In Vitro Lab
by Prof. Stefaan Werbrouck
Topolin cytokinins enhanced shoot proliferation, reduced hyperhydricity and altered cytokinin metabolism in Pistacia vera L. seedling explants.
Dhekra Abdouli (UGent) , Lenka Plačková, Karel Doležal, Taoufik Bettaieb and Stefaan Werbrouck (UGent) (2022)
PLANT SCIENCE. 322.
Abstract The effect of 10 mu M meta-topolin (mT) and meta-topolin riboside (mTR) on in vitro proliferation and anomalies of Pistacia vera L. were evaluated and compared to that of 6-benzylaminopurine (BA). The highest proliferation rate (15.6) was recorded in the mT-medium, with a value 6 times higher than in BA-medium. Moreover, the lowest percentage of hyperhydric usable shoots (58,9%) and callus weight (46,9%) were found in mTR-treated shoots. Shoot tip as well as leaf necrosis were not influenced by cytokinin (CK) type. Image analysis was used to evaluate photosynthetic efficiency as well as anthocyanin index. Photosynthesis was more efficient with BA and mTR but the higher anthocyanin accumulation in BA-treated shoots suggests more stress. Endogenous CKs and their metabolites were determined in seedlings and, for the first time, the metabolism of exogenous BA, mT and mTR was studied in pistachio. The stimulating effect on cis-zeatin and its riboside and the appearance of BA and traces of ortho-topolin and para-topolin as natural CKs are discussed. The quantitative and qualitative CK metabolite analyses provides some initial clues as to why topolin would be superior to BA in terms of proliferation rate and avoiding hyperhydricity and allowed a better understanding of the effect of exogenous administration of CK. Keywords Pistachio, Endogenous cytokinin, meta-topolin riboside, Shoot tip necrosis, IN-VITRO, ENDOGENOUS CYTOKININ, AROMATIC CYTOKININ, WILLIAMS BANANAS, PLANT-TISSUES, TIP NECROSIS, PROFILES, MICROPROPAGATION, IDENTIFICATION, ORGANOGENESIS
Fluorine containing topoloin cytokinins for Phalaenopsis amabilis (L.) Blume micropropagation.
Murvanidze, N., Doležal, K., & Werbrouck, S. (2019).
PROPAGATION OF ORNAMENTAL PLANTS, 19(2), 48-51.
Abstract The effect of 10 mu M meta-topolin (mT) and meta-topolin riboside (mTR) on in vitro proliferation and anomalies of Pistacia vera L. were evaluated and compared to that of 6-benzylaminopurine (BA). The highest proliferation rate (15.6) was recorded in the mT-medium, with a value 6 times higher than in BA-medium. Moreover, the lowest percentage of hyperhydric usable shoots (58,9%) and callus weight (46,9%) were found in mTR-treated shoots. Shoot tip as well as leaf necrosis were not influenced by cytokinin (CK) type. Image analysis was used to evaluate photosynthetic efficiency as well as anthocyanin index. Photosynthesis was more efficient with BA and mTR but the higher anthocyanin accumulation in BA-treated shoots suggests more stress. Endogenous CKs and their metabolites were determined in seedlings and, for the first time, the metabolism of exogenous BA, mT and mTR was studied in pistachio. The stimulating effect on cis-zeatin and its riboside and the appearance of BA and traces of ortho-topolin and para-topolin as natural CKs are discussed. The quantitative and qualitative CK metabolite analyses provides some initial clues as to why topolin would be superior to BA in terms of proliferation rate and avoiding hyperhydricity and allowed a better understanding of the effect of exogenous administration of CK. Keywords Pistachio, Endogenous cytokinin, meta-topolin riboside, Shoot tip necrosis, IN-VITRO, ENDOGENOUS CYTOKININ, AROMATIC CYTOKININ, WILLIAMS BANANAS, PLANT-TISSUES, TIP NECROSIS, PROFILES, MICROPROPAGATION, IDENTIFICATION, ORGANOGENESIS
Diphenylurea-derived cytokinin oxidase/dehydrogenase inhibitors for biotechnology and agriculture
Abstract Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10–8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture. Keywords Plant Science, Physiology, Agriculture, biotechnology, CKX inhibitor, crystal structure, cytokinin, cytokinin oxidase/dehydrogenase, diphenylurea, plant tissue culture, stress, yield, ARABIDOPSIS-THALIANA, ZEA-MAYS, BIOCHEMICAL-CHARACTERIZATION, SEED YIELD, OXIDASE, DEHYDROGENASE, EXPRESSION, THIDIAZURON, MAIZE, PURIFICATION
Cytokinin oxidase/dehydrogenase inhibitors stimulate 2iP to induce direct somatic embryogenesis in Coffea arabica
Abstract Somatic embryogenesis in Coffea spp. is important for mass production and genetic engineering. Coffee is an exceptional case, as somatic embryogenesis can be induced by applying a cytokinin such as 2iP as the only plant growth regulator. 1-(2-(2-Hydroxyethyl)phenyl)-3-(3-(trifluoromethoxy)phenyl)urea (3TFM-2HE) and 1-(3-bromo-5-(trifluoromethoxy)phenyl)-3-(2-(2-hydroxyethyl)phenyl)urea (3TFM,5Br-2HE) are two newly designed cytokinin oxidase/dehydrogenase inhibitors (CKX) from the diphenylurea group. We used a Coffea arabica leaf disks bioassay to demonstrate the indirect somatic embryo induction potential of these compounds. The leaf disks were incubated on modified Murashige and Skoog (MS) semi-solid medium in which 3TFM-2HE or 3TFM,5Br-2HE were combined with N-6-(2-isopentenyl)adenine (2iP). Although these compounds do not possess intrinsic cytokinin activity, they enhanced the activity of 2iP, resulting in direct somatic embryogenesis after seven weeks. The best results were obtained with 1 mu M 3TFM-2HE and 5 or 10 mu M 2iP. Maturation of somatic embryos into fully developed plants took place on medium supplemented with 0.5 mu M kinetin and the somatic embryos developed true leaves and a root system. Keywords Agronomy and Crop Science, Plant Science, Physiology, Micropropagation, CKX inhibitor, Somatic embryogenesis, Coffee
Optimizing pear micropropagation and rooting with light emitting diodes and trans-cinnamic acid.
Abstract Meta-Topolin riboside was selected as cytokinin for these experiments. The effects of combinations of monochromatic blue, red and far red LED light were compared to fluorescent light during the micropropagation and rooting of a recalcitrant pear (Pyrus communis L.). During the micropropagation phase, red light gave some particular advantages: maximal shoot length and leaf area were obtained. Under blue light, callus weight quadrupled compared to fluorescent light. Although far red was advantageous for shoot number, shoot quality was inferior because of hyperhydricity and chlorosis as indicated by a low Chlorophyll a+b and carotenoid content. The smallest leaf area was detected under fluorescent light. Blue+red showed significant improvements. Shoot cluster weight and shoot weight/callus weight' ratio was maximal, as well as shoot number and shoot length. Leaves were also dark green, showing a maximal Chlorophyll a+b and carotenoids content. Adventitious rooting of in vitro cultured pear plants was highly affected by different light spectra and the addition of a new rooting compound: trans-cinnamic acid. Without trans-cinnamic acid, limited rooting was observed under red, blue and blue+red light. In combination with trans-cinnamic acid, 100% rooting was achieved under red light. Keywords LED, Shoot multiplication, Auxin, Pyrus, In vitro, IN-VITRO GROWTH, RED-LIGHT, SHOOT REGENERATION, FAR-RED, MORPHOGENESIS, QUALITY, PLANT, IRRADIATION, CULTIVARS, LEDS
Effects of meta–topolin derivatives and temporary immersion on hyperhydricity and in vitro shoot proliferation in Pyrus communis.
Abstract Although micropropagation in temporary immersion systems might increase plant growth and multiplication, it can also cause specific problems such as hyperhydricity and losses by contamination. A new commercial temporary immersion bioreactor, SETIS™, was used to micropropagate two Tunisian pear cultivars, ‘Arbi’ and ‘Mahdia 6’. The latter cultivar was endogenously contaminated by Sphingomonas. Hyperhydricity was inevitable when 5 µM benzyladenine was applied. However, the symptoms could be reduced by lowering the immersion frequency to 3 times per day. Applying 5 µM meta–Methoxy topolin riboside (MemTR) or meta–Topolin riboside (mTR) completely inhibited hyperhydric shoot formation. Moreover, the addition of Plant Preservative Mixture was effective to control Sphingomonas and allowed the plants to proliferate. For both pear cultivars, the highest number of shoots per explant was induced by 5 µM MemTR, whereas the highest leaf area was obtained with 5 µM mTR. The longest shoots were obtained with 5 µM mTR for ‘Arbi’ and 5 µM MemTR for ‘Mahdia 6’. Key message Methoxy topoline-riboside (MemTR) and meta-topoline-riboside (mTR) were used as alternatives to benzyladenine to prevent hyperhydricity during the micropropagation of pears in a new temporary immersion system. These cytokinins also increased the number of good quality shoots, characterized by large leaves and longer shoots. Keywords Horticulture, Cytokinins, Hyperhydricity, Temporary, Immersion, Bioreactor, Contamination, META-TOPOLIN, AROMATIC CYTOKININS, LIQUID-MEDIUM, MICROPROPAGATION, BIOREACTOR, GROWTH, L.
Constant and Intermittent Contact with the Volatile Organic Compounds of Serendipita indica Alleviate Salt Stress In Vitro Ocimum basilicum L.
Abstract Serendipita indica is a plant growth-promoting fungus. It is a natural soil dweller that can colonize the roots of a wide range of plants, including cultivated crops. S. indica has been reported to improve plant nutrient uptake and increase stress tolerance when inoculated into the soil. The present study was undertaken to study the effect of volatile organic compounds (VOCs) of S. indica on salt-stressed Ocimum basilicum 'Fin vert' in vitro, either in a culture vessel with a semi-solid medium or via a modified temporary immersion bioreactor system (SETIS). For all salt concentrations, VOCs of S. indica significantly improved plant growth in both semi-solid medium and SETIS bioreactors. This resulted in heavier and taller plants, more shoots per plant, and longer roots. This was even observed for the control without salt. At 9 g/L NaCl, plants with Serendipita were able to give longer roots than those without (1.2 cm vs. 0.0 and 1.7 cm vs. 1.7 cm) in the semi-solid medium and SETIS, respectively. Nevertheless, the VOCs were not able to make the plant salt tolerant to this high concentration. The increase in total phenolic and flavonoid content and radical scavenging suggest that the antioxidant defense system is triggered by the S. indica VOCs. In the semi-solid system, without VOCs, 1 g/L NaCl led to an increase in total chlorophyll content (TCC) and a significant decrease in TCC was further measured only at 6 g/L NaCl or more. However, when VOCs were added, the bleaching effect of the salt was partially restored, even at 6 and 9 g/L NaCl. A significant decrease in TCC was also measured in the SETIS system at 6 g/L NaCl or more and treatment with VOC did not make any difference. An exception was 9 g/L, where the VOC-treated plants produced more than three times more chlorophyll than the non-treated plants. These findings will encourage the application of Serendipita indica for stress reduction. In addition, the proposed original adaptation of a temporary immersion system will be instrumental to investigate stress reduction associated with volatile compounds and better understand their mechanism of action. Keywords semi-solid medium, SETIS, NaCl, VOCs, basil, ENDOPHYTE PIRIFORMOSPORA-INDICA, PLANT-GROWTH, ANTIOXIDANT ACTIVITY, ARABIDOPSIS-THALIANA, BACTERIAL VOLATILES, FUSARIUM-OXYSPORUM, MEDICINAL-PLANT, TOLERANCE, SALINITY, BARLEY
Effects of salicylic acid on the production of polyphenols and the reducing power of Theobroma cacao calli.
Abstract Cocoa (Theobroma cacao) contains high levels of phenolic compounds with pharmacologically interesting activ-ities. Prior research has not sufficiently investigated obtaining these compounds through in vitro culture by using metabolic elicitation and quantifying polyphenols in undifferentiated cell biomass samples in this spe-cies. In vitro calli obtained from staminodes and petals were elicited with 0.01 and 0.1 mg L-1 salicylic acid (SA). Secondary metabolites were determined in the calli and their culture medium, from the immature and mature flowers, leaves, and fruits of field-grown plants. The most abundant compounds were free amino acids, alkaloids, reducing carbohydrates, phenols, pyrocatecholic tannins, and flavonoids. Resins, triterpenes, ster-oids, saponins, coumarins, and quinones were absent in the calli. A high concentration of total polyphenols (58.4 mu g g(-1)) was obtained from staminode-derived calli elicited with 0.1 mg L-1 SA. With these conditions, the total polyphenol content was 40.22 mu g g(-1) greater than that in the control. In general, SA also stimulated the reducing power activity; a better value (5.87 mu mol mL(-1) Fe2+) was obtained in callus extracts from petals with salicylic acid (0.1 mg L-1). This research was based on a full factorial study design with two levels, a method that allows the desired information to be obtained at the lowest possible cost. Keywords Biotechnology, Theobroma cacao, Salicylic acid, Callus, Polyphenols production, Reducing power
Production of tetraploid plants from cotyledons of diploid Melia volkensii Gurke
Abstract Polyploidy was induced in Melia volkensii (Mukau), a valuable native tree from the semi-arid regions of East Africa. Cotyledons of diploid M. volkensii (2n = 2x = 28) were treated with oryzalin for 0 (control), 1, 2, or 3 h with or without pretreatment with 1.1 & mu;M thidiazuron. Cotyledons treated with 10 mg & BULL;L-1 oryzalin for three hours yielded 40% tetraploids. Pretreatment of cotyledons with thidiazuron for 18 days followed by treatment with oryzalin increased tetraploid plant production to 52.5%, but this also yielded more mixoploids. Compared to diploid M. volkensii, the tetraploid in vitro and young potted plants were compacter, with thicker stems, wider leaves, and a low density of longer and wider stomata. In the coming years, tetraploid M. volkensii plants will be observed in field trials and serve as a basis for further breeding efforts. Keywords in vitro chromosome doubling, Melia volkensii, oryzalin, polyploidy, regeneration, POLYPLOIDIZATION, INDUCTION, REGENERATION, TREE, SEEDLINGS, EXPLANTS, STRESS, FIBER, LINUM
