induced in treatment where 40 μM oryzalin was applied for 48 h. The obtained polyploids exhibited significantly larger, thick, dark green leaves with elongated stomata compared with the control genotype. The polyploid plants also displayed higher moisture content and vigorous lateral growth. The essential oil yield of hexaploid M. spicata exhibited a 48.85 % increase, rising from 1.74 % in control to 2.59 % (v/w) in genotype P3. The major components of essential oils were carvone and limonene, which also increased significantly compared with the control genotype. The nutrient profile of the studied genotypes was influenced vastly and showed significant variations. Overall, the employed methodology using oryzalin as an antimitotic agent for polyploid induction in M. spicata was found to be effective. Despite relatively low polyploid induction frequency, the obtained polyploid genotypes demonstrated various superior agronomical traits. The genotypes obtained could serve to aid the commercial demands.
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•Oryzalin as an antimitotic agent was tested for the first time in Mentha spicata.•Induced polyploid plants showed stable ploidy levels and grew normally over time.•The hexaploid plants displayed superior morphological parameters.•Enhanced levels of essential oil yield were obtained in the polyploid genotype.
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•A protocol for in vitro polyploidization of Thymus vulgaris using oryzalin is proposed.•Tetraploid plants expressed enhanced morphological characteristics.•Essential oil yield ...increased in tetraploid plants.•Thymol and carvacrol content increased in the new genetic material.•Obtained autotetraploid genotype could be used for commercial essential oil production.
Garden thyme (Thymus vulgaris L.) is well known for its aromatic, anti-pathogenic and antioxidant properties and is commonly cultivated for ornamental, culinary and medicinal uses. The aim of this study was to induce ployploidy in T. vulgaris in order to obtain tetraploid plants (2n = 4x = 60) with enhanced morphological traits and increased essential oil content from diploid plants (2n = 2x = 30) using in vitro-induced polyploidization. Nodal segments were treated with four different concentrations of oryzalin (20, 40, 60 and 80 μM) for 24 and 48 h. The ploidy levels of treated and regenerated plants were determined by flow cytometry analysis. The chemical composition of hydrodistilled essential oil was analysed by gas chromatography with quadrupole time-of-flight mass spectrometry systems. Oryzalin treatment resulted in three tetraploid plants at a concentration of 80 μM for 24 h. Obtained tetraploid plants presented several morphological changes (increased plant weight, height, leaf length, breadth and thickness) in comparison to diploid plants. Furthermore, polyploidization demonstrated an increase in the production of secondary metabolites; tetraploids yield of essential oil increased from 0.81 % to 1.19 % and the content of several main components increased, including thymol and carvacrol by 18.01 % and 0.49 %, respectively. The obtained genotype could be implemented as a new variety for commercial production of garden thyme.
•Polyploidization in wallflower created tetraploid Erysimum cheiri (2n = 4x = 24).•The best chromosome doubling result obtained by 150 mgl−1 oryzalin for 36 h.•The 4x wallflower had more flowers, ...longevity, size and flowering duration.•Anthocyanin and sugars of 4x plants were increased by 56% and 140%, respectively.•Tetraploids’ cytokinin, salicylic acid, jasmonic acid and abscisic acid increased.
Wallflower (Erysimum cheiri (L.) Crantz is one of the most important bedding flowers and potted plants in the floriculture industry. Its widespread cultivation is hindered due to self-incompatibility and limited breeding. The present study was conducted to investigate the effects of polyploidy induction on different characteristics of wallflower. Seeds were treated with colchicine (0, 0.5, 0.75 and 1%) (w/v) and oryzalin (0, 50, 100 and 150 mgl−1) along various exposure times (24, 36 and 48 h). The highest tetraploidy induction (53%) was seen in 150 mgl−1 oryzalin treatment for 36 h. The chromosome doubled plants indicated increase in trichome number, pollen size, stomata size and number of chloroplasts in the stomata guard cells, while stomata density and pollen number were decreased. The new tetraploid induced wallflowers showed compact growth habit with shorter internodes and roots, while leaves were bigger (60%) and purplish, flowering period was prolonged (65%), and flowers showed greater longevity (41%), diameter (47%) and number (78%), compared to diploid controls. Their anthocyanin and total soluble solids contents were increased by 56% and 140%, respectively. UPLC-MS analysis indicated that phytohormone contents of Ze, SA, JA and ABA were significantly increased in the tetraploid induced plants than diploid controls, while IAA, GA3 and BR were less accumulated. The new ornamental features developed for the first time in wallflower proves that polyploidization is a promising tool to introduce a commercial flowering pot plant cultivar and a new genetic resource for future breeding programs.
The organization of cellulose microfibrils is critical for the strength and growth of plant cell walls. Microtubules have been shown to play a key role in controlling microfibril organization by ...guiding cellulose synthase complexes 1–4. However, cellulose synthase trajectories can be maintained when microtubules are removed by drugs, suggesting a separate guidance mechanism is also at play 1, 5, 6. By slowing down microtubule dynamics, we reveal such a mechanism by showing that cellulose synthase complexes can interact with the trails left by other complexes, causing them to follow the trails or disappear. The stability of the trails, together with the sensitivity of their directions to cellulase treatment, indicates they most likely reflect nascent cellulose microfibrils. Over many hours, this autonomous mechanism alone can lead to a change in the dominant orientation of cellulose synthase trajectories. However, the mechanism can be overridden by the microtubule guidance system. Our findings suggest a dual guidance model, in which an autonomous system, involving interaction between cellulose synthases and microfibrils, can maintain aligned cellulose synthase trajectories, while a microtubule guidance system allows alignments to be steered by environmental and developmental cues.
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•A dual mechanism guides cellulose synthesis in plant cell walls•Cellulose synthase complexes autonomously follow trails left by previous complexes•Microtubule guidance is dominant over autonomous guidance•Microtubules can guide cellulose synthases directly or indirectly
Chan and Coen reveal a novel mechanism for guiding cellulose synthesizing complexes based on the complexes following trails left by previous complexes. They show that this autonomous mechanism can be overridden by microtubule guidance. This dual guidance system provides a flexible, yet robust control of plant cell wall structure.
Cannabis sativa L. is a diploid (2 x ) herbaceous plant that provides a wide variety of products such as essential oils, fiber, and medicine. Hemp was defined in the 2018 Farm Bill as a Cannabis ...plant with a delta-9 tetrahydrocannabinol concentration of not more than 0.3% on a dry-weight basis. Polyploidy is frequently used in plant breeding to manipulate vigor, reproductive fertility, and biochemistry. By inducing polyploidy/chromosome doubling, we may increase the compounds of interest, principally CBD (cannabidiol), produced by hemp. The purpose of this experiment was to evaluate the efficacy of different treatments of colchicine and oryzalin applied in vivo and in vitro to induce polyploidy in ‘I3’ hemp. After treating vegetative cuttings with colchicine or oryzalin, we had a 31% survival rate. Of the 85 survivors, we recovered two tetraploids: one from the 12-h 0.05% colchicine treatment group and the other from the 12-h 0.2% colchicine treatment group. For the in vitro portion of the experiment, the 12-h 50-µM oryzalin treatment yielded one tetraploid and the 36 h 50 µM oryzalin treatment yielded one cytochimera (mixoploid). The relative efficiency of some treatments showed potential for a simple method to induce tetraploids in clonal hemp for breeding.
Blackberries are widely used as a fresh fruit, while being highlighted as an anti-inflammatory and antioxidant source. Polyploidization is considered as a valuable and fast breeding method to develop ...new cultivars with enhanced characteristics. In this research, a procedure was adapted in vitro to generate polyploid plants (3x, 4x, 6x and 8x) in seven genotypes assigned to five blackberry species, including Rubus sanctus (3 genotypes), R. hirtus, R. caecious, R. persicus and R. discolor. Nodal segments were cultured in Murashige and Skoog media supplemented with oryzalin (0, 125, 250 and 375 mgL−1) or colchicine (0, 5, 10 and 15 mgL−1) for different durations (24, 48, 72 and 96 h). Results of cytological analysis showed no significant differences between the ultimate effects of the two anti-mitotic agents (i.e. colchicine and oryzalin), whereas each species reacted differently to polyploidy induction. The best result was obtained in the R. sanctus genotype F (Sari), using 10 mgL−1 oryzalin for 48 h. Manipulative treatments led to the release of octaploid blackberry for the first time. A morphometric analysis of plant leaves showed that polyploidy affected the guard cells, increased the cell size and reduced cell count. The greenness, petiole length and chlorophyll content of leaves were significantly higher in the hexaploid R. sanctus genotype C (Lahijan), while the thickness and size of leaves were significantly larger and the spine length was significantly longer in the octaploid R. persicus. The developed in vitro procedure for polyploidy induction and detection is feasible and could be used as a promising tool to create blackberry cultivars with enhanced fruit characteristics, along with possible resistance to environmental stress.
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Oryzalin (ORY) is a dinitroaniline derivative that inhibits the microtubule polymerization in plants and parasitic protozoa by selectively binding to the α-tubulin subunit. This herbicidal agent ...exhibits good antiprotozoal activity against major human parasites, such as Toxoplasma gondii (toxoplasmosis), Leishmania mexicana (leishmaniasis), and Plasmodium falciparum (malaria). Previous chemical mutagenesis assays on T. gondii α-tubulin (TgAT) have identified key mutations that lead to ORY resistance. Herein, we employed alchemical free energy methods and molecular dynamics simulations to determine if the ORY resistance mutations either decrease the TgAT's affinity of the compound or increase the protein stability. Our results here suggest that L136F and V202F mutations significantly decrease the affinity of ORY to TgAT, while T239I and V252L mutations diminish TgAT's flexibility. On the other hand, protein stability predictors determined that R243S mutation reduces TgAT stability due to the loss of its salt bridge interaction with E27. Interestingly, molecular dynamics simulations confirm that the loss of this key interaction leads to ORY binding site closure. Our study provides a better insight into the TgAT-ORY interaction, further supporting our recently proposed ORY-binding site.
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•Toxoplasma gondii α-tubulin mutations alter either oryzalin affinity or tubulin stability.•L136F and V202F mutations affect ORY's affinity to T. gondii α-tubulin.•T239I and V252L mutations decrease T. gondii α-tubulin flexibility.•R243S mutation closes ORY-binding site by increasing the H7 kink angle.
Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of ...mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development‐specific requirements of B1‐type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1‐type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1‐type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA‐TUBULIN COMPLEX PROTEIN 3‐INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1‐type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.
Synopsis
Studying multiple mutant combinations, this study reveals the role of B1‐type cyclins in plant development and microtubule organization.
The B1‐type cyclin group is comprised of four true members in Arabidopsis thaliana.
B1‐type cyclins have a tissue‐specific, yet overlapping, role in plant development.
CYCB1;2 is the most central B1‐type cyclin.
Microtubule organization is regulated by B1‐type cyclins and likely achieved by phosphorylation of microtubule‐associated proteins.
Studying multiple mutant combinations, this study reveals the role of B1‐type cyclins in plant development and microtubule organization.
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